ORCID Profile
0000-0002-7194-2922
Current Organisations
University of California Davis Department of Plant Pathology
,
Australian National University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Plant Biology | Plant Cell and Molecular Biology | Bioinformatics and computational biology | Forestry Sciences | Evolutionary Biology | Horticultural crop protection (incl. pests diseases and weeds) | Sociology and social studies of science and technology | Sociology and Social Studies of Science and Technology | Epigenetics (incl. Genome Methylation and Epigenomics) | Host-Parasite Interactions | Plant Pathology | Genomics | Phylogeny and Comparative Analysis | Ecological applications | Forestry Pests, Health and Diseases | Horticultural Crop Improvement (Selection and Breeding) | Conservation and Biodiversity | Crop and Pasture Biochemistry and Physiology | Mycology | Biosecurity science and invasive species ecology | Genomics and transcriptomics |
ANZSRC Socio-Economic Objective (SEO)
Wheat | Control of Animal Pests, Diseases and Exotic Species in Farmland, Arable Cropland and Permanent Cropland Environments | Essential Oil Crops (e.g. Tea Tree, Eucalyptus, Lavender, Peppermint, Boronia, Sandalwood) | Expanding Knowledge in Technology | Winter Grains and Oilseeds not elsewhere classified | Control of Plant Pests, Diseases and Exotic Species in Farmland, Arable Cropland and Permanent Cropland Environments | Resourcing of Education and Training Systems | Forest and Woodlands Flora, Fauna and Biodiversity
Related Links
Publications
DNA Quality Control by Agarose Gel Electrophoresis
Publisher: ZappyLab, Inc.
Date: 12-02-2023
DOI: 10.17504/PROTOCOLS.IO.N92LDPQW7L5B/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a step by step guide to assess DNA quality via agarose gel electrophoresis. Agarose gel electrophoresis lets you assess the size of DNA molecules. In addition, you can estimate DNA concentrations and impurities of RNA. During agarose gel electrophoresis DNA gets linearised. Larger molecules take longer to migrate through the gel when migration is driven by a electric potential at the specific pH of the buffer. When running a standard DNA ladder with bands of known molecular size, one can compare the DNA size of ones DNA or PCR s le with the known standards. The final goal is to achieve the following: To assess DNA length of molecules in DNA stock s les and for all PCR reactions. To approximately estimate relative DNA concentrations. To test if negative PCR controls did not lify anything as appropriate. To test if whole DNA extracts contain RNA in addition to DNA. This protocol is applicable for week 5. Protocols progress overview: Week 5 Run a 1% Agarose Gel electrophoresis for all s les of each research group. Some useful explainers and resources: mc/articles/PMC4846332/ iki/Agarose_gel_electrophoresis atch?v=ZDZUAleWX78
High molecular weight gDNA extraction after Mayjonade et al. optimised for eucalyptus for nanopore sequencing v9
Publisher: ZappyLab, Inc.
Date: 29-10-2017
DOI: 10.17504/PROTOCOLS.IO.KHKCT4W
Abstract: Extraction of high quality DNA for long read sequencing e.g. the Oxford Nanopore Optimized for DNA extraction from eucalyptus grandis and eucalyptus pauciflora. This protocol contains an optional Chloroform clean up step which is necessary for eucalyptus but might not be for other tissue. For long DNA fragments don't vortex the DNA s le.
The draft nuclear genome assembly of Eucalyptus pauciflora: new approaches to comparing de novo assemblies
Publisher: Cold Spring Harbor Laboratory
Date: 21-06-2019
DOI: 10.1101/678730
Abstract: Selecting the best genome assembly from a collection of draft assemblies for the same species remains a difficult task. Here, we combine new and existing approaches to help to address this, using the non-model plant Eucalyptus pauciflora (snow gum) as a test case. Eucalyptus pauciflora is a long-lived tree with high economic and ecological importance. Currently, little genomic information for Eucalyptus pauciflora is available. We generated high coverage of long-(Nanopore, 174x) and short-(Illumina, 228x) read data from a single Eucalyptus pauciflora in idual and compared assemblies from four assemblers with a variety of settings: Canu, Flye, Marvel, and MaSuRCA. A key component of our approach is to keep a randomly selected collection of ~10% of both long- and short-reads separate from the assemblies to use as a validation set with which to assess the assemblies. Using this validation set along with a range of existing tools, we compared the assemblies in eight ways: contig N50, BUSCO scores, LAI scores, assembly ploidy, base-level error rate, computing genome assembly likelihoods, structural variation and genome sequence similarity. Our result showed that MaSuRCA generated the best assembly, which is 594.87 Mb in size, with a contig N50 of 3.23 Mb, and an estimated error rate of ~0.006 errors per base. We report a draft genome of Eucalyptus pauciflora , which will be a valuable resource for further genomic studies of eucalypts. These approaches for assessing and comparing genomes should help in assessing and choosing among many potential genome assemblies for a single species.
A Bacterial Tyrosine Phosphatase Inhibits Plant Pattern Recognition Receptor Activation
Publisher: American Association for the Advancement of Science (AAAS)
Date: 28-03-2014
Abstract: Receptors on plant cell surfaces are tuned to recognize molecular patterns associated with pathogenic bacteria. Macho et al. (p. 1509 published online 13 March) found that activation of one of these receptors in Arabidopsis results in phosphorylation of a specific tyrosine residue, which in turn triggers the plant's immune response to the phytopathogen Pseudomonas syringae. P. syringae counters by secreting a specifically targeted phosphatase, thus stalling the plant's immune response.
DNA size selection (>3-4kb) and purification of DNA using an improved homemade SPRI beads solution. v1
Publisher: ZappyLab, Inc.
Date: 03-04-2018
DOI: 10.17504/PROTOCOLS.IO.N7HDHJ6
Abstract: Most noncommercial tradition DNA extraction protocols result in a crude DNA preparation. If the DNA is intended to be used for a high-end application like Nanopore sequencing, it requires a thorough clean-up and size selection before it could be used for sequencing. Solid Phase Reversible Immobilisation (SPRI) magnetic beads is a quick and convenient way of purifying and size selecting intact double-stranded DNA from crude DNA. Most commercially available SPRI beads based DNA purification mix is quite expensive so our lab endeavored to develop an inexpensive beads mix which is as good as the commercially available ones. In this effort, our lab has optimized a beads mix for purifying and size selecting crude DNA extracted from eucalyptus and posted on protocol.io,www.protocols.io/edit/high-purity-high-molecular-weight-dna-extraction-f-n5ydg7w?step=16. However, this solution was not very effective in purifying crude DNA extracted from fungal material. DNA extracted from fungal material is highly viscous which is indicative of high levels of impurities in the DNA preparation. I tried to improve the beads mix for purifying rust DNA by adding 0.25 % (v/v) Tween-20 into the beads mix. I tested beads mix with Tween-20 to see if adding tween into the solution makes any difference in the recovery, purity and size selection. Turns out that bead solutions with Tween-20 make big difference in the size selection and recovery of the DNA compared to the bead solutions without. I calibrated/tested the bead solution with and without Tween-20 on the 1 kb DNA ladder to establish which DNA solution to beads volume ratio gives optimal recovery and size selection. We found that beads mix with 0.25 % Tween-20 works much better in size selection and recovery than beads mix without Tween-20. The best DNA to beads volume ratios were 1.0: 0.9 and 1.0: 1.0.
Blurred lines: integrating emerging technologies to advance plant biosecurity
Publisher: Elsevier BV
Date: 08-2020
Multiplexed effector screening for recognition by endogenous resistance genes using positive defense reporters in wheat protoplasts
Publisher: Cold Spring Harbor Laboratory
Date: 30-04-2023
DOI: 10.1101/2023.04.30.538885
Abstract: A key goal in agriculture is to create pathogen-resistant crop species via breeding or biotechnological approaches. Plant resistance ( R ) and pathogen avirulence ( Avr ) gene interactions play a vital role in pathogen resistance. Efficient molecular screening tools for crops lack far behind their model organism counterparts, yet they are essential to rapidly identify agriculturally important molecular interactions that trigger host resistance. To fill this important gap, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to fifty Avr candidates. Our assay is based on newly identified Avr/R-induced defense genes which we used to create promoter-luciferase reporter constructs. We combined these reporter constructs with a synthetic biology based dual-color ratiometric reporter system that normalizes read-outs accounting for experimental variability and Avr/R-induced cell-death. In addition, we introduced a replicon-based plasmid derived from the wheat dwarf virus, which self-replicates in wheat reducing the amount of plasmid used in the assay. Our new assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that the uptake of our assay by the community will significantly accelerate Avr identification for many wheat pathogens, leading to improved genome-guided pathogen surveillance and breeding of disease-resistant crops.
De Novo Assembly and Phasing of Dikaryotic Genomes from Two Isolates of Puccinia coronata f. sp. avenae , the Causal Ag
Publisher: American Society for Microbiology
Date: 07-03-2018
Abstract: Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae , is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype ersity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed ergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenae . IMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae , which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae , resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic ersity in populations of P. coronata f. sp. avenae .
Brassinosteroids inhibit pathogen-associated molecular pattern–triggered immune signaling independent of the receptor kinase BAK1
Publisher: Proceedings of the National Academy of Sciences
Date: 27-10-2011
Abstract: Plants and animals use innate immunity as a first defense against pathogens, a costly yet necessary tradeoff between growth and immunity. In Arabidopsis , the regulatory leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 combines with the LRR-RLKs FLS2 and EFR in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and the LRR-RLK BRI1 in brassinosteroid (BR)-mediated growth. Therefore, a potential tradeoff between these pathways mediated by BAK1 is often postulated. Here, we show a unidirectional inhibition of FLS2-mediated immune signaling by BR perception. Unexpectedly, this effect occurred downstream or independently of complex formation with BAK1 and associated downstream phosphorylation. Thus, BAK1 is not rate-limiting in these pathways. BRs also inhibited signaling triggered by the BAK1-independent recognition of the fungal PAMP chitin. Our results suggest a general mechanism operative in plants in which BR-mediated growth directly antagonizes innate immune signaling.
Antagonistic regulation of growth and immunity by the arabidopsis basic helix-loop-helix transcription factor HOMOLOG OF BRASSINOSTEROID ENHANCED EXPRESSION2 INTERACTING WITH INCREASED LEAF INCLINATION1 BINDING bHLH1
Publisher: Oxford University Press (OUP)
Date: 17-01-2014
Abstract: Plants need to finely balance resources allocated to growth and immunity to achieve optimal fitness. A tradeoff between pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and brassinosteroid (BR)-mediated growth was recently reported, but more information about the underlying mechanisms is needed. Here, we identify the basic helix-loop-helix (bHLH) transcription factor HOMOLOG OF BRASSINOSTEROID ENHANCED EXPRESSION2 INTERACTING WITH IBH1 (HBI1) as a negative regulator of PTI signaling in Arabidopsis (Arabidopsis thaliana). HBI1 expression is down-regulated in response to different PAMPs. HBI1 overexpression leads to reduced PAMP-triggered responses. This inhibition correlates with reduced steady-state expression of immune marker genes, leading to increased susceptibility to the bacterium Pseudomonas syringae. Overexpression of the HBI1-related bHLHs BRASSINOSTEROID ENHANCED EXPRESSION2 (BEE2) and CRYPTOCHROME-INTERACTING bHLH (CIB1) partially inhibits immunity, indicating that BEE2 and CIB1 may act redundantly with HBI1. In contrast to its expression pattern upon PAMP treatment, HBI1 expression is enhanced by BR treatment. Also, HBI1-overexpressing plants are hyperresponsive to BR and more resistant to the BR biosynthetic inhibitor brassinazole. HBI1 is nucleus localized, and a mutation in a conserved leucine residue within the first helix of the protein interaction domain impairs its function in BR signaling. Interestingly, HBI1 interacts with several inhibitory atypical bHLHs, which likely keep HBI1 under negative control. Hence, HBI1 is a positive regulator of BR-triggered responses, and the negative effect of PTI is likely due to the antagonism between BR and PTI signaling. This study identifies a novel component involved in the complex tradeoff between innate immunity and BR-regulated growth.
Wheat disease symptoms observation, capture, description, and evaluation
Publisher: ZappyLab, Inc.
Date: 06-02-2023
DOI: 10.17504/PROTOCOLS.IO.KXYGX9O44G8J/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a step by step guide to capture and describe to the disease symptoms the provided wheat plants display in week 2 and 3. You can then compare the observed disease symptoms with the provided compendium and other online sources to try to identify the causal infective agent. The final goal is to achieve the following: obtain high quality images of your diseased leaves for each treatment group that are representative. These pictures can be taken with your mobile phone for the complete attached leaves lined up on black cardboard paper or via the stereo microscope as demonstrated by the support staff. provide a detailed description of the symptoms observed on the leaves. This includes the following: size, shape, form, color, microscopic structure, spore shape and color, and timing of disease symptom development post infection. Compare your observed disease symptoms with the uninfected negative control, the provided compendium, and online sources. This protocol is applicable for week 2 and 3. Protocols progress overview: Week 2 observe, capture, and describe wheat leaf disease symptoms. Week 3 observe, capture, describe, and evaluate wheat disease symptoms.
Culturing of infective agents from infected wheat leaves
Publisher: ZappyLab, Inc.
Date: 02-02-2023
DOI: 10.17504/PROTOCOLS.IO.6QPVR4RKPGMK/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a step by step guide to culture infective agents out of infected wheat leaves. This process will take three weeks to complete, therefore the protocol is applicable for week 2-4. The final goal is to isolate the disease causing agent, if at all possible, via culture media. Of course not all pathogens are culturable and the protocol is geared towards filamentous pathogens which are the most severe pathogens of wheat in Australia. So the specific approach to culturing and the identity of the disease causing agent defines the possibility of the organism being cultured. Something to keep in mind when interpreting the results. Protocols progress overview: Week 2 you will identify suitable infected leaf material, surface sterilise it, and culture cut leaf pieces on water agar to enable microbial growth. Week 3 you will identify organisms that have grown out of the leaf onto the agar and subculture them onto high nutrient agar plates. Week 4 you will take pictures of the subcutlured organisms and try to classify it them based on morphological features.
Uncovering the microbiome of invasive sympatric European brown hares and European rabbits in Australia
Publisher: Cold Spring Harbor Laboratory
Date: 06-11-2019
DOI: 10.1101/832477
Abstract: European brown hares ( Lepus europaeus ) and European rabbits ( Oryctolagus cuniculus ) are invasive pest species in Australia, with rabbits having a substantially larger environmental impact than hares. As their spatial distribution in Australia partially overlaps, we conducted a comparative microbiome study to determine how the composition of the gastrointestinal microbiota varies between these species, since this may indicate species differences in diet, physiology, and other internal and external factors. We analysed the faecal microbiome of wild hares and rabbits from a sympatric environment, additionally comparing Illumina and Nanopore sequencing platforms. The faecal microbiomes varied significantly between hares and rabbits, despite both species occupying a similar habitat. Moreover, we identified significantly more variation in faecal microbiome composition between in idual rabbits compared to hares. The faecal microbiome in both species was dominated by the phyla Firmicutes and Bacteroidetes , typical of many vertebrates. Many phyla, including Actinobacteria , Proteobacteria , and Patescibacteria , were shared between rabbits and hares. In contrast, bacteria from phylum Verrucomicrobia were present only in rabbits, while phyla Lentisphaerae and Synergistetes were represented only in hares. We did not identify phylum Spirochetes in Australian hares this phylum was previously shown to be present at high relative abundance in European hare faecal s les. These differences in the faecal microbiota between hares and rabbits in Australia may be associated with differences in diet, and potentially behaviour, of the host species in their non-native range, which may influence the environmental impacts that these species have in Australia.
How postdocs benefit from building a union.
Publisher: eLife Sciences Publications, Ltd
Date: 21-11-2014
DOI: 10.7554/ELIFE.05614
Abstract: Members of UAW 5810—the union for postdoctoral researchers at the University of California—describe how their union has led to improved terms and conditions for postdocs.
De novo assembly and phasing of dikaryotic genomes from two isolates of Puccinia coronata f. sp. avenae, the causal agent of oat crown rust
Publisher: Cold Spring Harbor Laboratory
Date: 25-08-2017
DOI: 10.1101/179226
Abstract: Oat crown rust, caused by the fungus Puccinia coronata f. sp. avenae ( Pca ), is a devastating disease that impacts worldwide oat production. For much of its life cycle, Pca is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype ersity in this species. We generated highly contiguous de novo genome assemblies of two Pca isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for a total assembly length of 99.16 Mbp for 12SD80 and 777 primary contigs with a total length of 105.25 Mbp for 12NC29, and approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of co-expressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed ergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in Pca . Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae ( Pca ), which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding Pca , resources to study the molecular mechanisms underpinning pathogenicity and emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of Pca as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic ersity in populations of Pca .
Phosphorylation-Dependent Differential Regulation of Plant Growth, Cell Death, and Innate Immunity by the Regulatory Receptor-Like Kinase BAK1
Publisher: Public Library of Science (PLoS)
Date: 28-04-2011
Author response: A community-led initiative for training in reproducible research
Publisher: eLife Sciences Publications, Ltd
Date: 26-01-2021
The Arabidopsis Leucine-Rich Repeat Receptor–Like Kinases BAK1/SERK3 and BKK1/SERK4 Are Required for Innate Immunity to Hemibiotrophic and Biotrophic Pathogens
Publisher: Oxford University Press (OUP)
Date: 06-2011
Abstract: Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.
DNA clean-up and size selection for long-read sequencing v2
Publisher: ZappyLab, Inc.
Date: 09-11-2019
DOI: 10.17504/PROTOCOLS.IO.87EHZJE
Abstract: DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. Finally, a Short Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This has been trialled for the sorghum rot fungus Macrophomina phaseolina, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results are expected across many organisms, but have not been tested.
Cooperative anion binding and electrochemical sensing by modular podands
Publisher: Proceedings of the National Academy of Sciences
Date: 09-04-2002
Abstract: A series of podands based on two or three hydrogen bonding “arms” situated in mutually ortho , meta , or para relationships about an aryl core have been prepared, and their affinities for simple inorganic anions were measured. Of the two-arm hosts the meta compound and to a lesser extent the ortho host exhibit a cooperative anion binding effect. The two arms function essentially independently in the para derivative. The mutually meta three-arm host shows dramatically enhanced cooperative binding. Conformational changes within the meta two-arm host result in significantly enhanced electrochemical anion sensing compared with the more conformationally rigid three-arm host.
A community-led initiative for training in reproducible research
Publisher: eLife Sciences Publications, Ltd
Date: 21-06-2021
DOI: 10.7554/ELIFE.64719
Abstract: Open and reproducible research practices increase the reusability and impact of scientific research. The reproducibility of research results is influenced by many factors, most of which can be addressed by improved education and training. Here we describe how workshops developed by the Reproducibility for Everyone (R4E) initiative can be customized to provide researchers at all career stages and across most disciplines with education and training in reproducible research practices. The R4E initiative, which is led by volunteers, has reached more than 3000 researchers worldwide to date, and all workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at www.repro4everyone.org/ .
A Common Signaling Process that Promotes Mycorrhizal and Oomycete Colonization of Plants
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.CUB.2012.09.043
Abstract: The symbiotic association between plants and arbuscular mycorrhizal fungi is almost ubiquitous within the plant kingdom, and the early stages of the association are controlled by plant-derived strigolactones acting as a signal to the fungus in the rhizosphere and lipochito-oligosaccharides acting as fungal signals to the plant. Hyphopodia form at the root surface, allowing the initial invasion, and this is analogous to appressoria, infection structures of pathogenic fungi and oomycetes. Here, we characterize RAM2, a gene of Medicago truncatula required for colonization of the root by mycorrhizal fungi, which is necessary for appropriate hyphopodia and arbuscule formation. RAM2 encodes a glycerol-3-phosphate acyl transferase (GPAT) and is involved in the production of cutin monomers. Plants defective in RAM2 are unable to be colonized by arbuscular mycorrhizal fungi but also show defects in colonization by an oomycete pathogen, with the absence of appressoria formation. RAM2 defines a direct signaling function, because exogenous addition of the C16 aliphatic fatty acids associated with cutin are sufficient to promote hyphopodia/appressoria formation. Thus, cutin monomers act as plant signals that promote colonization by arbuscular mycorrhizal fungi, and this signaling function has been recruited by pathogenic oomycetes to facilitate their own invasion.
DNA clean-up and size selection for long-read sequencing v4
Publisher: ZappyLab, Inc.
Date: 14-07-2021
DOI: 10.17504/PROTOCOLS.IO.BWKDPCS6
Abstract: DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. An optional needle shearing is described which can help create a more uniform DNA length to maximise sequencing output. Finally, polymer and salt based solutions are used to selectively precipitate DNA based on size, which also appears to further clean the DNA. This was trialled for the sorghum rot fungus Macrophomina phaseolina, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results have been obtained with other fungi, plants, reptiles and insects.
DNA clean-up and size selection for long-read sequencing v3
Publisher: ZappyLab, Inc.
Date: 08-04-2020
DOI: 10.17504/PROTOCOLS.IO.BETDJEI6
Abstract: DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. An optional needle shearing is described which can help create a more uniform DNA length to maximise sequencing output. Finally, a Short-Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This was trialled for the sorghum rot fungus Macrophomina phaseolina, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results have been obtained with other fungi, plants, reptiles and insects.
Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans
Publisher: Springer Science and Business Media LLC
Date: 09-2009
DOI: 10.1038/NATURE08358
Abstract: Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.
Changing SERKs and priorities during plant life.
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.TPLANTS.2015.06.006
Abstract: SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES (SERKs) are coreceptors for erse extracellular signals. SERKs are involved in a wide array of developmental and immune related processes first discovered in Arabidopsis. Recent work demonstrates the evolutionary conservation of SERKs in all multicellular plants, and highlights their functional conservation in monocots and dicots.
A Chromosome Scale Assembly of an Australian Puccinia striiformis f. sp. tritici Isolate of the PstS1 Lineage
Publisher: Scientific Societies
Date: 03-2022
Long-Reads-Based Metagenomics in Clinical Diagnosis With a Special Focus on Fungal Infections
Publisher: Frontiers Media SA
Date: 06-01-2022
DOI: 10.3389/FMICB.2021.708550
Abstract: Identification of the causative infectious agent is essential in the management of infectious diseases, with the ideal diagnostic method being rapid, accurate, and informative, while remaining cost-effective. Traditional diagnostic techniques rely on culturing and cell propagation to isolate and identify the causative pathogen. These techniques are limited by the ability and the time required to grow or propagate an agent in vitro and the facts that identification based on morphological traits are non-specific, insensitive, and reliant on technical expertise. The evolution of next-generation sequencing has revolutionized genomic studies to generate more data at a cheaper cost. These are ided into short- and long-read sequencing technologies, depending on the length of reads generated during sequencing runs. Long-read sequencing also called third-generation sequencing emerged commercially through the instruments released by Pacific Biosciences and Oxford Nanopore Technologies, although relying on different sequencing chemistries, with the first one being more accurate both platforms can generate ultra-long sequence reads. Long-read sequencing is capable of entirely spanning previously established genomic identification regions or potentially small whole genomes, drastically improving the accuracy of the identification of pathogens directly from clinical s les. Long-read sequencing may also provide additional important clinical information, such as antimicrobial resistance profiles and epidemiological data from a single sequencing run. While initial applications of long-read sequencing in clinical diagnosis showed that it could be a promising diagnostic technique, it also has highlighted the need for further optimization. In this review, we show the potential long-read sequencing has in clinical diagnosis of fungal infections and discuss the pros and cons of its implementation.
The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that direct genome-wide DNA methylation
Publisher: Cold Spring Harbor Laboratory
Date: 14-11-2018
DOI: 10.1101/469338
Abstract: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici ( Pgt ), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (∼250 kb) that are highly erse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally ersified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5’ uracil derived from genes. In contrast, the late wave sRNAs are mainly 22 nt sRNAs with a 5’ adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. We conclude that rust fungi use an epigenetic silencing pathway that resembles RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.
Risk of suicide and repeat self-harm after hospital attendance for non-fatal self-harm in Sri Lanka: a cohort study.
Publisher: Elsevier BV
Date: 08-2019
Draft genome resource for Macrophomina phaseolina associated with charcoal rot in sorghum
Publisher: Scientific Societies
Date: 05-2020
DOI: 10.1094/MPMI-12-19-0356-A
Abstract: Macrophomina phaseolina is a soil-borne phytopathogenic fungus that causes charcoal rot in several plant species, including sorghum. We constructed a draft genome of M. phaseolina isolate BRIP 70780a from sorghum, using long-read native DNA from MinION sequencing, which was error-corrected using short-read Illumina MiSeq reads. The draft genome, consisting of 22 contigs with an N 50 of 4,257,441 bp, 99.3% complete benchmarking universal single-copy orthologs, and 14,471 genes, is a valuable resource to aid future studies in population genomics and molecular diagnostic marker development for rapid detection of the pathogen.
High purity, high molecular weight DNA extraction from rust spores via CTAB based DNA precipitation for long read sequencing v1
Publisher: ZappyLab, Inc.
Date: 30-03-2018
DOI: 10.17504/PROTOCOLS.IO.N5YDG7W
Abstract: Nanopore sequencing (Oxford Nanopore Technologies MinION instrument) requires high quality, high molecular weight DNA (HMW) to produce long sequence reads. Extracting high purity, HMW DNA is a difficult challenge, especially for a biotrophic rust fungus. This is because the biomass for DNA extraction is restricted to spores which are physically tough and rich in complex polysaccharides and lipids. When DNA extracted from spores precipitated with ethanol or isopropanol, many other impurities like polysaccharides and lipids get coprecipitated along with DNA which renders DNA purity unsuitable for Nanopore sequencing. These impurities cannot be removed with any subsequent DNA purification with either paramagnetic beads (SPRI) or re-precipitation with ethanol and isopropanol. These impurities always co-precipitated with the DNA which absorbs light at 230 nm. In an attempt to improve purity, I tried precipitating DNA with cationic detergent CTAB (Hexadecyl-Trimethyl-Ammonium Bromide) which has been used to precipitate DNA in the past but not employed extensively. CTAB is a cationic surfactant which selectively complex with DNA and precipitates it out of the solution in the presence low salt concentration (0.4M NaCl) while leaving other impurities. Unlike isopropanol or ethanol precipitated DNA which looks highly viscous, the CTAB precipitated DNA lack similar viscosity and therefore indicative less or no impurities. Moreover, with CTAB precipitation I get ~ 20-25 ug crude DNA per 100 mg of spores which is almost double the amount I get with isopropanol and ethanol precipitation. The crude DNA once washed with 1 V homemade SPRI beads solution, gives a 260/280 ratio ~1.8-2 and 260/230 2.0-2.2.
Focus issue on plant immunity: from model systems to crop species
Publisher: Frontiers Media SA
Date: 26-03-2015
Cost-conscious generation of multiplexed short-read DNA libraries for whole-genome sequencing v2
Publisher: ZappyLab, Inc.
Date: 04-02-2021
DOI: 10.17504/PROTOCOLS.IO.14EGNX27ZL5D/V2
Abstract: Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. By preparing libraries in high-throughput with small reaction volumes that use 1/50th the amount of transposome compared to Illumina DNA Prep tagmentation protocols, the cost per library can be substantially reduced, by approximately 1/20th. Furthermore, we optimised the protocol to minimise magnetic bead-based clean-ups between steps, further reducing cost, time and DNA input requirements. By developing our own dual index primers to multiplex nine 96-well microplates, up to 864 s les can be placed on a single flow cell. This enables efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell.
Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides
Publisher: Wiley
Date: 19-09-2016
DOI: 10.1111/MPP.12444
Sequence analysis of Nanopore based fungal and bacterial metabarcode DNA reads
Publisher: ZappyLab, Inc.
Date: 08-04-2023
DOI: 10.17504/PROTOCOLS.IO.BP2L6969ZLQE/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a general description of how we analysed the bacterial and fungal metabarcode derived Nanopore reads for you and how you can summarise the data. You will be able to use this protocol as a reference in your methods section. The 16S primers target bacteria and long-ITS primers target fungi (or so we thought). Check the protocols "PCR reaction of marker regions (a.k.a metabarcodes) for two kingdoms V.1" and "Sequencing fungal and bacterial metabarcodes with native barcoding and Nanopore" for how the sequences were generated including details on the primers used and specific Nanopore sequencing chemistry. Long-ITS analysis In brief, we mapped the long-ITS reads against two databases using minimap2 with a winner takes all approach only reporting the best hit in the databases for each read. Not all reads might have a hit in the database as we learned that the primers also lify plant and human DNA. The "in-house" database is part of a larger project the Schwessinger group is involved to improve the identification of fungal spores from air-s led material. This is a collaboration with DAFF, UC, SARDI, and USyd. You can check out the spore traps here as part of the iMapPests project. The database includes about 80 species with TG1 and TG2 being part of it but not TG3 and TG4. The entries in the database were generated using the same primer pairs as we used in class and hence have they same length as the ITS reads we generated. The UNITE database is a large public database for fungal ITS sequences which mostly focuses on ITS2. It has about 500,000 'specie' entries in the database. You can read up more on the database here (see also the reference section). The database entries are mostly around 600-700 bp long. This database contains pathogens for TG1, TG3, and TG4. The final goal is to achieve the following: Basecall the fast5 files to fastq files. Filter fastq files on quality using NanoFilt. Assess overall read lengths and quality after filtering using NanoPlot. Map ITS reads separately against the 'in-house' long-ITS and the UNITE ITS2 database using minimap2 and a "winner takes all approach". Perform taxonomic assignment of 16S sequences using EMU. Critically evaluate taxonomic assignments and summaries them using Excel (shown in class) or R. Software used for analysis: Guppy basecaller ow-it-works/basecalling NanoFilt decoster/nanofilt NanoPlot decoster/NanoPlot seqtk h3/seqtk Minimap2 h3/minimap2 EMU 16S analysis reangenlab/emu References: Curry, Kristen D., Qi Wang, Michael G. Nute, Alona Tyshaieva, Elizabeth Reeves, Sirena Soriano, Qinglong Wu, et al. “Emu: Species-Level Microbial Community Profiling of Full-Length 16S RRNA Oxford Nanopore Sequencing Data.” Nature Methods 19, no. 7 (July 2022): 845–53. 0.1038/s41592-022-01520-4. De Coster, Wouter, Svenn D’Hert, Darrin T Schultz, Marc Cruts, and Christine Van Broeckhoven. “NanoPack: Visualizing and Processing Long-Read Sequencing Data.” Bioinformatics 34, no. 15 (August 1, 2018): 2666–69. 0.1093/bioinformatics/bty149. Li, Heng. “Minimap2: Pairwise Alignment for Nucleotide Sequences.” Bioinformatics 34, no. 18 (September 15, 2018): 3094–3100. 0.1093/bioinformatics/bty191. Nilsson, Rolf Henrik, Karl-Henrik Larsson, Andy F S Taylor, Johan Bengtsson-Palme, Thomas S Jeppesen, Dmitry Schigel, Peter Kennedy, et al. “The UNITE Database for Molecular Identification of Fungi: Handling Dark Taxa and Parallel Taxonomic Classifications.” Nucleic Acids Research 47, no. D1 (January 8, 2019): D259–64. 0.1093/nar/gky1022.
Vitamin D in ankylosing spondylitis: Review and meta-analysis
Publisher: Elsevier BV
Date: 2015
DOI: 10.1016/J.CCA.2014.08.040
Abstract: The role of vitamin D in ankylosing spondylitis (AS) is largely unknown. This paper aims to examine the association between serum vitamin D levels and susceptibility and disease activity of AS. We searched the relevant literatures in PubMed, Elsevier Science Direct, Chinese Biomedical Database (CBM), Chinese National Knowledge Infrastructure (CNKI) and Wanfang (Chinese) Database published before June 2014. Eight independent case-control studies with a total of 533 AS patients and 478 matching controls were selected into this meta-analysis. Standard mean differences (SMDs) with 95% confidence intervals (CIs) were used to assess the levels of serum vitamin D, parathyroid hormone (PTH), serum calcium and alkaline phosphatase (ALP) in cases and controls, respectively. Correlation coefficients (CORs) have been performed to value the correlationship between vitamin D and disease activity (erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)) of AS patients. Meta-analysis results suggested that vitamin D may play a protective role in AS (for total vitamin D: SMD=-0.71, P<0.001 for 25OHD: SMD=-0.66, P=0.002 for 1,25OHD: SMD=-0.72, P=0.19). Differences in PTH and serum calcium levels were not significant in AS (SMD=-0.10, P=0.67 SMD=0.12, P=0.17 respectively), while ALP was associated with AS susceptibility (SMD=0.20, P=0.04). The relationship between serum vitamin D levels and disease activity was statistically significant except for 25OHD versus (vs.) CRP or BASDAI (for CRP vs. 25OHD: COR=-0.22, P=0.08 for BASDAI vs. 25OHD: COR=-0.20, P=0.06, respectively). The higher levels of serum vitamin D were associated with a decreased risk of AS, and showed an inverse relationship with AS activity.
Pulse Field Gel Electrophoresis for Long Read Sequencing v1
Publisher: ZappyLab, Inc.
Date: 06-11-2019
DOI: 10.17504/PROTOCOLS.IO.83JHYKN
Abstract: Pulsefield gel electrophoresis is an easy and affordable method of quality control for high molecular weight DNA extractions, particularly for use in long-read Oxford Nanopore sequencing. This method is currently used in the Borevitz Lab (ANU) and is adapted from the method used by Benjamin Schwessinger (ANU). For more details on the PFGE hardware, refer to the manual at ebroot/web df/lsr/literature/M1703690.pdf You may need to use different settings for different DNA size ranges - see pg. 25 of the manual ("5.2 Pulsed Field Conditions by DNA Size")
Reproducibility for everyone: a community-led initiative with global reach in reproducible research training
Publisher: Center for Open Science
Date: 31-10-2020
Abstract: Reproducibility is a cornerstone of the scientific method and sets apart science from pseudoscience. Unfortunately, a majority of scientists have experienced difficulties in reproducing their own or someone else’s results. This inability to confirm scientific findings negatively impacts in idual scientists, funding bodies, academic journals, pharmaceutical drug development and the public’s perception of science. Factors causing irreproducible results can arise from nearly every aspect of the scientific process, and typically reflect a lack of in-depth training in reproducible research practices. Here, we present the Reproducibility for Everyone (R4E) initiative, a collaboration between researchers from erse scientific disciplines and industry partners who aspire to promote open and reproducible research practices. We have developed a customizable workshop series targeting researchers at all levels and across disciplines. Our workshop series covers the conceptual framework of reproducible research practices followed by an overview of actionable research practices. To date, we have reached more than 2000 researchers through over 25 workshops held at international conferences and local meetings. By incorporating further contributions from the scientific community, we hope to expand this valuable resource for teaching transparent and reproducible research practices. Our initiative demonstrates how a shared set of materials may form the basis for a global initiative to improve reproducibility in science. The workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at www.repro4everyone.org.
Changing scientific meetings for the better
Publisher: Springer Science and Business Media LLC
Date: 15-03-2021
Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens
Publisher: American Society for Microbiology
Date: 29-10-2019
Abstract: This work dissects the tripartite horizontal transfer of ToxA , a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.
DNA size selection (>1kb) and clean up using an optimized SPRI beads mixture v1
Publisher: ZappyLab, Inc.
Date: 26-06-2017
DOI: 10.17504/PROTOCOLS.IO.IDMCA46
Abstract: This protocol describesa clean up and size selection method for nucleic acids (tested on DNA) to deplete and remove fragments below 1 - 2 kb.The success of this depends on the cleanliness of your s le (it doesn't have to be super clean but a whole lot of contaminants make working with the beads more difficult, diluting the s le out before usage can help with that). The concentrations of PEG and NaCl and the volume of the beads solution are crutial for recovery and proper removal of small fragments. As a basic guidline it can be said: more PEG and NaCl - higher recovery but hence less removal of small fragments and the other way round. I found for my s les (eucalyptus) that with 1 volume of the beads solution respectively to DNA s le I'm on the safe side recovery wise, but if I want to make sure to get rid of more smaller fragments I use 0.8 volumes. So in numbers that means: Final reaction concentration of PEG8000: 1 V: 5.5% 0.8 V: 4.8% Final reaction concentration of NaCl: 1V: 0.8 M 0.8V: 0.7 M
Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case
Publisher: Springer Science and Business Media LLC
Date: 12-2018
Control of the pattern-recognition receptor EFR by an ER protein complex in plant immunity
Publisher: Wiley
Date: 17-09-2009
SARS-CoV-2 consensus genome reconstruction, quality control, and lineage analysis
Publisher: ZappyLab, Inc.
Date: 30-04-2023
DOI: 10.17504/PROTOCOLS.IO.14EGN2KP6G5D/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #2 "An SARS-COV2 incursion scenario: Genomics, phylogenetics, and incursions." This mini-research project is modeled on the yearly Quality Assurance Program of The Royal College of Pathologists of Australia (RCPAQAP), we take part in together with ACT Pathology. This research project is split into two major parts, identical to how the official RCPAQAP is run every year. Part #1 is focusing on the 'wet- lab' by sequencing SARS-COV2 from real world RNA s les provided by ACT Pathology especially for our ANU biosecurity course (Thank YOU!). Here you will lify and sequence five (5) RNA s les per research group. You will assess the SARS-COV2 genome sequences for their lineage assignments using online programs, put sequences into a global context, estimate the collection date based on genetic information, and describe mutations in the spike protein. Part #2 is focusing on the 'dry-lab' by investigating a hypothetical incursion scenario in the so-called city Fantastica. You will combine genomic surveillance of SARS-COV2 with case interview data to trace the spread into of SARS-COV2 in the community and into high risk settings. We will provide you with real publicly available SARS-COV2 genome and fantasized case interviews. You will put these two together to trace the spread and suggest potential improvements in containment strategies with a focus on high risk settings. This protocol describes the analysis component of Part #1. The metrics you are suppose to report for each of your s les are mostly borrowed from the official SARS-CoV-2 QAP. Don't worry if not all of these mean something to you at the moment as we will explain them again during the prac. In case all/most of your s les have 50% genome coverage please also include the analysis of MakeUp for points 1 to 7 and TimeMakeUp for point 8. You can access the MakeUp data here (ANU only). Make sure to read the README file so you understand what each item relates to. The metrics you have to report for each of our s les (or MakeUps) include the following. Consensus genome coverage. Average read depth You might want to include detailed read depth plots here as well. Pangolin Lineage. NextClade Lineage. Base pair differences relative to the original SARS-CoV-2 genome. Amino acid replacements and deletions in the S (spike) protein sequence. Evaluation if your and/or the MakeUp s les would make the QC cut-off 90% genome coverage and other metrics that you deem important for QC. Would you 'flag' any of your s les as standing out e.g. being negative control? Approximate s ling date of your sequences for month and year. You must report the versions of all tools used in your report and the day the analysis was performed. This is extremely important for reporting as lineage naming and such change VERY frequently during the pandemic and any outbreak. This protocol is applicable for week 9. The following links might be useful for your report: The original publication that describes the sequencing protocol is here: iomethods/article/5/1/bpaa014/5873518?login=true Original sources describing the consensus reconstruction from raw reads are here: artic.network/ncov-2019, artic.network/ncov-2019/ncov2019-bioinformatics-sop.html, labs.epi2me.io/, rticles/s41467-020-20075-6 Other websites and resources used in the protocol: igv.org/, clades.nextstrain.org/, pangolin.cog-uk.io/, gi-bin/hgPhyloPlace
Austropuccinia psidii, causing myrtle rust, has a gigabase-sized genome shaped by transposable elements
Publisher: Cold Spring Harbor Laboratory
Date: 20-03-2020
DOI: 10.1101/2020.03.18.996108
Abstract: Austropuccinia psidii , originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in transposable elements and a very low overall GC content of 33.8%. The overall gene content is highly conserved, when compared to other closely related Pucciniales, yet the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how transposable elements shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.
High-molecular weight DNA extraction from challenging fungi using CTAB for lysis and precipitation v3
Publisher: ZappyLab, Inc.
Date: 21-07-2021
DOI: 10.17504/PROTOCOLS.IO.BWR8PD9W
Abstract: Extracting pure high-molecular weight DNA from fungi is often difficult due to the presence of polysaccharides and potentially other compounds, which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, making them difficult to remove from solution. Some contaminants may even be undetected by spectrophotometers or fluorometric devices, however they still substantially interferes with long-read DNA sequencing. In attempt to resolve these challenges, a protocol is presented that utilses cetrimonium bromide (CTAB) for lysis and precipitation. CTAB in the lysis solution acts as a cationic detergent to dissolve cell membranes and remove polysaccharides by the formation of CTAB-polysaccharide complexes that co-precipitate during chloroform phase separation. Using CTAB for DNA precipitation under low salt, low ionic conditions, CTAB-DNA complexes form while leaving polysaccharides in solution, resulting in significantly less co-precipitation than alcohol methods. The presented protocol also includes some updates to current strategies and incorporates two different options for DNA clean-up and size selection. Using this protocol, we have been successfully sequencing various fungi with a MinION (Oxford Nanopore Technologies). For wheat stripe rust Puccinia striiformis and wheat leaf rust Puccinia triticina, we performed the presented method followed by size selection with an automated gel purification system, with sequencing yielding 5.88 Gbp with an N50 of 34.79 kb and 4.88 Gbp with an N50 of 27.61 kb, respectively for the two fungi. These fungi are notorious for being recalcitrant and these results have been positive improvements on many other methods. To increase sequencing output for these s les, more work is needed to identify and remove the elusive contaminants. Other fungi, such as sorghum rot fungus Macrophomina phaseolina, we chose an alternative size selection method, precipitation with a polymer and salt solution. This sequencing yielded 13.71 Gbp with an N50 of 21.75 kb, while another strain yielded 9.72 Gbp with an N50 of 43.50 kb.
Distinct life histories impact dikaryotic genome evolution in the rust fungus Puccinia striiformis causing stripe rust in wheat
Publisher: Cold Spring Harbor Laboratory
Date: 29-11-2019
DOI: 10.1101/859728
Abstract: Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici , is a major threat to wheat production world-wide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst . This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole genome comparative genomics of two P . striiformis f. sp . tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural and allele levels. At the whole genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates while 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall this detailed comparative analysis of two haplotype-aware assemblies of P . striiformis f. sp . tritici are the first steps in understanding the evolution of dikaryotic rust fungi at a whole genome level.
High quality DNA from Fungi for long read sequencing e.g. PacBio
Publisher: ZappyLab, Inc.
Date: 16-05-2019
DOI: 10.17504/PROTOCOLS.IO.2YFGFTN
Abstract: Extraction of high quality DNA for long read sequencing e.g. PacBio Optimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust. Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months. Critical steps to obtain high quality DNA: Do NOT heat s les during DNA extractions! Perform all steps at RT or 4oC as indicated. Do NOT incubate s les with KAc for prolonged time periods Perform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites. DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.
High-molecular weight DNA extraction and small fragment removal ofAscochyta lentis v1
Publisher: ZappyLab, Inc.
Date: 28-05-2020
DOI: 10.17504/PROTOCOLS.IO.BGW5JXG6
Abstract: Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, being difficult to separate fom the DNA. Although the contaminant may not be detected by spectrophotometers or fluorometric devices, it substantially interferes with long-read DNA sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol is presented with some updates to current strategies and incorporates a small fragment removal step using Polyethylene Glycol. Using this protocol, we have been successfully sequencing the lentil pathogen Ascochyta lentis with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 13 gigabases with an N50 of approximately 15 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants. DNA extraction modified from: www.protocols.io/view/high-molecular-weight-dna-extraction-from-challeng-5isg4ee PEG small fragment elimination after: www.protocols.io/view/size-selective-precipitation-of-dna-using-peg- -7erhjd6
Cautionary Notes on the Use of C-Terminal BAK1 Fusion Proteins for Functional Studies
Publisher: Oxford University Press (OUP)
Date: 11-2011
Fundamental wheat stripe rust research in the 21st century.
Publisher: Wiley
Date: 30-08-2017
DOI: 10.1111/NPH.14159
Abstract: In the 21 st century, the wheat stripe rust fungus has evolved to be the largest biotic limitation to global wheat production. New pathogen genotypes are more aggressive and able to infect previously resistant wheat varieties, leading to rapid pathogen migration across and between continents. We now know the full life cycle, microevolutionary relationships and past migration routes on a global scale. Current sequencing technologies have provided the first fungal draft genomes and simplified plant resistance gene cloning. Yet, we know nothing about the molecular and microevolutionary mechanisms that facilitate the infection process and cause new devastating pathogen races. These are the questions that need to be addressed by exploiting the synergies between novel 21 st century biology tools and decades of dedicated pathology work. Contents Summary 1625 I. Introduction 1625 II. Wheat stripe rust can be controlled with genetics 1626 III. The Puccinia striformis f. sp. tritici life cycle enables genetic ersity and rapid adaptation 1626 IV. Puccinia striformis f. sp. tritici evolves and migrates rapidly on a global scale 1626 V. Puccinia striformis . sp. tritici genomes are highly heterozygous and encode over 1000 candidate effectors 1628 VI. Novel 21 st century tools to provide insight into Puccinia striformis f. sp. tritici biology 1629 VII. Conclusion 1629 Acknowledgements 1630 References 1630
High-molecular weight DNA extraction, cleanup and size selection for long-read sequencing
Publisher: Public Library of Science (PLoS)
Date: 15-07-2021
DOI: 10.1371/JOURNAL.PONE.0253830
Abstract: Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30–50 kb, with reads exceeding 200 kb and outputs ranging from 15–30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria s les.
Evolution and Adaptation of Forest and Crop Pathogens in the Anthropocene
Publisher: Scientific Societies
Date: 2021
DOI: 10.1094/PHYTO-08-20-0358-FI
Abstract: Anthropocene marks the era when human activity is making a significant impact on earth, its ecological and biogeographical systems. The domestication and intensification of agricultural and forest production systems have had a large impact on plant and tree health. Some pathogens benefitted from these human activities and have evolved and adapted in response to the expansion of crop and forest systems, resulting in global outbreaks. Global pathogen genomics data including population genomics and high-quality reference assemblies are crucial for understanding the evolution and adaptation of pathogens. Crops and forest trees have remarkably different characteristics, such as reproductive time and the level of domestication. They also have different production systems for disease management with more intensive management in crops than forest trees. By comparing and contrasting results from pathogen population genomic studies done on widely different agricultural and forest production systems, we can improve our understanding of pathogen evolution and adaptation to different selection pressures. We find that in spite of these differences, similar processes such as hybridization, host jumps, selection, specialization, and clonal expansion are shaping the pathogen populations in both crops and forest trees. We propose some solutions to reduce these impacts and lower the probability of global pathogen outbreaks so that we can envision better management strategies to sustain global food production as well as ecosystem services.
A microbially derived tyrosine‐sulfated peptide mimics a plant peptide hormone
Publisher: Wiley
Date: 30-05-2017
DOI: 10.1111/NPH.14609
Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses
Publisher: Cold Spring Harbor Laboratory
Date: 11-06-2014
DOI: 10.1101/006155
Abstract: Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.
Physical separation of haplotypes in dikaryons allows benchmarking of phasing accuracy in Nanopore and HiFi assemblies with Hi-C data
Publisher: Springer Science and Business Media LLC
Date: 25-03-2022
DOI: 10.1186/S13059-022-02658-2
Abstract: Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false-positive signals. We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false-positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30–40 times using HiFi sequencing is required for phasing of the leaf rust genome, with 0.7% heterozygosity, and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.
Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics
Publisher: Oxford University Press (OUP)
Date: 23-11-2019
DOI: 10.1093/MMY/MYZ109
Abstract: The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical s les. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) s les from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized s le preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.
Species-specific basecallers improve actual accuracy of nanopore sequencing in plants
Publisher: Springer Science and Business Media LLC
Date: 14-12-2022
DOI: 10.1186/S13007-022-00971-2
Abstract: Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii , to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT’s improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads. Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy’s plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific). The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.
The Rice Xa3 Gene Confers Resistance to Xanthomonas oryzae pv. oryzae in the Model Rice Kitaake Genetic Background
Publisher: Frontiers Media SA
Date: 06-02-2020
A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection
Publisher: Oxford University Press (OUP)
Date: 2016
DOI: 10.1039/C5IB00232J
Abstract: The synthetic biological production of posttranslationally modified proteins enables control of biological processes in plants and animals.
On the value of preprints: An early career researcher perspective
Publisher: Public Library of Science (PLoS)
Date: 21-02-2019
Cost-conscious generation of multiplexed short-read DNA libraries for whole-genome sequencing.
Publisher: Public Library of Science (PLoS)
Date: 27-01-2023
DOI: 10.1371/JOURNAL.PONE.0280004
Abstract: Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. We prepare libraries in high-throughput with small reaction volumes that use 1/50 th the amount of transposome compared to Illumina DNA Prep tagmentation protocols. By reducing transposome usage and optimising the protocol to circumvent magnetic bead-based clean-ups between steps, we reduce costs, labour time and DNA input requirements. Developing our own dual index primers further reduced costs and enables up to nine 96-well microplate combinations. This facilitates efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell. The protocol presented substantially reduces the cost per library by approximately 1/20 th compared to conventional Illumina methods.
Variation and inheritance of theXanthomonasraxX‐raxSTABgene cluster required for activation of XA21‐mediated immunity
Publisher: Wiley
Date: 18-02-2019
DOI: 10.1111/MPP.12783
Genomic DNA extraction from animal faecal tissues using DNeasy blood and tissue kit v1
Publisher: ZappyLab, Inc.
Date: 09-08-2019
DOI: 10.17504/PROTOCOLS.IO.6BBHAIN
Abstract: Faecal tissues are difficult to lyse and contain lower amount of DNA compared to other animal tissues such as liver and duodenum. This protocol was optimised to extract maximum amount of DNA using 50 mg of faecal tissues following Qiagen DNeasy blood and tissue kit.
Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses
Publisher: Public Library of Science (PLoS)
Date: 30-03-2015
Genome biology and evolution of mating type loci in four cereal rust fungi
Publisher: Cold Spring Harbor Laboratory
Date: 02-03-2023
DOI: 10.1101/2023.03.02.530769
Abstract: Sex defining loci or chromosomes often contain large non-recombining regions. In Basidiomycota, two distinct gene loci confer mate compatibility. These loci encode for homeodomain (HD) transcription factors and pheromone receptor (PR)-ligand pairs. In some fungi these two loci are physically linked and give rise to large evolutionary strata with extensive recombination suppression. To date genome level mating type ( MAT ) loci analysis is lacking for obligate biotrophic basidiomycetes that depend on their host plants for survival, this includes the order Pucciniales . These economically important plant pathogens have long-term dikaryotic life-stages in which matched haploid nuclei must carry compatible MAT loci. Here we focus on four Puccinia cereal rust species, including Puccinia graminis f.sp tritici , P. striiformis f.sp tritici , P. triticina and P. coronata f.sp avenae , which infect major cereals including wheat and oat. We identified MAT locations on two separate chromosomes that supports previous hypotheses of a tetrapolar mating system in the Pucciniales . HD loci displayed largely conserved macrosynteny among species and were not suppressed for recombination. Consistently, HD transcription factor genes showed no trans-specific polymorphisms between species. Regions, surrounding the PR loci however, displayed extensive genomic degeneration, signs of recombination suppression and were found to be linked to centromeres in some species. Expression analysis suggests that both MAT loci are involved in nuclear pairing during spore formation in the asexual cycle. Together, our study provides insights into the evolution of MAT loci since speciation of key pathogenic Puccinia species. This detailed understanding is important to predict the possible combinations of nuclear pairs that can arise via sexual reproduction, somatic recombination, or di-om mating to enable the evolution of newly virulent isolates of these agriculturally important plant pathogens.
Uncovering the microbiome of invasive sympatric European brown hares and European rabbits in Australia
Publisher: PeerJ
Date: 18-08-2020
DOI: 10.7717/PEERJ.9564
Abstract: European brown hares ( Lepus europaeus ) and European rabbits ( Oryctolagus cuniculus ) are invasive pest species in Australia, with rabbits having a substantially larger environmental impact than hares. As their spatial distribution in Australia partially overlaps, we conducted a comparative microbiome study to determine how the composition of gastrointestinal microbiota varies between these species, since this may indicate species differences in diet, physiology, and other internal and external factors. We analysed the faecal microbiome of nine wild hares and twelve wild rabbits from a sympatric periurban reserve in Canberra, Australia, using a 16S rRNA licon-based sequencing approach. Additionally, we compared the concordance between results from Illumina and Nanopore sequencing platforms. We identified significantly more variation in faecal microbiome composition between in idual rabbits compared to hares, despite both species occupying a similar habitat. The faecal microbiome in both species was dominated by the phyla Firmicutes and Bacteroidetes , typical of many vertebrates. Many phyla, including Actinobacteria , Proteobacteria and Patescibacteria , were shared between rabbits and hares. In contrast, bacteria from phylum Verrucomicrobia were present only in rabbits, while phyla Lentisphaerae and Synergistetes were represented only in hares. We did not identify phylum Spirochaetes in Australian hares this phylum was previously shown to be present at high relative abundance in European hare faecal s les. These differences in the composition of faecal microbiota may be indicative of less discriminate foraging behaviour in rabbits, which in turn may enable them to adapt quicker to new environments, and may reflect the severe environmental impacts that this species has in Australia.
News from the frontline: recent insights into PAMP-triggered immunity in plants
Publisher: Elsevier BV
Date: 08-2008
DOI: 10.1016/J.PBI.2008.06.001
Abstract: Plants have developed a complex defence network to fight off invading pathogens. In recent years, the full importance of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) within this network became apparent. Several new PAMPs have been isolated and new pattern-recognition receptors (PRRs) identified. The discovery of the PRR-interacting protein BAK1 sheds light on the immediate downstream signalling events. Further, transcriptomic analyses identified a core set of approximately 100 PAMP-responsive genes. These studies also revealed a significant overlap with genes regulated during effector-triggered immunity (ETI). Strikingly, ETI seems to operate by alleviating the negative feedback regulation of PTI, leading to stronger defences. This review discusses recent findings in PTI recognition and signalling, and illustrates the need to discover new regulators of PTI responses for a full understanding of plant innate immunity.
High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification v2
Publisher: ZappyLab, Inc.
Date: 16-07-2019
DOI: 10.17504/PROTOCOLS.IO.5ISG4EE
Abstract: Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, being difficult to separate fom the DNA. Although the contaminant may not be detected by spectrophotometers or fluorometric devices, it substantially interferes with long-read DNA sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol is presented with some updates to current strategies and incorporates a gel purification with a Pippin Prep (Sage Science). Using this protocol, we have been successfully sequencing the wheat stripe rust Puccinia striiformis and leaf rust Puccinia triticina with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 4 gigabases with an N50 of approximately 30 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants.
A near complete haplotype-phased genome of the dikaryotic wheat stripe rust fungusPuccinia striiformisf. sp.triticireveals high inter-haplotype diversity
Publisher: Cold Spring Harbor Laboratory
Date: 22-09-2017
DOI: 10.1101/192435
Abstract: A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here we present a diploidaware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long-reads using the FALCON-Unzip assembler. RNA-seq datasets were used to infer high quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high inter-haplotype ersity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When investigating genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors, and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales . In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant pathogenic fungus previously hidden in collapsed and fragmented genome assemblies. Current representations of eukaryotic microbial genomes are haploid, hiding the genomic ersity intrinsic to diploid and polyploid life forms. This hidden ersity contributes to the organism’s evolutionary potential and ability to adapt to stress conditions. Yet it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which leads to allelic variation and the presence of genes lacking allelic counterparts. Non-allelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic ersity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.
A high-quality pseudo-phased genome forMelaleuca quinquenerviashows allelic diversity of NLR-type resistance genes
Publisher: Cold Spring Harbor Laboratory
Date: 28-04-2023
DOI: 10.1101/2023.04.27.538497
Abstract: The coastal wetland tree species Melaleuca quinquenervia (Cav.) S.T.Blake (Myrtaceae), commonly named the broad-leaved paperbark, is a foundation species in eastern Australia, Indonesia, Papua New Guinea, and New Caledonia. The species has been widely grown as an ornamental, becoming invasive in areas such as Florida in the United States. Long-lived trees must respond to a wide range pests and pathogens throughout their lifespan, and immune receptors encoded by the nucleotide- binding domain and leucine-rich repeat containing (NLR) gene family play a key role in plant stress responses. Expansion of this gene family is driven largely by tandem duplication, resulting in a clustering arrangement on chromosomes. Due to this clustering and their highly repetitive domain structure, comprehensive annotation of NLR encoding genes within genomes has been difficult. Additionally, as many genomes are still presented in their haploid, collapsed state, the full allelic ersity of the NLR gene family has not been widely published for outcrossing tree species. We assembled a chromosome-level pseudo-phased genome for M . quinquenervia and describe the full allelic ersity of plant NLRs using the novel FindPlantNLRs pipeline. Analysis reveals variation in the number of NLR genes on each haplotype, differences in clusters and in the types and numbers of novel integrated domains. We anticipate that the high quality of the genome for M. quinquenervia will provide a new framework for functional and evolutionary studies into this important tree species. Our results indicate a likely role for maintenance of NLR allelic ersity to enable response to environmental stress, and we suggest that this allelic ersity may be even more important for long-lived plants.
Genome Sequence of the Fungus Nannizziopsis barbatae, an Emerging Reptile Pathogen
Publisher: American Society for Microbiology
Date: 07-01-2021
DOI: 10.1128/MRA.01213-20
Abstract: Nannizziopsis barbata is an emerging fungal pathogen capable of causing contagious dermatomycosis in reptiles. Here, we report a 31.54-Mb draft genome sequence of an isolate originating from an infected eastern water dragon in Brisbane, Australia.
DNA clean-up and size selection for long-read sequencing v1
Publisher: ZappyLab, Inc.
Date: 16-08-2019
DOI: 10.17504/PROTOCOLS.IO.6KAHCSE
Abstract: DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. Finally, a Short Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This has been trialled for the sorghum rot fungus Macrophomina phaseolina, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results are expected across many organisms, but have not been tested.
Factors associated with purchasing pesticide from shops for intentional self‐poisoning in Sri Lanka
Publisher: Wiley
Date: 05-08-2020
DOI: 10.1111/TMI.13469
The First Annotated Genome Assembly of Macrophomina tecta Associated with Charcoal Rot of Sorghum
Publisher: Oxford University Press (OUP)
Date: 31-05-2022
DOI: 10.1093/GBE/EVAC081
Abstract: Charcoal rot is an important soilborne disease caused by a range of Macrophomina species, which affects a broad range of commercially important crops worldwide. Even though Macrophomina species are fungal pathogens of substantial economic importance, their mechanism of pathogenicity and host spectrum are poorly understood. There is an urgent need to better understand the biology, epidemiology, and evolution of Macrophomina species, which, in turn, will aid in improving charcoal rot management strategies. Here, we present the first high-quality genome assembly and annotation of Macrophomina tecta strain BRIP 70781 associated with charcoal rot symptoms on sorghum. Hybrid assembly integrating long reads generated by Oxford Nanopore Technology and short Illumina paired-end reads resulted in 43 contigs with a total assembly size of ∼54 Mb, and an N50 of 3.4 Mb. In total, 12,926 protein-coding genes and 7,036 repeats were predicted. Genome comparisons detected accumulation of DNA transposons in Macrophomina species associated with sorghum. The first reference genome of M. tecta generated in this study will contribute to more comparative and population genomics studies of Macrophomina species.
Scalable high-molecular weight DNA extraction for long-read sequencing v1
Publisher: ZappyLab, Inc.
Date: 19-10-2020
DOI: 10.17504/PROTOCOLS.IO.BNJHMCJ6
Abstract: With rapid advances in long-read DNA sequencing technologies, it is becoming possible to resolve complex genomes, including repetitive, polyploid plant genomes. Despite the technology being available, a challenge persists: the extraction of pure high molecular weight DNA suitable for long-read sequencing. This is particularly true of native plants, crops and fungi. To resolve this, we optimised a gentle magnetic bead based high-molecular weight DNA extraction free of columns and high-centrifugation, to limit DNA fragmentation. A protocol that is scalable based on tissue input is presented, that can be used on many species of plants, fungi, reptiles, insects and bacteria. An optional sorbitol wash is listed and is highly recommended for plant tissues. To remove any remaining contaminants such as phenols and polysaccharides, two optional DNA clean-up and size selection strategies are given. Sequencing with Oxford Nanopore Technologies MinION, we can approximately obtain over 15-30 Gbp of sequencing from a single MinION flow cell with N50 values 30-50 kb. This has been routinely achieved with eucalypts, acacias, rice, themeda, wheat, wheat rusts, various other fungi, geckos, skinks, ticks, ladybird beetles, caterpillars and E. coli.
Revisiting the β-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach
Publisher: American Society for Microbiology
Date: 11-2019
DOI: 10.1128/AAC.01319-19
Abstract: The suboptimal effectiveness of β-lactam antibiotics against Mycobacterium tuberculosis has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a β-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R.
Exploring polymorphic interspecies structural variants in Eucalyptus: Unravelling Their Role in Reproductive Isolation and Adaptive Divergence
Publisher: Cold Spring Harbor Laboratory
Date: 21-10-2023
Multi-step high purity high molecular weight DNA extraction protocol from challenging fungal tissues v1
Publisher: ZappyLab, Inc.
Date: 26-07-2018
DOI: 10.17504/PROTOCOLS.IO.RZKD74W
Abstract: This protocol is based on the fact that we struggled for a long time to obtain high purity DNA from the fungal material, especially from several rust species. We finally thought we made it when we obtained DNA with perfect QC measures using a CTAB based DNA precipitation. Yet we still got low yields caused by high amount of 'active feedback' during the sequencing run. We and many others had similar observations that awesome looking DNA doesn't sequence well (www.protocols.io/groups/awesome-DNA-from-all-kingdoms-of-life/discussions/awesome-dna-purity-measures-but-quickly-dying-pores). In this protocol, we combined several ideas we accumulated over the last 1.5 years since we started working on HMW DNA extractions for Nanopore sequencing. - Different precipitants have different affinities for different contaminants. - Some contaminants may have a higher affinity and lower solubility in NaCl/PEG/SPRI 'clean' up steps. - Adding enzyme cocktails during the extraction may help to get rid of some contaminants. We combined all these three steps in the current protocol as combinatorial testing is currently cost prohibitive. It may well be that one of these steps is already enough. These ideas are laid out in more detail below and in publication soon to come. Our general recommendation is to test different buffer conditions and precipitants and if necessary combine them in a sequential manner. We hypothesize that different precipitants, e.g. NaCl/PEG, isopropanol, ethanol, or CTAB, display varying affinities for precipitating different contaminants. By applying them in a sequential manner it may be possible to obtain clean DNA via preferential precipitation of DNA over contaminants. In addition, in this newly developed protocol, we add enzyme mixes to the extraction buffer containing pectinases and cellulases reducing the amount of co-purifying contaminants from the fungal tissue. In case of other tissue types, different enzymes may have to be tested. It is important to add these enzymes during the extraction and not apply them to the final DNA suspension as most are not completely pure enzyme preparations and contain traces of DNAase activity that degrades the DNA when applied in simple solutions like TE buffer. We (see above) and many others have reported that NaCl/PEG-SPRI bead solutions are not always ideally suited to clean up DNA as contaminants simply co-precipitate. Following a similar logic of preferential precipitation, We hypothesize that is possible to first precipitate contaminants onto SPRI beads at low NaCl/PEG concentrations when HMW DNA stays in solution. In a subsequent step, DNA can be precipitated out of the remaining supernatant by increasing NaCl/PEG concentration adding more of the initial NaCl/PEG-SPRI beads solution. Contaminants with higher affinity to SPRI beads and lower solubility than DNA can thereby be removed from the solution. It is important to mention that we have had DNA preparations that fulfilled all our recommended quality control criteria but did not sequence well on the MinION. This was likely caused by ‘invisible’ contaminants. However, applying a combination of the above-suggested approaches enabled us to overcome this problem with our latest protocol.
DNA extraction from infected or not wheat leaf tissue
Publisher: ZappyLab, Inc.
Date: 10-02-2023
DOI: 10.17504/PROTOCOLS.IO.EWOV1O7DPLR2/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a step by step guide to s le representative leaf material for each treatment group and to extract DNA from these s les with the Qiagen DNeasy Plant Mini Kit. We use a kit in the course to speed up the process and make it reliable. Though it is important to understand the rough principles of DNA extraction as this process might influence the outcome of pathogen detection and identification in a biosecurity setting. We provide a short description of each step and its function in the protocol. In addition, please consult the handbook of the DNeasy Plant Mini Kit provider for further details as required. The final goal is to achieve the following: s le representative leaves for each treatment group that contain enough infective agent for DNA based pathogen identification. extract pure and high quality DNA that allows for PCR lification of marker gene regions for two kingdoms (bacteria and fungi). This protocol is applicable for week 2 and 3. Protocols progress overview: Week 2 S ling of two representative leaves and subsections thereof. Wee 2-3 tissue rupture with metal beads when tissues is frozen (performed by demonstrator). Week 3 DNA extraction from ruptured leaf tissue. The whole process follows the following major steps. S ling - Tissue rupture - Tissue lysis - Clearance of material - DNA precipitation - DNA binding to silica matrix - Washing of impurities - DNA elution from column and collection.
Socioeconomic position and suicidal behaviour in rural Sri Lanka: a prospective cohort study of 168,000+ people.
Publisher: Springer Science and Business Media LLC
Date: 21-02-2019
Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor
Publisher: Oxford University Press (OUP)
Date: 08-07-2016
DOI: 10.1105/TPC.15.00893
XA21-specific induction of stress-related genes followingXanthomonasinfection of detached rice leaves
Publisher: PeerJ
Date: 28-09-2016
DOI: 10.7717/PEERJ.2446
Abstract: The rice XA21 receptor kinase confers robust resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae ( Xoo ). We developed a detached leaf infection assay to quickly and reliably measure activation of the XA21-mediated immune response using genetic markers. We used RNA sequencing of elf18 treated EFR:XA21:GFP plants to identify candidate genes that could serve as markers for XA21 activation. From this analysis, we identified eight genes that are up-regulated in both in elf18 treated EFR:XA21:GFP rice leaves and Xoo infected XA21 rice leaves. These results provide a rapid and reliable method to assess bacterial-rice interactions.
High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing v1
Publisher: ZappyLab, Inc.
Date: 26-02-2021
DOI: 10.17504/PROTOCOLS.IO.BSS7NEHN
Abstract: Rapid advancements in long-read sequencing technologies have transformed sequencing read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles, insects and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale at low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. This has been routinely achieved with eucalypts, acacias, rice, themeda, wheat, wheat rusts, various other fungi, geckos, skinks, ticks, ladybird beetles, caterpillars and E. coli.
Whole‐mitochondrial genomes of Nannizziopsis provide insights in evolution and detection
Publisher: Wiley
Date: 03-2023
DOI: 10.1002/ECE3.9955
Abstract: Infectious fungal diseases can have devastating effects on wildlife health and a detailed understanding of the evolution of related emerging fungal pathogen along with the ability to detect them in the wild is considered indispensable for effective management strategies. Several fungi from the genera Nannizziopsis and Paranannizziopsis are emerging pathogens of reptiles and have been observed to cause disease in a wide range of taxa. Nannizziopsis barbatae has become a particularly important pathogen of Australian reptiles with an increasing number of herpetofauna being reported with cases of infection from across the country. Here, we present the mitochondrial genome sequences and phylogenetic analysis for seven species in this group of fungi uncovering new information on the evolutionary relationship of these emerging pathogens. From this analysis, we designed a species‐specific qPCR assay for the rapid detection of N. barbatae and demonstrate its application in a wild urban population of a dragon lizard.
Activation loop phosphorylation of a non-RD receptor kinase initiates plant innate immune signaling
Publisher: Proceedings of the National Academy of Sciences
Date: 16-09-2021
Abstract: Protein kinases lacking Arg in the catalytic loop HxD motif (i.e., non-RD kinases) are associated with innate immune signaling across kingdoms. Phosphorylation activates plant immune receptor kinases (RKs), but the mechanistic details of activation are limited. Using the non-RD immune RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we investigated the role of the receptor cytoplasmic domain in immune signaling and found that the catalytic activity of EFR is dispensable for antibacterial immunity. Nevertheless, ligand-induced EFR-mediated signaling is initiated by activation loop phosphorylation, but not via the catalytic activity of the receptor protein kinase domain. We propose that leucine-rich repeat-receptor kinase complexes containing a non-RD kinase are activated through phosphorylation-dependent conformational changes of the receptor cytoplasmic domain.
Transcriptional acclimation to warming temperatures of the Australian alpine herbWahlenbergia ceracea
Publisher: Cold Spring Harbor Laboratory
Date: 28-09-2023
Distinct Life Histories Impact Dikaryotic Genome Evolution in the Rust Fungus Puccinia striiformis Causing Stripe Rust in Wheat
Publisher: Oxford University Press (OUP)
Date: 09-04-2020
DOI: 10.1093/GBE/EVAA071
Abstract: Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f.sp. tritici, is a major threat to wheat production worldwide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst. This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole-genome comparative genomics of two P. striiformis f.sp. tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural, and allele levels. At the whole-genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates, whereas 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall, this detailed comparative analysis of two haplotype-aware assemblies of P. striiformis f.sp. tritici is the first step in understanding the evolution of dikaryotic rust fungi at a whole-genome level.
Sorbitol washing complex homogenate for improved DNA extractions v1
Publisher: ZappyLab, Inc.
Date: 09-04-2020
DOI: 10.17504/PROTOCOLS.IO.BEUVJEW6
Abstract: The extraction of pure DNA can be challenging due to the presence of sugars, oils and other endogenous chemicals present within biological s les. This is particularly true for many plants and fungi due to the presence of secondary metabolites such as polyphenols and polysaccharides. Polyphenols within the cytosol can become irreversibly DNA-bound after cell lysis and polysaccharides can co-precipitate with DNA during the extraction. Sorbitol is an osmotically active sugar alcohol and washing homogenate with sorbitol before cell lysis has been shown to significantly improve the purity of DNA extractions. Sorbitol does not pass cell membranes and likely acts by drawing the cytosol out of the cell. Therefore polyphenols and polyscacharides would be removed.
Plant Innate Immunity: Perception of Conserved Microbial Signatures
Publisher: Annual Reviews
Date: 02-06-2012
DOI: 10.1146/ANNUREV-ARPLANT-042811-105518
Abstract: Plants and animals sense conserved microbial signatures through receptors localized to the plasma membrane and cytoplasm. These receptors typically carry or associate with non-arginine-aspartate (non-RD) kinases that initiate complex signaling networks cumulating in robust defense responses. In plants, coregulatory receptor kinases have been identified that not only are critical for the innate immune response but also serve an essential function in other regulatory signaling pathways.
Tyrosine sulfation in a Gram-negative bacterium
Publisher: Springer Science and Business Media LLC
Date: 23-10-2012
DOI: 10.1038/NCOMMS2157
PCR reaction of marker regions (a.k.a metabarcodes) for two kingdoms
Publisher: ZappyLab, Inc.
Date: 12-02-2023
DOI: 10.17504/PROTOCOLS.IO.81WGBYQ4NVPK/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a step by step guide to perform PCR reactions on the extracted DNA s les of the five TG. The two PCR reaction target conserved marker regions (a.k.a. metabarcodes) for two kingdoms. With 16S primers targeting bacteria and ITS primers targeting fungi. The simplified idea of metabarcodes is that one can lify a region that is conserved enough to generate primers that allow one to lify this region from "all" members of the kindgom somewhat representatively. Within the licon though there are located hypervariable regions that are distinct between different species or genera. Matching these licons against a databases let's one identify the organism of this specific kingdom present in the s le. At least this is the simple theory, however as you will see in the lectures there are many issues and biases to consider when applying these techniques in general and specifically to pathogen diagnostics for biosecurity purposes. The final goal is to achieve the following: To measure and adjust DNA concentration for each s le to allow for equal amount of DNA being added to each s le. Perform PCR reaction for the fungal kingdom metabarcode using published ITS (Internal transcribed spacer) primers. Perform PCR reaction for the bacterial kingdom metabarcode using published 16S primers. This protocol is applicable for week 4. Protocols progress overview: Week 4 PCR reaction for 16S and ITS. You will measure double stranded DNA concentration with a dye based method. Here the dye binds to double stranded DNA only and this will change the fluorescent of the dye in a way directly proportional to the DNA concentration. You will use the pre-mixed broad range dye kit rder/catalog roduct/Q32853. The primers for the ITS PCR reactions are called ITSFor and ITSRev. These are based on White et. al., 1989 and have the following sequences. ITSFor (a.k.a. NS5): 5'-AACTTAAAGGAATTGACGGAAG-3' ITSRev (a.k.a.LR6): 5'-CGCCAGTTCTGCTTACC-3' The expected licon size is around 2.5 to 3 kb (kilobase pairs). The primers for the 16S PCR reaction are called 16SFor and 16SRev. These are based on Wuyts et. al., 2002, Baker et. al., 2003, and Youssef et. al., 2009. 16SFor (a.k.a. 27F, E8F, or E8Fa): 5'-AGAGTTTGATCCTGGCTCAG-3' 16SRev (a.k.a. 1492R): 5'-ACCTTGTTACGACTT-3' Importantly, these 16S primers also partially bind and lify 16S regions found in plant mitochondria and chloroplast. The lification of the plant 16S region has to be therefore inhibited with sequence specific probes called PNAs (Peptide mediated Nucleic Acids) roducts/PCR_blocker.htm. We will use the following PNAs for the 16S PCR reactions. These are based on Lundberg et a. 2013. The anti-mitochondrial PNA (mPNA): 5′-GGCAAGTGTTCTTCGGA-3′ The anti-plastid PNA (pPNA): 5′-GGCTCAACCCTGGACAG-3′ References: White, Bruns, Lee and Taylor, 1989, 'Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics', in PCR - Protocols and Applications - A Laboratory Manual, Academic Press Wuyts, Van de Peer, Winkelmans and De Wachter, 2002, 'The European database on small subunit ribosomal RNA', Nuc. Ac. Res., vol. 30 (1), pp. 183-185 Baker, Smith and Cowan, 2003, 'Review and re-analysis of domain-specific 16S primers', J. Microbiol. Meth., vol. 55 (3), pp. 541-555 Youssef, Sheik, Krumholz, Najar, Roe and Elshahed, 2009, 'Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys', App. Env. Microbiol., vol. 75 (16) Lundberg, Derek S., Scott Yourstone, Piotr Mieczkowski, Corbin D. Jones, and Jeffery L. Dangl. “Practical Innovations for High-Throughput Amplicon Sequencing.” Nature Methods 10, no. 10 (October 2013): 999–1002.
Non-arginine-aspartate (non-RD) kinases are associated with innate immune receptors that recognize conserved microbial signatures
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.PBI.2012.05.002
Abstract: An important question in the field of plant-pathogen interactions is how the detection of pathogens is converted into an effective immune response. In recent years, substantial insight has been gained into the identities of both the plant receptors and the microbial molecules they recognize. Likewise, many of the downstream signaling proteins and transcriptions factors that activate defense responses have been characterized. However, the early molecular events that comprise 'recognition' and how defense signaling specificity is achieved are not as well understood. In this review we discuss the significance of non-arginine-aspartate (non-RD) kinases, a subclass of kinases that are often found in association with pattern recognition receptors (PRRs).
Sequencing fungal and bacterial metabarcodes with native barcoding and Nanopore
Publisher: ZappyLab, Inc.
Date: 13-02-2023
DOI: 10.17504/PROTOCOLS.IO.EWOV1O7XYLR2/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol describes the molecular biology and a step-by-step guide for native barcoding and Nanopore sequencing of the metabarcode regions for fungi (Internal Transcribed Spacer, ITS) and bacteria (16S locus). Most of the steps will have been conducted by your demonstrators due to time limitations. During class we will have the final library ready to load. This protocol is applicable for week 5. Conceptual overview: Clean up of PCR products with magnetic beads to remove buffer, enzyme, and primers. Measure of PCR product concentration after clean up. Start of library preparation end-prep to repair the ends of each licon for ligation of barcodes including clean up with magnetic beads. Barcode ligation. Each PCR licon from each group will receive a unique DNA barcode. This barcode can be used to demultiplex each s le after the sequencing run while being able to run up to 96 s les within the same reaction. Clean up with magnetic beads to remove buffer, enzyme, and non-ligated barcodes. Pooling of all barcoded licons. Ligation of sequencing adapters. Clean up with magnetic beads to remove buffer, enzyme, and non-ligated sequencing adapters. Final library preparation for loading. Flow-cell priming to be ready to load. Library loading. Start of sequencing run. Demonstrators will perform step 1-8. In class we will perform step 9-12 for two libraries. One library is multiplexed to contain all ITS licons from class, and the other library contains all 16S licons from class. You can cite this protocol in the methods section of your report as for all other protocols. No need to write it all up again :).
Genomics-informed responses in the elimination of COVID-19 in Victoria, Australia: an observational, genomic epidemiological study
Publisher: Elsevier BV
Date: 08-2021
Transposon mediated horizontal transfer of the host-specific virulence protein ToxA between three fungal wheat pathogens
Publisher: Cold Spring Harbor Laboratory
Date: 17-06-2019
DOI: 10.1101/671446
Abstract: Most known ex les of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there ex les of HGT where non-coding DNA is also reported. The gene encoding the wheat virulence protein ToxA and surrounding 14 kb is one of these rare ex les. ToxA has been horizontally transferred between three fungal wheat pathogens ( Parastagonospora nodorum, Pyrenophora tritici-repentis and Bipolaris sorokiniana ) as part of a conserved ∼14kb element, which contains coding and non-coding regions. Here we use long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal level assemblies enabled identification of terminal inverted repeats on either end of the 14kb region, typical of a Type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-250 kb) transposon-rich genomic island which is absent in toxA- isolates. We demonstrate that the horizontal transfer of ToxhAT between Pyrenophora tritici-repentis and P. nodorum occurred as part of a large ∼80kb HGT which is now undergoing extensive decay. In contrast, in B. sorokiniana ToxhAT and its resident genomic island are mobile within the genome. Together these data provide insight into the non-coding regions that facilitate HGT between eukaryotes and the genomic processes which mask the extent of HGT between these species. This work dissects the tripartite horizontal transfer of ToxA a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues as to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a Type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point-mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts. All raw sequencing data is available under NCBI BioProject PRJNA505097. The P. nodorum SN15 Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SSHU00000000. The version SSHU01000000 is described in this paper. The P. nodorum SN79-1087 Whole Genome Shotgun project has been deposited under the accessions CP039668 - CP039689. The Whole Genome shotgun project and accession numbers for B. sorokiniana isolates are as follows: CS10 SRZH00000000, CS27 SRZG00000000, WAI2406 SRZF00000000, WAI2411 SRZE00000000. Transposon annotations, CS10 and CS27 gene annotations are available at egancamilla/Transposon-Mediated-transfer-of-ToxA
A Near-Complete Haplotype-Phased Genome of the Dikaryotic Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici Reveals High Interhaplotype Diversity
Publisher: American Society for Microbiology
Date: 07-03-2018
Abstract: A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N 50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype ersity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales . In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies. IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic ersity intrinsic to diploid and polyploid life forms. This hidden ersity contributes to the organism’s evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic ersity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.
TheXanthomonasAx21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles
Publisher: PeerJ
Date: 07-01-2014
DOI: 10.7717/PEERJ.242
The rice XA21 ectodomain fused to the Arabidopsis EFR cytoplasmic domain confers resistance to Xanthomonas oryzae pv. oryzae
Publisher: PeerJ
Date: 09-05-2018
DOI: 10.7717/PEERJ.4456
Abstract: Rice ( Oryza sativa ) plants expressing the XA21 cell-surface receptor kinase are resistant to Xanthomonas oryzae pv. oryzae (Xoo) infection. We previously demonstrated that expressing a chimeric protein containing the ELONGATION FACTOR Tu RECEPTOR (EFR) ectodomain and the XA21 endodomain (EFR:XA21) in rice does not confer robust resistance to Xoo . To test if the XA21 ectodomain is required for Xoo resistance, we produced transgenic rice lines expressing a chimeric protein consisting of the XA21 ectodomain and EFR endodomain (XA21:EFR) and inoculated these lines with Xoo . We also tested if the XA21:EFR rice plants respond to a synthetic sulfated 21 amino acid derivative (RaxX21-sY) of the activator of XA21-mediated immunity, RaxX. We found that five independently transformed XA21:EFR rice lines displayed resistance to Xoo as measured by lesion length analysis, and showed that five lines share characteristic markers of the XA21 defense response (generation of reactive oxygen species and defense response gene expression) after treatment with RaxX21-sY. Our results indicate that expression of the XA21:EFR chimeric receptor in rice confers resistance to Xoo . These results suggest that the endodomain of the EFR and XA21 immune receptors are interchangeable and the XA21 ectodomain is the key determinant conferring robust resistance to Xoo .
Small ubiquitin-like modifier proteases Overly Tolerant To Salt1 and -2 regulate salt stress responses in Arabidopsis
Publisher: Oxford University Press (OUP)
Date: 10-2008
Abstract: Understanding salt stress signaling is key to producing salt-tolerant crops. The small ubiquitin-like modifier (SUMO) is a crucial regulator of signaling proteins in eukaryotes. Attachment of SUMO onto substrates is reversible, and SUMO proteases, which specifically cleave the SUMO–substrate linkages, play a vital regulatory role during SUMOylation. We have identified two SUMO proteases, OVERLY TOLERANT TO SALT1 (OTS1) and OTS2, which are localized in the nucleus and act redundantly to regulate salt stress responses in Arabidopsis thaliana. ots1 ots2 double mutants show extreme sensitivity to salt. However, under low-salt conditions, ots1 ots2 double mutants are phenotypically similar to wild-type plants. We demonstrate that salt stress induces a dose-dependent accumulation of SUMO1/2-conjugated proteins in Arabidopsis. ots1 ots2 double mutants constitutively accumulate high levels of SUMO1/2-conjugated proteins even under nonstress conditions and show a further dramatic increase in SUMO1/2-conjugated proteins in response to salt stress. Transgenic lines overexpressing OTS1 have increased salt tolerance and a concomitant reduction in the levels of SUMOylated proteins. Conversely, the ectopic expression of the mutant ots1(C526S) protein lacking SUMO protease activity fails to produce a salt-tolerant phenotype. We show that salt directly affects OTS1-dependent signaling by inducing OTS1 protein degradation. Our results indicate a requirement for OTS1 deSUMOylation activity in plant salt tolerance responses.
Proteome profile of the endomembrane of developing coleoptiles from switchgrass (Panicum virgatum)
Publisher: Wiley
Date: 16-03-2015
Abstract: The cost-effective production of biofuels from lignocellulosic material will likely require manipulation of plant biomass, specifically cell walls. The North American native prairie grass Panicum virgatum (switchgrass) is seen as a potential biofuel crop with an array of genetic resources currently being developed. We have characterized the endomembrane proteome of switchgrass coleoptiles to provide additional information to the switchgrass community. In total, we identified 1750 unique proteins from two biological replicates. These data have been deposited in the ProteomeXchange with the identifier PXD001351 (ataset/PXD001351).
Editorial overview: Biotic interactions - from single molecules to complex ecosystems.
Publisher: Elsevier BV
Date: 08-2020
The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that direct genome-wide DNA methylation
Publisher: Springer Science and Business Media LLC
Date: 15-09-2021
DOI: 10.1186/S12915-021-01123-Z
Abstract: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici ( Pgt ), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly erse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally ersified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5′ uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5′ adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.
Empowering Early Career Researchers to Improve Science
Publisher: Center for Open Science
Date: 11-06-2021
Abstract: Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in the conduct and communication of science. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve science. The event was focused on why ECRs are needed to improve science and the obstacles they face when trying to promote reform. Our discussions also highlighted the additional obstacles that ECRs in countries with limited research funding experience when working to improve the scientific system. We provide the lessons learned from successful ECR-led or ECR-focused initiatives and outline actions that in iduals and organizations can take to further support ECRs who are working to improve research culture and practice.
Public health interventions successfully mitigated multiple incursions of SARS-CoV-2 Delta variant in the Australian Capital Territory
Publisher: Cold Spring Harbor Laboratory
Date: 24-08-2022
DOI: 10.1101/2022.08.23.22278828
Abstract: The Australian Capital Territory rapidly responded to an incursion of the SARS-CoV-2 Delta (B.1.617.2) variant on 12 August 2021 with several public health interventions, including a territory-wide lockdown and genomic sequencing. Prior to this date, SARS-CoV-2 had been eliminated locally since July 7, 2020. Sequencing of % of cases identified at least 13 independent incursions with onwards spread in the community during the study period, between 12 August and 11 November 2021. Two incursions resulted in the majority of community transmission during this period, with persistent transmission in vulnerable sections of the community. Ultimately, both major incursions were successfully mitigated through public health interventions, including COVID-19 vaccines. In this study we explore the demographic factors that contributed to the spread of these incursions. The high rates of SARS-CoV-2 sequencing in the Australian Capital Territory and the relatively small population size facilitated detailed investigations of the patterns of virus transmission. Genomic sequencing was critical to disentangling complex transmission chains to target interventions appropriately. Despite a strict lockdown and interstate travel restrictions, the Australian Capital Territory experienced at least 13 incursions of SARS-CoV-2 Delta (B.1.617.2) with onwards spread in the community between 12 August and 11 November 2021. This level of detail was only accessible because of the high rate of SARS-CoV-2 sequencing, with sequencing attempted on 1438/1793 (80%) of cases. Transmission chains varied in size and duration, with two dominant incursions (ACT.19 and ACT.20) comprising 35% and 53% of all sequenced cases during the study period, respectively. The ACT.20 outbreak persisted longer, due to specific challenges with implementing public health interventions in the affected populations. Both major incursions were successfully curbed through stringent public health measures, including the widespread acceptance of COVID-19 vaccines ( % of the eligible population by the end of the study period).
Austropuccinia psidii, causing myrtle rust, has a gigabase-sized genome shaped by transposable elements
Publisher: Oxford University Press (OUP)
Date: 27-11-2021
DOI: 10.1093/G3JOURNAL/JKAA015
Abstract: Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in TEs and a very low overall GC content of 33.8%. Compared to other Pucciniales, the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how TEs shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.
"Midnight" SARS-CoV2 genome sequencing protocol using 1200bp amplicon primer set v2 and the Nanopore Rapid library kit
Publisher: ZappyLab, Inc.
Date: 08-04-2023
DOI: 10.17504/PROTOCOLS.IO.14EGN2Q2YG5D/V1
Abstract: This protocols is part of the ANU Biosecurity mini-research project #2 "An SARS-COV2 incursion scenario: Genomics, phylogenetics, and incursions." This mini-research project is modeled on the yearly Quality Assurance Program of The Royal College of Pathologists of Australia (RCPAQAP), we take part in together with ACT Pathology. This research project is split into two major parts, identical to how the official RCPAQAP is run every year. Part #1 is focusing on the 'wet- lab' by sequencing SARS-COV2 from real world RNA s les provided by ACT Pathology especially for our ANU biosecurity course (Thank YOU!). Here you will lify and sequence five (5) RNA s les per research group. You will assess the SARS-COV2 genome sequences for their lineage assignments using online programs, put sequences into a global context, estimate the collection date based on genetic information, and describe mutations in the spike protein. Part #2 is focusing on the 'dry-lab' by investigating a hypothetical incursion scenario in the so-called city Fantastica. You will combine genomic surveillance of SARS-COV2 with case interview data to trace the spread into of SARS-COV2 in the community and into high risk settings. We will provide you with real publicly available SARS-COV2 genome and fantasized case interviews. You will put these two together to trace the spread and suggest potential improvements in containment strategies with a focus on high risk settings. This protocol describes the wet-lab component of Part #1. It is an adaption (fork) of a previous protocol published by Nikki Freed and Olin Silander during the early days of the pandemic here and here. They designed the protocol to enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than previous methods, we use multiplexed 1200 base pair PCR licons with the Oxford Nanopore RAPID barcoding kit. The original publication that describes this protocol is here along with important considerations is here: iomethods/article/5/1/bpaa014/5873518?login=true Primers were all designed using Primal Scheme: primal.zibraproject.org/, described here rticles/nprot.2017.066. You will be using the midnight primer set v2 described here and purchased here ages roducts/next-generation-sequencing/workflow/xgen-ngs- licon-sequencing redesigned- licon-panels/sars-cov-2-midnight- -panel. The final goal is to achieve the following: Week 7: cDNA reverse transcription of RNA into DNA. Pooled lification of the SARS-COV2 genomes chunked into ~1200bp licons with non-overlapping pools A and B. Week 8: Library preparation using the Nanopore Rapid barcoding kit. Sequencing your own library on a Nanopore flongel. This protocol is applicable for week 7 and 8.
Inferring species compositions of complex fungal communities from long- and short-read sequence data
Publisher: Cold Spring Harbor Laboratory
Date: 02-05-2021
DOI: 10.1101/2021.05.02.442318
Abstract: The kingdom fungi is crucial for life on earth and is highly erse. Yet fungi are challenging to characterize. They can be difficult to culture and may be morphologically indistinct in culture. They can have complex genomes of over 1 Gb in size and are still underrepresented in whole genome sequence databases. Overall their description and analysis lags far behind other microbes such as bacteria. At the same time, classification of species via high throughput sequencing without prior purification is increasingly becoming the norm for pathogen detection, microbiome studies, and environmental monitoring. However, standardized procedures for characterizing unknown fungi from complex sequencing data have not yet been established. We compared different metagenomics sequencing and analysis strategies for the identification of fungal species. Using two fungal mock communities of 44 phylogenetically erse species, we compared species classification and community composition analysis pipelines using shotgun metagenomics and licon sequencing data generated from both short and long read sequencing technologies. We show that regardless of the sequencing methodology used, the highest accuracy of species identification was achieved by sequence alignment against a fungi-specific database. During the assessment of classification algorithms, we found that applying cut-offs to the query coverage of each read or contig significantly improved the classification accuracy and community composition analysis without significant data loss. Overall, our study expands the toolkit for identifying fungi by improving sequence-based fungal classification, and provides a practical guide for the design of metagenomics analyses.
An XA21-Associated Kinase (OsSERK2) Regulates Immunity Mediated by the XA21 and XA3 Immune Receptors
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1093/MP/SSU003
A comprehensive toolkit to enable MinION sequencing in any laboratory
Publisher: Cold Spring Harbor Laboratory
Date: 27-03-2018
DOI: 10.1101/289579
Abstract: Long-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realising their full potential relies crucially on extracting high quality, high molecular weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which potentiates all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time consuming and costly. Here we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimising DNA extractions from a challenging plant tissue as a case study. Following the workflow illustrated we were able to reliably and repeatedly obtain 8.5 Gb of long read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open-source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls, will be useful to others who plan to establish long-read sequencing in their own laboratories.
The draft nuclear genome assembly of Eucalyptus pauciflora: a pipeline for comparing de novo assemblies
Publisher: Oxford University Press (OUP)
Date: 2020
DOI: 10.1093/GIGASCIENCE/GIZ160
Abstract: Eucalyptus pauciflora (the snow gum) is a long-lived tree with high economic and ecological importance. Currently, little genomic information for E. pauciflora is available. Here, we sequentially assemble the genome of Eucalyptus pauciflora with different methods, and combine multiple existing and novel approaches to help to select the best genome assembly. We generated high coverage of long- (Nanopore, 174×) and short- (Illumina, 228×) read data from a single E. pauciflora in idual and compared assemblies from 5 assemblers (Canu, SMARTdenovo, Flye, Marvel, and MaSuRCA) with different read lengths (1 and 35 kb minimum read length). A key component of our approach is to keep a randomly selected collection of ∼10% of both long and short reads separated from the assemblies to use as a validation set for assessing assemblies. Using this validation set along with a range of existing tools, we compared the assemblies in 8 ways: contig N50, BUSCO scores, LAI (long terminal repeat assembly index) scores, assembly ploidy, base-level error rate, CGAL (computing genome assembly likelihoods) scores, structural variation, and genome sequence similarity. Our result showed that MaSuRCA generated the best assembly, which is 594.87 Mb in size, with a contig N50 of 3.23 Mb, and an estimated error rate of ∼0.006 errors per base. We report a draft genome of E. pauciflora, which will be a valuable resource for further genomic studies of eucalypts. The approaches for assessing and comparing genomes should help in assessing and choosing among many potential genome assemblies from a single dataset.
Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling
Publisher: Springer Science and Business Media LLC
Date: 09-2018
MinIONQC: fast and simple quality control for MinION sequencing data
Publisher: Oxford University Press (OUP)
Date: 23-07-2018
DOI: 10.1093/BIOINFORMATICS/BTY654
Abstract: MinIONQC provides rapid diagnostic plots and quality control data from one or more flowcells of sequencing data from Oxford Nanopore Technologies’ MinION instrument. It can be used to assist with the optimisation of extraction, library preparation, and sequencing protocols, to quickly and directly compare the data from many flowcells, and to provide publication-ready figures summarising sequencing data. MinIONQC is implemented in R and released under an MIT license. It is available for all platforms from oblanf/minion_qc.
Amplicon sequencing using MinION optimized from 1D native barcoding genomic DNA v1
Publisher: ZappyLab, Inc.
Date: 08-01-2018
DOI: 10.17504/PROTOCOLS.IO.MHKC34W
Abstract: This is a protocol optimized from Oxford Nanopore Technology 1D native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) for sequencing licons of fungal marker genes (ITS, EF1a) using PCR. Thanks to Dr. Benjamin Schwesinger for his suggestions about the protocol. Thanks to Dr. Leon Smith (ANU, Linde lab), Prof. Wieland Meyer (USydney) and Dr. Laszlo Irinyi for their contribution of fungal s les.
Running a research group in the next generation: combining sustainable and reproducible research with values-driven leadership
Publisher: Oxford University Press (OUP)
Date: 19-12-2022
DOI: 10.1093/JXB/ERAC407
Evaluating features of scientific conferences: A call for improvements
Publisher: Cold Spring Harbor Laboratory
Date: 03-04-2020
DOI: 10.1101/2020.04.02.022079
Abstract: Scientific conferences provide valuable opportunities for researchers across career stages and disciplines to present their latest work, and to network with their peers. These meetings have largely been held in-person with rapid proliferation in the number of meetings and attendees. Yet the format and quality of their organization lag behind what is possible and as a result, the current experience of attending conferences in many disciplines remains unchanged in many respects. We created a database of 270 national and international academic conferences held in-person during 2018-2019 in various disciplines and examined them for their features, costs and impact on the community. We found that many meetings could still be improved significantly in terms of ersity, inclusivity, promoting early career researcher (ECR) networking and career development, venue accessibility, and importantly, reducing the meetings’ carbon footprint. It is important to accelerate and mandate efforts to improve conferences so that researchers in all disciplines, in particular ECRs, consistently benefit from scientific gatherings, for years to come. We discuss a combination of approaches and recommendations to make conferences more modern, effective, equitable and intellectually productive for the research community and environmentally sustainable for our planet. “They always say time changes things, but you actually have to change them yourself . ” — Andy Warhol
High molecular weight gDNA extraction after Mayjonade et al. optimised for eucalyptus for nanopore sequencing v8
Publisher: ZappyLab, Inc.
Date: 14-10-2017
DOI: 10.17504/PROTOCOLS.IO.KA2CSGE
Abstract: Extraction of high quality DNA for long read sequencing e.g. the Oxford Nanopore Optimized for DNA extraction from eucalyptus grandis and eucalyptus pauciflora. This protocol contains an optional Chloroform clean up step which is necessary for eucalyptus but might not be for other tissue. For long DNA fragments don't vortex the DNA s le.
OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae
Publisher: Wiley
Date: 16-11-2014
DOI: 10.1111/JIPB.12290
Public health interventions successfully mitigated multiple incursions of SARS-CoV-2 Delta variant in the Australian Capital Territory
Publisher: Cambridge University Press (CUP)
Date: 2023
DOI: 10.1017/S0950268823000201
Abstract: The COVID-19 pandemic has presented a unique opportunity to understand how real-time pathogen genomics can be used for large-scale outbreak investigations. On 12 August 2021, the Australian Capital Territory (ACT) detected an incursion of the SARS-CoV-2 Delta (B.1.617.2) variant. Prior to this date, SARS-CoV-2 had been eliminated locally since 7 July 2020. Several public health interventions were rapidly implemented in response to the incursion, including a territory-wide lockdown and comprehensive contact tracing. The ACT has not previously used pathogen genomics at a population level in an outbreak response therefore, this incursion also presented an opportunity to investigate the utility of genomic sequencing to support contact tracing efforts in the ACT. Sequencing of % of the 1793 laboratory-confirmed cases during the 3 months following the initial notification identified at least 13 independent incursions with onwards spread in the community. Stratification of cases by genomic cluster revealed that distinct cohorts were affected by the different incursions. Two incursions resulted in most of the community transmission during the study period, with persistent transmission in vulnerable sections of the community. Ultimately, both major incursions were successfully mitigated through public health interventions, including COVID-19 vaccines. The high rates of SARS-CoV-2 sequencing in the ACT and the relatively small population size facilitated detailed investigations of the patterns of virus transmission, revealing insights beyond those gathered from traditional contact tracing alone. Genomic sequencing was critical to disentangling complex transmission chains to target interventions appropriately.
Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case
Publisher: Cold Spring Harbor Laboratory
Date: 11-05-2018
DOI: 10.1101/320085
Abstract: Chloroplasts are organelles that conduct photosynthesis in plant and algal cells. Chloroplast genomes code for around 130 genes, and the information they contain is widely used in agriculture and studies of evolution and ecology. Correctly assembling complete chloroplast genomes can be challenging because the chloroplast genome contains a pair of long inverted repeats (10–30 kb). The advent of long-read sequencing technologies should alleviate this problem by providing sufficient information to completely span the inverted repeat regions. Yet, long-reads tend to have higher error rates than short-reads, and relatively little is known about the best way to combine long- and short-reads to obtain the most accurate chloroplast genome assemblies. Using Eucalyptus pauciflora , the snow gum, as a test case, we evaluated the effect of multiple parameters, such as different coverage of long (Oxford nanopore) and short (Illumina) reads, different long-read lengths, different assembly pipelines, and different genome polishing steps, with a view to determining the most accurate and efficient approach to chloroplast genome assembly. Hybrid assemblies combining at least 20x coverage of both long-reads and short-reads generated a single contig spanning the entire chloroplast genome with few or no detectable errors. Short-read-only assemblies generated three contigs representing the long single copy, short single copy and inverted repeat regions of the chloroplast genome. These contigs contained few single-base errors but tended to exclude several bases at the beginning or end of each contig. Long-read-only assemblies tended to create multiple contigs with a much higher single-base error rate, even after polishing. The chloroplast genome of Eucalyptus pauciflora is 159,942 bp, contains 131 genes of known function, and confirms the phylogenetic position of Eucalyptus pauciflora as a close relative of Eucalyptus regnans . Our results suggest that very accurate assemblies of chloroplast genomes can be achieved using a combination of at least 20x coverage of long- and short-reads respectively, provided that the long-reads contain at least ~5x coverage of reads longer than the inverted repeat region. We show that further increases in coverage give little or no improvement in accuracy, and that hybrid assemblies are more accurate than long-read-only or short-read-only assemblies.
Library preparation protocol to sequence V3-V4 region of 16S rRNA to run in Illumina MiSeq platform v1
Publisher: ZappyLab, Inc.
Date: 15-08-2019
DOI: 10.17504/PROTOCOLS.IO.6I7HCHN
Abstract: This is an optimised protocol for 16S library preparation of V3-V4 region for sequencing through Illumina MiSeq platform (2 x 300 bp V3 chemistry). This protocol uses Platinum™ SuperFi™ PCR Master Mix instead of 2x KAPA HiFi HotStart ReadyMix given in Illumina 16S protocol. This polymerase master mix has lower error rate than KAPA, making it more suitable for sequencing.
A chloroplast retrograde signal, 3’phosphoadenosine 5’-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination
Publisher: eLife Sciences Publications, Ltd
Date: 21-03-2017
DOI: 10.7554/ELIFE.23361
Abstract: Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3′-phosphoadenosine 5′- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1) priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+ up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.
Interspecies genome divergence is predominantly due to frequent small scale rearrangements in Eucalyptus
Publisher: Wiley
Date: 21-07-2022
DOI: 10.1111/MEC.16608
Abstract: Synteny, the ordering of sequences within homologous chromosomes, must be maintained within the genomes of sexually reproducing species for the sharing of alleles and production of viable, reproducing offspring. However, when the genomes of closely related species are compared, a loss of synteny is often observed. Unequal homologous recombination is the primary mechanism behind synteny loss, occurring more often in transposon rich regions, and resulting in the formation of chromosomal rearrangements. To examine patterns of synteny among three closely related, interbreeding, and wild Eucalyptus species, we assembled their genomes using long‐read DNA sequencing and de novo assembly. We identify syntenic and rearranged regions between these genomes and estimate that ~48% of our genomes remain syntenic while ~36% is rearranged. We observed that rearrangements highly fragment microsynteny. Our results suggest that synteny between these species is primarily lost through small‐scale rearrangements, not through sequence loss, gain, or sequence ergence. Further examination of identified rearrangements suggests that rearrangements may be altering the phenotypes of Eucalyptus species. Our study also underscores that the use of single reference genomes in genomic variation studies could lead to reference bias, especially given the scale at which we show potentially adaptive loci have highly erged, deleted, duplicated and/or rearranged. This study provides an unbiased framework to look at potential speciation and adaptive loci among a rapidly radiating foundation species of woodland trees that are free from selective breeding seen in most crop species.
Recommendations for empowering early career researchers to improve research culture and practice
Publisher: Public Library of Science (PLoS)
Date: 07-07-2022
DOI: 10.1371/JOURNAL.PBIO.3001680
Abstract: Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and ex les of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian.
Variation and inheritance of theXanthomonasgene cluster required for activation of XA21-mediated immunity
Publisher: Cold Spring Harbor Laboratory
Date: 14-06-2017
DOI: 10.1101/149930
Abstract: The rice XA21-mediated immune response is activated upon recognition of the RaxX peptide produced by the bacterium Xanthomonas oryzae pv. oryzae ( Xoo ). The 60 residue RaxX precursor is posttranslationally modified to form a sulfated tyrosine peptide that shares sequence and functional similarity with the plant sulfated tyrosine (PSY) peptide hormones. The five kb raxX-raxSTAB gene cluster of Xoo encodes RaxX, the RaxST tyrosylprotein sulfotransferase, and the RaxA and RaxB components of a predicted type one secretion system. The identified the complete raxX-raxSTAB gene cluster is present only in Xanthomonas spp., in five distinct lineages in addition to X. oryzae . The phylogenetic distribution of the raxX-raxSTAB gene cluster is consistent with the occurrence of multiple lateral transfer events during Xanthomonas speciation. RaxX variants representing each of the five lineages, and three Xoo RaxX variants, fail to activate the XA21-mediated immune response yet retain peptide hormone activity. These RaxX variants contain a restricted set of missense mutations, consistent with the hypothesis that selection acts to maintain peptide hormone-like function. These observations are also consistent with the hypothesis that the XA21 receptor evolved specifically to recognize Xoo RaxX.
Plant genome evolution in the genusEucalyptusdriven by structural rearrangements that promote sequence divergence
Publisher: Cold Spring Harbor Laboratory
Date: 20-04-2023
DOI: 10.1101/2023.04.19.537464
Abstract: Genomes have a highly organised architecture (non-random organisation of functional and non-functional genetic elements within chromosomes) that is essential for many biological functions, particularly, gene expression and reproduction. Despite the need to conserve genome architecture, a surprisingly high level of structural variation has been observed within species. As species separate and erge, genome architecture also erges, becoming increasingly poorly conserved as ergence time increases. However, within plant genomes, the processes of genome architecture ergence are not well described. Here we use long-read sequencing and de novo assembly of 33 phylogenetically erse, wild and naturally evolving Eucalyptus species, covering 1-50 million years of erging genome evolution to measure genome architectural conservation and describe architectural ergence. The investigation of these genomes revealed that following lineage ergence genome architecture is highly fragmented by rearrangements. As genomes continue to erge, the accumulation of mutations and subsequent ergence beyond recognition of rearrangements becomes the primary driver of genome ergence. The loss of syntenic regions also contribute to genome ergence, but at a slower pace than rearrangements. We hypothesise that duplications and translocations are potentially the greatest contributors to genome ergence.
Inferring Species Compositions of Complex Fungal Communities from Long- and Short-Read Sequence Data
Publisher: American Society for Microbiology
Date: 26-04-2022
Abstract: Our study is unique in that it provides an in-depth comparative study of a real-life complex fungal community analyzed with multiple long- and short-read sequencing approaches. These technologies and their application are currently of great interest to erse biologists as they seek to characterize the community compositions of microbiomes.
Pathogen Detection and Microbiome Analysis of Infected Wheat Using a Portable DNA Sequencer
Publisher: Scientific Societies
Date: 2019
DOI: 10.1094/PBIOMES-01-19-0004-R
Abstract: Fungal diseases of plants are responsible for major losses in agriculture, highlighting the need for rapid and accurate identification of plant pathogens. Disease outcomes are often defined not only by the main pathogen but are influenced by erse microbial communities known as the microbiome at sites of infection. Here we present the first use of whole genome shot-gun sequencing with a portable DNA sequencing device as a method for the detection of fungal pathogens from wheat (Triticum aestivum) in a standard molecular biology laboratory. The data revealed that our method is robust and applicable to the diagnosis of fungal diseases including wheat stripe rust (caused by Puccinia striiformis f. sp. tritici), Septoria tritici blotch (caused by Zymoseptoria tritici), and yellow leaf spot (caused by Pyrenophora tritici repentis). We also identified the bacterial genus Pseudomonas co-present with Puccinia and Zymoseptoria but not Pyrenophora infections. One limitation of the method is the over-representation of redundant wheat genome sequences from s les. This could be addressed by long-range licon-based sequencing approaches in future studies, which specifically target nonhost organisms. Our work outlines a new approach for detection of a broad range of plant pathogens and associated microbes using a portable sequencer in a standard laboratory, providing the basis for future development of an on-site disease monitoring system. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora
Publisher: Wiley
Date: 05-10-2018
Library preparation protocol to sequence full length 16S rRNA gene in Nanopore MinION sequencer v1
Publisher: ZappyLab, Inc.
Date: 16-08-2019
DOI: 10.17504/PROTOCOLS.IO.6J6HCRE
Abstract: This protocol was optimised from an existing protocol to achieve maximum pore occupancy in a MinION flow cell. This protocol was performed using Ligation sequencing kit (ONT) in combination with native barcoding kit (ONT). Using this protocol, I was able to achieve ~12 Gbp of data from one MinION run from s les with very low input concentration.
On the value of preprints: an early career researcher perspective
Publisher: PeerJ
Date: 12-2018
DOI: 10.7287/PEERJ.PREPRINTS.27400V1
Abstract: Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent, public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. ECRs face changes in the academic landscape including the increased interdisciplinarity of life sciences research, expansion of the researcher population and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal managed peer-review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback and facilitating collaborations. While there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting needs to be addressed. We provide a perspective from an interdisciplinary group of early career researchers on the value of preprints and advocate the wide adoption of preprints to advance knowledge and facilitate career development.
Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity
Publisher: Frontiers Media SA
Date: 02-08-2017
Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-7249-4_5
Abstract: Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.
Related Organisations
University Of California Davis Department Of Plant Pathology
Location: United States of America
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