Peter Solomon

Ubiquity of ToxA and absence of ToxB in Australian populations ofPyrenophora tritici-repentis

Publisher: Springer Science and Business Media LLC

Date: 2010

DOI: 10.1071/AP09056

A metabolomic approach to dissecting osmotic stress in the wheat pathogen Stagonospora nodorum

Publisher: Elsevier BV

Date: 11-2008

DOI: 10.1016/J.FGB.2008.08.006

Abstract: A non-targeted metabolomics approach was used to identify significant changes in metabolism upon exposure of the wheat pathogen Stagonospora nodorum to 0.5M NaCl. The polyol arabitol, and to a lesser extent glycerol, was found to accumulate in response to the osmotic stress treatment. Amino acid synthesis was strongly down-regulated whilst mannitol levels were unaffected. A reverse genetic approach was undertaken to dissect the role of arabitol metabolism during salt stress. Strains of S. nodorum lacking a gene encoding an l-arabitol dehydrogenase (abd1), a xylitol dehydrogenase (xdh1) and a double-mutant lacking both genes (abd1xdh1) were exposed to salt and the intracellular metabolites analysed. Arabitol levels were significantly up-regulated upon salt stress in the xdh1 strains but were significantly lower than the wild-type. Arabitol was not significantly different in either the abd1 or the abd1xdh1 strains during osmotic stress but the concentration of glycerol was significantly higher indicating a compensatory mechanism in operation. Genome sequence analysis identified a second possible enzyme capable of synthesizing arabitol explaining the basal level of arabitol present in the abd1xdh1 strains. This study identified that arabitol is the primary compatible solute in S. nodorum but in-built levels of redundancy are present allowing the fungus to tolerate osmotic stress.

A genome-wide survey of the secondary metabolite biosynthesis genes in the wheat pathogen Parastagonospora nodorum.

Publisher: Informa UK Limited

Date: 24-06-2014

DOI: 10.1080/21501203.2014.928386

Just the surface: advances in the discovery and characterization of necrotrophic wheat effectors

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.MIB.2018.01.019

Abstract: For many years pathogens of wheat have remained poorly understood. Hindered by an inaccessible host and the obligate nature of many of the pathogens, our understanding of these interactions has been limited compared to other more amenable pathosystems. However, breakthroughs over recent years have shed new light on diseases of wheat, particularly those caused by the genetically tractable necrotrophic pathogens. We now understand that many of the necrotrophic fungal pathogens do interact with wheat in a strict gene-for-gene relationship, and that pathogen and host partners in these interactions have now been identified. This improved understanding of necrotrophic effector biology has fundamentally changed the way we consider these important wheat diseases.

Emergence of Tan Spot Disease Caused by Toxigenic Pyrenophora tritici-repentis in Australia Is Not Associated with Increased Deployment of Toxin-Sensitive Cultivars

Publisher: Scientific Societies

Date: 05-2008

DOI: 10.1094/PHYTO-98-5-0488

Abstract: The wheat disease tan (or yellow leaf) spot, caused by Pyrenophora tritici-repentis, was first described in the period 1934 to 1941 in Canada, India, and the United States. It was first noted in Australia in 1953 and only became a serious disease in the 1970s. The emergence of this disease has recently been linked to the acquisition by P. tritici-repentis of the ToxA gene from the wheat leaf and glume blotch pathogen, Stagonospora nodorum. ToxA encodes a host-specific toxin that interacts with the product of the wheat gene Tsn1. Interaction of ToxA with the dominant allele of Tsn1 causes host necrosis. P. tritici-repentis races lacking ToxA give minor indistinct lesions on wheat lines, whereas wheat lines expressing the recessive tsn1 are significantly less susceptible to the disease. Although the emergence and spread of tan spot had been attributed to the adoption of minimum tillage practices, we wished to test the alternative idea that the planting of Tsn1 wheat lines may have contributed to the establishment of the pathogen in Australia. To do this, wheat cultivars released in Australia from 1911 to 1986 were tested for their sensitivity to ToxA. Prior to 1941, 16% of wheat cultivars were ToxA-insensitive and hence, all other factors being equal, would be more resistant to the disease. Surprisingly, only one of the cultivars released since 1940 was ToxA insensitive, and the area planted to ToxA-insensitive cultivars varied from 0 to a maximum of only 14% in New South Wales. Thus, the majority of the cultivars were ToxA-sensitive both before and during the period of emergence and spread of the disease. We therefore conclude that the spread of P. tritici-repentis in Australia cannot be causally linked to the deployment of ToxA-sensitive cultivars.

Limited formal education is strongly associated with lower cognitive status, functional disability and frailty status in older adults

Publisher: FapUNIFESP (SciELO)

Date: 06-2019

DOI: 10.1590/1980-57642018DN13-020011

Abstract: ABSTRACT. Limited formal education is still common in ageing populations. Although limited formal education seems to be independently and negatively associated with cognition, functional abilities and frailty in ageing, no studies have examined whether the gradient of limited formal education has an impact on health in later life. Objective: to examine the relationship of limited formal education with cognitive status, functional abilities, and frailty status. Methods: a cross-sectional study was conducted involving 540 older adults stratified into groups: no formal education, 12-24 months of education, and 25-48 months of education. Cognitive screening (MMSE), functional abilities (Lawton Index), and frailty (CHS criteria) were measured. Regression analyses were performed. Results: 27% had no formal education, 21% had 12-24 months of formal education, and 55% had 25-48 months of formal education. Limited formal education has a clear gradient of negative impact: No formal education was associated with scoring below MMSE cut-off scores (OR = 7.9), being totally artially dependent for IADLs (OR = 2.5) and frail (OR = 2.0). Having 12-24 months of education was associated with scoring below MMSE cut-off scores (OR = 5.2) and with being frail (OR = 2.0). The No formal education group was 10.1 times more likely to have worse cognitive scores, worse functional abilities and frailty re-frailty status concomitantly (CCoFF), while older adults who had 12-24 months of education had a 4.6 times greater chance of having CCoFF. Conclusion: limited education had a gradient association with cognitive performance, functional disability and frailty. These findings clearly emphasize the importance of prevention through education from childhood to older age.

A review on South Asian wheat blast: The present status and future perspective

Publisher: Wiley

Date: 13-08-2020

DOI: 10.1111/PPA.13250

Combining Protein Ratio p-Values as a Pragmatic Approach to the Analysis of Multirun iTRAQ Experiments

Publisher: American Chemical Society (ACS)

Date: 13-01-2015

DOI: 10.1021/PR501091E

Abstract: iTRAQ labeling of peptides is widely used for quantitative comparison of biological s les using mass spectrometry. However, iTRAQ determined protein ratios have varying credibility depending on the number and quality of the peptide ratios used to generate them, and accounting for this becomes problematic particularly in the multirun scenario needed for larger scale biological studies. One approach to this problem relies on the use of sophisticated statistical global models using peptide ratios rather than working directly with the protein ratios, but these yield complex models whose solution relies on computational approaches such as stage-wise regression, which are nontrivial to run and verify. Here we evaluate an alternative pragmatic approach to finding differentially expressed proteins based on combining protein ratio p-values across experiments in a fashion similar to running a meta-analysis across different iTRAQ runs. Our approach uses the well-established Stouffer's Z-transform for combining p-values, alongside a ratio trend consistency measure, which we introduce. We evaluate this method with data from two iTRAQ experiments using plant and animal models. We show that in the specific context of iTRAQ data analysis this method has advantages of simplicity, high tolerance of run variability, low false discovery rate, and emphasis on proteins identified with high confidence.

δ-Aminolaevulinic acid synthesis is required for virulence of the wheat pathogen Stagonospora nodorum

Publisher: Microbiology Society

Date: 05-2006

DOI: 10.1099/MIC.0.28556-0

Abstract: δ -Aminolaevulinic acid (ALA) is synthesized in fungi by ALA synthase, a key enzyme in the synthesis of haem. The requirement for ALA synthase in Stagonospora nodorum to cause disease in wheat was investigated. The single gene encoding ALA synthase ( Als1 ) was cloned and characterized. Expression analysis determined that Als1 transcription was up-regulated during germination and also towards the latter stages of the infection. The Als1 gene was further characterized by homologous gene replacement. The inactivation of Als1 resulted in strains producing severely stunted germ tubes leading quickly to death. The strains could be recovered by supplementation with 33 μM ALA. Pathogenicity assays revealed the als1 strains were essentially non-pathogenic, inferring a key role for the synthesis of ALA during in planta growth. Supplementing the strains with ALA restored growth in vitro and also pathogenicity for up to 5 days after inoculation. Further examination by inoculating the als1 strains onto wounded leaves found that pathogenicity was only partially restored, suggesting that host-derived in planta levels of ALA are not sufficient to support growth. This study has identified a key role for fungal ALA synthesis during infection and revealed its potential as an antifungal target.

A comparative analysis of the heterotrimeric G-protein Gα, Gβ and Gγ subunits in the wheat pathogen Stagonospora nodorum

Publisher: Springer Science and Business Media LLC

Date: 03-07-2012

DOI: 10.1186/1471-2180-12-131

Abstract: It has been well established that the Gα subunit of the heterotrimeric G-protein in the wheat pathogen Stagonospora nodorum is required for a variety of phenotypes including pathogenicity, melanisation and asexual differentiation. The roles though of the Gγ and Gβ subunits though were unclear. The objective of this study was to identify and understand the role of these subunits and assess their requirement for pathogenicity and development. G-protein Gγ and Gβ subunits, named Gga1 and Gba1 respectively, were identified in the Stagonospora nodorum genome by comparative analysis with known fungal orthologues. A reverse genetics technique was used to study the role of these and revealed that the mutant strains displayed altered in vitro growth including a differential response to a variety of exogenous carbon sources. Pathogenicity assays showed that Stagonospora nodorum strains lacking Gba1 were essentially non-pathogenic whilst Gga1- impaired strains displayed significantly slower growth in planta . Subsequent sporulation assays showed that like the previously described Gα subunit mutants, both Gba1 and Gga1 were required for asexual sporulation with neither mutant strain being able to differentiate either pycnidia nor pycnidiospores under normal growth conditions. Continued incubation at 4°C was found to complement the mutation in each of the G-protein subunits with nearly wild-type levels of pycnidia recovered. This study provides further evidence on the significance of cAMP-dependent signal transduction for many aspects of fungal development and pathogenicity. The observation that cold temperatures can complement the G-protein sporulation defect now provides an ideal tool by which asexual differentiation can now be dissected.

The relationship between sensory attributes and wine composition for Australian Cabernet Sauvignon wines

Publisher: Hindawi Limited

Date: 15-08-2011

DOI: 10.1111/J.1755-0238.2011.00155.X

Is Zymoseptoria tritici a hemibiotroph?

Publisher: Elsevier BV

Date: 06-2015

DOI: 10.1016/J.FGB.2015.04.001

Abstract: The growth of microorganisms in planta is often categorized based on their methods of nutrient acquisition and the physical appearance of symptoms on the host. For ex le, biotrophs thrive on living tissue while necrotrophic pathogens often quickly lyse cells to access nutrients. Hemibiotrophs are pathogens that initially feed on living host tissue without causing visible symptoms prior to switching to necrotrophy. During infection of wheat, the pathogen Zymoseptoria tritici undergoes a prolonged and asymptomatic phase during which it grows slowly and protects itself from host defenses prior to eliciting a strong necrotic response. However careful analyses of the asymptomatic phase indicate that the pathogen does not alter host growth, casting doubt on the biotrophic nature of this asymptomatic period. Consequently, we question whether Z. tritici is correctly defined as a hemibiotroph.

Origins of Grape and Wine Aroma. Part 2. Chemical and Sensory Analysis

Publisher: American Society for Enology and Viticulture

Date: 23-01-2014

DOI: 10.5344/AJEV.2013.13106

An In Planta -Expressed Polyketide Synthase Produces ( R )-Mellein in the Wheat Pathogen Parastagonospora nodorum

Publisher: American Society for Microbiology

Date: 2015

DOI: 10.1128/AEM.02745-14

Abstract: Parastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 ( SN477 ), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as ( R )-mellein and ( R )- O -methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression system, we successfully demonstrated that SN477 is the only enzyme required for the production of ( R )-mellein. This is the first identification of a fungal PKS that is responsible for the synthesis of ( R )-mellein. The P. nodorum Δ SN477 mutant did not show any significant difference from the wild-type strain in its virulence against wheat. However, ( R )-mellein at 200 μg/ml inhibited the germination of wheat ( Triticum aestivum ) and barrel medic ( Medicago truncatula ) seeds. Comparative sequence analysis identified the presence of mellein synthase (MLNS) homologues in several Dothideomycetes and two sodariomycete genera. Phylogenetic analysis suggests that the MLNSs in fungi and bacteria evolved convergently from fungal and bacterial 6-methylsalicylic acid synthases.

Host-specific toxins: effectors of necrotrophic pathogenicity

Publisher: Hindawi Limited

Date: 07-2008

DOI: 10.1111/J.1462-5822.2008.01153.X

Abstract: Host-specific toxins (HSTs) are defined as pathogen effectors that induce toxicity and promote disease only in the host species and only in genotypes of that host expressing a specific and often dominant susceptibility gene. They are a feature of a small but well-studied group of fungal plant pathogens. Classical HST pathogens include species of Cochliobolus, Alternaria and Pyrenophora. Recent studies have shown that Stagonospora nodorum produces at least four separate HSTs that interact with four of the many quantitative resistance loci found in the host, wheat. Rationalization of fungal phylogenetics has placed these pathogens in the Pleosporales order of the class Dothideomycetes. It is possible that all HST pathogens lie in this order. Strong evidence of the recent lateral gene transfer of the ToxA gene from S. nodorum to Pyrenophora tritici-repentis has been obtained. Hallmarks of lateral gene transfer are present for all the studied HST genes although definitive proof is lacking. We therefore suggest that the Pleosporales pathogens may have a conserved propensity to acquire HST genes by lateral transfer.

Protoplast Isolation from Zymoseptoria tritici v1

Publisher: ZappyLab, Inc.

Date: 11-12-2017

DOI: 10.17504/PROTOCOLS.IO.K8GCZTW

Abstract: This is a basic protocol for protoplasting the fungal pathogen Zymoseptoria tritici

The global regulator of pathogenesis PnCon7 positively regulates Tox3 effector gene expression through direct interaction in the wheat pathogen Parastagonospora nodorum

Publisher: Wiley

Date: 21-05-2018

DOI: 10.1111/MMI.13968

Abstract: To investigate effector gene regulation in the wheat pathogenic fungus Parastagonospora nodorum, the promoter and expression of Tox3 was characterised through a series of complementary approaches. Promoter deletion and DNase I footprinting experiments identified a 25 bp region in the Tox3 promoter as being required for transcription. Subsequent yeast one-hybrid analysis using the DNA sequence as bait identified that interacting partner as the C2H2 zinc finger transcription factor PnCon7, a putative master regulator of pathogenesis. Silencing of PnCon7 resulted in the down-regulation of Tox3 demonstrating that the transcription factor has a positive regulatory role on gene expression. Analysis of Tox3 expression in the PnCon7 silenced strains revealed a strong correlation with PnCon7 transcript levels, supportive of a direct regulatory role. Subsequent pathogenicity assays using PnCon7-silenced isolates revealed that the transcription factor was required for Tox3-mediated disease. The expression of two other necrotrophic effectors (ToxA and Tox1) was also affected but in a non-dose dependent manner suggesting that the regulatory role of PnCon7 on these genes was indirect. Collectively, these data have advanced our fundamental understanding of the Con7 master regulator of pathogenesis by demonstrating its positive regulatory role on the Tox3 effector in P. nodorum through direct interaction.

Prevalence of ToxA-sensitive alleles of the wheat gene Tsn1 in Australian and Chinese wheat cultivars

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/CP08259

Abstract: A recent survey of worldwide isolates of Stagonospora nodorum showed that all Australian isolates expressed the host-specific toxin ToxA (Stukenbrock and McDonald 2007). In contrast, very few Chinese isolates did. All the Australian Pyrenophora tritici-repentis isolates that were tested expressed ToxA. We therefore postulated that the wheat gene that confers sensitivity to ToxA, Tsn1, would vary in prevalence in wheat cultivars in use in the two countries. Contrary to expectation, 10 out of 21 Chinese cultivars responded to ToxA as did 26 out of 46 Australian cultivars. The result suggests that ToxA has not had a determining effect on the survival of wheat cultivars in either country. They also suggest that despite the widespread use of Tsn1 markers in Australia, sensitive alleles are still commonplace. The removal of sensitive alleles from breeders’ lines could be readily achieved and could significantly affect the resistance of wheat to both diseases.

Surveying the potential of secreted antimicrobial peptides to enhance plant disease resistance

Publisher: Frontiers Media SA

Date: 27-10-2015

DOI: 10.3389/FPLS.2015.00900

Functional redundancy of necrotrophic effectors – consequences for exploitation for breeding

Publisher: Frontiers Media SA

Date: 08-07-2015

DOI: 10.3389/FPLS.2015.00501

The past, present and future of secondary metabolite research in the Dothideomycetes

Publisher: Wiley

Date: 07-07-2014

DOI: 10.1111/MPP.12162

Recent advances in the Zymoseptoria tritici–wheat interaction: insights from pathogenomics

Publisher: Frontiers Media SA

Date: 24-02-2015

DOI: 10.3389/FPLS.2015.00102

Development of a sensitive non-targeted method for characterizing the wine volatile profile using headspace solid-phase microextraction comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

Publisher: Elsevier BV

Date: 2011

DOI: 10.1016/J.CHROMA.2010.11.008

Abstract: Future understanding of differences in the composition and sensory attributes of wines require improved analytical methods which allow the monitoring of a large number of volatiles including those present at low concentrations. This study presents the optimization and application of a headspace solid-phase microextraction (HS-SPME) method for analysis of wine volatiles by comprehensive two-dimensional gas chromatography (GC×GC) time-of-flight mass spectrometry (TOFMS). This study demonstrates an important advancement in wine volatile analysis as the method allows for the simultaneous analysis of a significantly larger number of compounds found in the wine headspace compared to other current single dimensional GC-MS methodologies. The methodology allowed for the simultaneous analysis of over 350 different tentatively identified volatile and semi-volatile compounds found in the wine headspace. These included potent aroma compound classes such as monoterpenes, norisoprenoids, sesquiterpenes, and alkyl-methoxypyrazines which have been documented to contribute to wine aroma. It is intended that wine aroma research and wine sensory research will utilize this non-targeted method to assess compositional differences in the wine volatile profile.

Wheat PR ‐1 proteins are targeted by necrotrophic pathogen effector proteins

Publisher: Wiley

Date: 10-08-2016

DOI: 10.1111/TPJ.13228

Abstract: Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum are required to cause disease on wheat. These effectors interact in a gene-for-gene manner with host-dominant susceptibilty loci, resulting in disease. However, whilst the requirement of these effectors for infection is clear, their mechanisms of action remain poorly understood. A yeast-two-hybrid library approach was used to search for wheat proteins that interacted with the necrotrophic effector SnTox3. Using this strategy we indentified an interaction between SnTox3 and the wheat pathogenicity-related protein TaPR-1-1, and confirmed it by in-planta co-immunprecipitation. PR-1 proteins represent a large family (23 in wheat) of proteins that are upregulated early in the defence response however, their function remains ellusive. Interestingly, the P. nodorum effector SnToxA has recently been shown to interact specifically with TaPR-1-5. Our analysis of the SnTox3-TaPR-1 interaction demonstrated that SnTox3 can interact with a broader range of TaPR-1 proteins. Based on these data we utilised homology modeling to predict, and validate, regions on TaPR-1 proteins that are likely to be involved in the SnTox3 interaction. Precipitating from this work, we identified that a PR-1-derived defence signalling peptide from the C-terminus of TaPR-1-1, known as CAPE1, enhanced the infection of wheat by P. nodorum in an SnTox3-dependent manner, but played no role in ToxA-mediated disease. Collectively, our data suggest that P. nodorum has evolved unique effectors that target a common host-protein involved in host defence, albeit with different mechanisms and potentially outcomes.

Acquisition and Loss of Secondary Metabolites Shaped the Evolutionary Path of Three Emerging Phytopathogens of Wheat

Publisher: Oxford University Press (OUP)

Date: 21-02-2019

DOI: 10.1093/GBE/EVZ037

Stagonospora nodorum: From Pathology to Genomics and Host Resistance

Publisher: Annual Reviews

Date: 08-09-2012

DOI: 10.1146/ANNUREV-PHYTO-081211-173019

Abstract: Stagonospora nodorum is a major necrotrophic pathogen of wheat that causes the diseases S. nodorum leaf and glume blotch. A series of tools and resources, including functional genomics, a genome sequence, proteomics and metabolomics, host-mapping populations, and a worldwide collection of isolates, have enabled the dissection of pathogenicity mechanisms. Metabolic and signaling genes required for pathogenicity have been defined. Interaction with the host is dominated by interplay of fungal effectors that induce necrosis on wheat lines carrying specific sensitivity loci. As such, the pathogen has emerged as a model for the Pleosporales group of pathogens.

Proteomic identification of extracellular proteins regulated by the Gna1 Gα subunit in Stagonospora nodorum

Publisher: Elsevier BV

Date: 05-2009

DOI: 10.1016/J.MYCRES.2009.01.004

Abstract: The fungus Stagonospora nodorum is the causal agent of stagonospora nodorum blotch (syn. leaf and glume blotch) disease of wheat. The Gna1-encoded Galpha protein is an important signal transduction component in the fungus, which is required for full pathogenicity, sporulation and extracellular depolymerase production. In this study, we sought to gain a better understanding of defects associated with the gna1 mutant by using two-dimensional gel electrophoresis to analyse the extracellular proteome for differences to the wildtype. Mass spectrometry analysis of altered abundant protein spots and peptide matching to the Stagonospora nodorum genome database have led to the identification of genes implicated in cell wall degradation, proteolysis, RNA hydrolysis and aromatic compound metabolism. In addition, quantitative RT-PCR has demonstrated that some of the encoding genes showed differential expression throughout host infection. Implications of these proteins and their corresponding genes in fungal virulence are discussed.

The nitrogen content of the tomato leaf apoplast increases during infection by Cladosporium fulvum

Publisher: Springer Science and Business Media LLC

Date: 06-2001

DOI: 10.1007/S004250000500

Abstract: To address the problem of the nutritional requirements of phyto-pathogenic fungi growing in planta, the environment for the intercellular biotrophic pathogen, Cladosporium fulvum Cooke, of tomato (Lycopersicon esculentum Mill.) was analysed. Using a novel technique for infiltrating the intercellular space, we measured the concentrations of 21 amino acids, nitrate and ammonia in the apoplast of the tomato leaf during infection. The concentrations of most amino acids, and total nitrogen content, increased during infection. The levels of nearly all amino acids remained relatively unchanged during an incompatible interaction. All protein amino acids were detected during infection, except cysteine and tryptophan. Most amino acids were present at a concentration between 0.1-0.7 mM. The non-protein amino acid gamma-aminobutyric acid was detected at the highest concentration (up to 2.5 mM) during the compatible interaction. Preliminary investigations on the source of the amino acids revealed that protease activity within the apoplast increased during infection and that infection induced the expression of the pathogenicity-related extracellular serine protease P69B. The nitrogen status of the infecting fungus and sources for the additional amino acids are discussed.

Xanthine dehydrogenase from the phototrophic purple bacteriumRhodobacter capsulatusis more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes

Publisher: Wiley

Date: 02-1998

DOI: 10.1046/J.1365-2958.1998.00733.X

Abstract: Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis. Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases. DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase. The deduced amino acid sequence of XDHA contains binding sites for two [2Fe-2S] clusters and FAD, whereas XDHB is predicted to contain the molybdopterin cofactor. In contrast to R. capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases. The amino acid sequence of xanthine dehydrogenase from R. capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase-related aldehyde oxidoreductase from Desulphovibrio gigas. The expression of an xdhA-lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source. Mutations in nifR1 (ntrC) and nifR4 (rpoN, encoding sigma54) had no influence on xdh gene expression. A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine. The transcriptional start site of xdhA was mapped by primer extension analysis. Comparison with known promoter elements revealed no significant homology. Xanthine dehydrogenase from R. capsulatus was purified to homogeneity. The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively. N-terminal amino acid sequencing of both subunits confirmed the predicted start codons. The molecular mass of the native enzyme was determined to be 275 kDa, indicating an alpha2beta2-subunit structure. Analysis of the molybdenum cofactor of xanthine dehydrogenase from R. capsulatus revealed that it contains the molybdopterin cofactor and not a molybdopterin dinucleotide derivative.

Pathogen effectors shed light on plant diseases

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/FPV37N10_FO

Transition from heterothallism to homothallism is hypothesised to have facilitated speciation among emerging Botryosphaeriaceae wheat-pathogens

Publisher: Elsevier BV

Date: 12-2017

DOI: 10.1016/J.FGB.2017.10.005

Abstract: White grain disorder (WGD) is a recently emerged wheat disease in Australia caused by three Botryosphaeriaceae fungi, from the genus Eutiarosporella. These species are E. tritici-australis, E. darliae, and E. pseudodarliae. Characterisation of the mating type genes for the WGD-species show that the genome sequence of a single E. darliae and E. pseudodarliae isolate both harbour MAT1-2-1 and MAT1-1-1, which suggests that these species are homothallic. However, unlike most other characterised mating-type loci from other homothallic Dothideomycetes, these species' MAT1-1-1 are located at a separate locus, inserted within the coding region of another gene. The sequenced strain of E. tritici-australis analysed did not harbour MAT1-1-1. Including the sequenced strain, we screened the mating type genes present in 16 E. tritici-australis in iduals isolated from infected grain from fields in South Australia. Of these 16, 11 harbour MAT1-1-1 and the other five harbour MAT1-2-1. The genome of a MAT1-1-1 harbouring isolate was re-sequenced, which demonstrated that MAT1-1-1 was present at the MAT locus. We examined non-coding DNA surrounding the MAT1-1-1 gene in E. pseudodarliae and observed fragments of the MAT locus both up and downstream. These fragments and their orientation around MAT1-1-1 is similar to characterised heterothallic Botryosphaeriaceae. Based on these gene arrangements, we conclude that the new MAT1-1-1 containing locus likely originated from a cryptic DNA integration event between two heterothallic in iduals. We hypothesise that this integration event led to the formation of a homothallic lineage, which is the common ancestor of E. darliae and E. pseudodarliae.

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

Publisher: Wiley

Date: 19-09-2016

DOI: 10.1111/MPP.12444

Next-generation re-sequencing as a tool for rapid bioinformatic screening of presence and absence of genes and accessory chromosomes across isolates of Zymoseptoria tritici

Publisher: Elsevier BV

Date: 06-2015

DOI: 10.1016/J.FGB.2015.04.012

Abstract: The wheat pathogen Zymoseptoria tritici possesses a large number of accessory chromosomes that may be present or absent in its genome. The genome of the reference isolate IPO323 has been assembled to a very high standard and contains 21 full length chromosome sequences, 8 of which represent accessory chromosomes. The IPO323 reference, when combined with low-cost next-generation sequencing and bioinformatics, can be used as a powerful tool to assess the presence or absence of accessory chromosomes. We present an outline of a range of bioinformatics techniques that can be applied to the analysis of presence-absence variation among accessory chromosomes across 13 novel isolates of Z. tritici.

Evidence that γ-aminobutyric acid is a major nitrogen source during Cladosporium fulvum infection of tomato

Publisher: Springer Science and Business Media LLC

Date: 15-08-2001

DOI: 10.1007/S004250100632

Abstract: The growth of the biotrophic pathogen Cladosporium fulvum within the tomato (Lycopersicon esculentum Mill.) leaf is restricted to the intercellular space. Previous studies from this laboratory have demonstrated that gamma-aminobutyric acid (GABA) accumulates to millimolar concentrations in the apoplast during a compatible interaction. We decided to further investigate the role of GABA during infection. A gene encoding a required enzyme for GABA metabolism, GABA transaminase (Gat1), was cloned and sequenced from C. fulvum. The predicted protein sequence of Gat1 had high homology to other fungal GABA transaminases, particularly from Aspergillus nidulans. In vitro expression experiments revealed Gat1 to be strongly expressed during fungal growth on both GABA and glutamate whereas nearly no expression was evident during nitrogen starvation conditions. Expression of Gat1 was also apparent during infection, suggesting for the first time that C. fulvum actively metabolises GABA during infection. This indicates that the fungus may be utilising the GABA in the apoplast as a nutrient source. Further analysis revealed that the expression of tomato glutamate decarboxylase, the enzyme responsible for GABA synthesis, appeared appreciably higher during a compatible interaction than in the incompatible interaction. These findings imply that the infecting fungus may alter the physiology of the tomato leaf with the result that a source of nitrogen is supplied.

Lolium perenne apoplast metabolomics for identification of novel metabolites produced by the symbiotic fungus Epichloë festucae

Publisher: Wiley

Date: 13-04-2020

DOI: 10.1111/NPH.16528

Abstract: Epichloë festucae is an endophytic fungus that forms a symbiotic association with Lolium perenne . Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae . A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from uninfected and infected L. perenne . Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem MS was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using MarVis in combination with the BioCyc and KEGG databases, and an in‐house Epichloë metabolite database. We were able to identify the known endophyte‐specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë ‐derived metabolites, we deleted a gene encoding a NRPS ( lgsA ) that is highly expressed in planta . Comparative MS analysis of apoplastic wash fluid from wild‐type‐ vs mutant‐infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

Have we finally opened the door to understanding Septoria tritici blotch disease in wheat?

Publisher: Wiley

Date: 20-03-2017

DOI: 10.1111/NPH.14502

The necrotrophic effector protein SnTox3 re-programs metabolism and elicits a strong defence response in susceptible wheat leaves

Publisher: Springer Science and Business Media LLC

Date: 15-08-2014

DOI: 10.1186/S12870-014-0215-5

Oral pharmacologic doses of cobalamin may not be as effective as parenteral cobalamin therapy in reversing hyperhomocystinemia and methylmalonic acidemia in apparently normal subjects

Publisher: Wiley

Date: 19-04-2006

DOI: 10.1111/J.1365-2257.2006.00783.X

Abstract: A postmenopausal female evaluated for thrombophilia because of bone infarcts had mild hyperhomocysteinemia, which increased when hormone replacement was discontinued. Serum folate, cobalamin and methylmalonic acid were normal. Compound heterozygosity for C677T/A1298C methylenetetrahydrofolate reductase polymorphisms was present but oral folic acid failed to lower homocysteine and actually increased methylmalonic acid. Oral cobalamin therapy increased serum cobalamin and partially decreased methylmalonic acid but had no effect on homocysteine. Homocysteine remained unchanged after 11 months of oral cobalamin, folic acid and pyridoxine therapy. However, intramuscular cobalamin promptly decreased both metabolites to normal. Thus, parenteral cobalamin therapy may have greater metabolic effects than oral vitamin therapy even in apparently normal subjects.

Rapid Parallel Evolution of Azole Fungicide Resistance in Australian Populations of the Wheat Pathogen Zymoseptoria tritici

Publisher: American Society for Microbiology

Date: 15-02-2019

DOI: 10.1128/AEM.01908-18

Abstract: Fungicides are essential to control diseases in agriculture because many crops are highly susceptible to pathogens. However, many pathogens rapidly evolve resistance to fungicides. A large body of studies have described specific mutations conferring resistance and have often made inferences about the origins of resistance based on sequencing data from the target gene alone. Here, we show the de novo acquisition of resistance to the ubiquitously used azole fungicides in genetically isolated populations of the wheat pathogen Zymoseptoria tritici in Tasmania, Australia. We confirm evidence for parallel evolution through genome-scale analyses of representative worldwide populations. The emergence of complex resistance haplotypes following a well-documented recent introduction of azoles into Australian farming practices demonstrates how rapidly chemical resistance evolves in agricultural ecosystems.

Dothideomycete-plant interactions illuminated by genome sequencing and EST analysis of the wheat pathogen Stagonospora nodorum

Publisher: Oxford University Press (OUP)

Date: 11-2007

DOI: 10.1105/TPC.107.052829

Abstract: Stagonospora nodorum is a major necrotrophic fungal pathogen of wheat (Triticum aestivum) and a member of the Dothideomycetes, a large fungal taxon that includes many important plant pathogens affecting all major crop plant families. Here, we report the acquisition and initial analysis of a draft genome sequence for this fungus. The assembly comprises 37,164,227 bp of nuclear DNA contained in 107 scaffolds. The circular mitochondrial genome comprises 49,761 bp encoding 46 genes, including four that are intron encoded. The nuclear genome assembly contains 26 classes of repetitive DNA, comprising 4.5% of the genome. Some of the repeats show evidence of repeat-induced point mutations consistent with a frequent sexual cycle. ESTs and gene prediction models support a minimum of 10,762 nuclear genes. Extensive orthology was found between the polyketide synthase family in S. nodorum and Cochliobolus heterostrophus, suggesting an ancient origin and conserved functions for these genes. A striking feature of the gene catalog was the large number of genes predicted to encode secreted proteins the majority has no meaningful similarity to any other known genes. It is likely that genes for host-specific toxins, in addition to ToxA, will be found among this group. ESTs obtained from axenic mycelium grown on oleate (chosen to mimic early infection) and late-stage lesions sporulating on wheat leaves were obtained. Statistical analysis shows that transcripts encoding proteins involved in protein synthesis and in the production of extracellular proteases, cellulases, and xylanases predominate in the infection library. This suggests that the fungus is dependant on the degradation of wheat macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing pycnidiospores.

Bipolenins K-N

Publisher: Beilstein Institut

Date: 26-08-2019

DOI: 10.3762/BJOC.15.198

Abstract: Chemical investigation of the barley and wheat fungal pathogen Bipolaris sorokiniana BRIP10943 yielded four new sativene-type sesquiterpenoid natural products, bipolenins K–N ( 1 – 4 ), together with seven related known analogues ( 5 – 11 ), and a sesterterpenoid ( 12 ). Their structures were determined by detailed analysis of spectroscopic data, supported by TDDFT calculations and comparison with previously reported analogues. These compounds were evaluated for their phytotoxic activity against wheat seedlings and wheat seed germination. The putative biosynthetic relationships between the isolated sesquiterpenoids were also explored.

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

DOI: 10.1128/MBIO.01515-19

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.

Co-creating value in online innovation communities

Publisher: Emerald

Date: 10-06-2019

DOI: 10.1108/EJM-12-2016-0780

Abstract: Online innovation communities are central for many organizations seeking to advance their innovation portfolio. While these communities rely on consumers to collaborate in the innovation process, it remains unclear what drives these consumers to perform value co-creation activities and what value dimensions they derive as a result. This paper aims to advance the understanding of value co-creation in the online collaborative innovation context. Specifically, it aims to examine social and in idual factors driving such activities, and the value derived from the perspective of the member. A self-administered online questionnaire was used to collect data from collaborative innovation community members yielding 309 complete responses. Structural equation modelling was used to analyse the data, using variance-based structural equation modelling with partial least squares path modelling in SmartPLS. Results confirm that distinct social and in idual factors facilitate in idual value co-creation activities, including the provision of feedback, helping, rapport building and information sharing. Furthermore, the research confirms the mediating role of learning on these relationships. This study contributes to the micro-foundation movement in marketing by undertaking an independent examination of value co-creation activities and their nomological network. A shift in the mindset of managing for collaborative innovation is required, from a focus on collaborative product development to the management of an online community where members derive value from their co-creation activities. This research is the first to offer insight into important in idual and social pre-conditions and subsequent value outcomes of four common value co-creation activities. It informs practice about how to facilitate value co-creation activities and contribute to the co-creation of value for online innovation community members.

The identification of a transposon affecting the asexual reproduction of the wheat pathogen Zymoseptoria tritici

Publisher: Wiley

Date: 05-05-2021

DOI: 10.1111/MPP.13064

Abstract: Zymoseptoria tritici , the causal agent of Septoria tritici blotch, is a fungal wheat pathogen that causes significant global yield losses. Within Z. tritici populations, quantitative differences in virulence among different isolates are commonly observed however, the genetic components that underpin these differences remain elusive. In this study, intraspecific comparative transcriptomic analysis was used to identify candidate genes that contribute to differences in virulence on the wheat cultivar WW2449. This led to the identification of a multicopy gene that was not expressed in the high‐virulence isolate when compared to the medium‐ and low‐virulence isolates. Further investigation suggested this gene resides in a 7.9‐kb transposon. Subsequent long‐read sequencing of the isolates used in the transcriptomic analysis confirmed that this gene did reside in an active Class II transposon, which is composed of four genes named REP9‐1 to ‐4 . Silencing and overexpression of REP9‐1 in two distinct genetic backgrounds demonstrated that its expression alone reduces the number of pycnidia produced by Z. tritici during infection. The REP9‐1 gene identified within a Class II transposon is the first discovery of a gene in a transposable element that influences the virulence of Z. tritici . This discovery adds further complexity to genetic loci that contribute to quantitative virulence in this important pathogen.

Origins of grape and wine aroma. Part 1. Chemical components and viticultural impacts

Publisher: American Society for Enology and Viticulture

Date: 10-12-2014

DOI: 10.5344/AJEV.2013.12070

The necrotrophic effector ToxA from Parastagonospora nodorum interacts with wheat NHL proteins to facilitate Tsn1‐mediated necrosis

Publisher: Wiley

Date: 20-02-2022

DOI: 10.1111/TPJ.15677

Abstract: The plant pathogen Parastagonospora nodorum secretes necrotrophic effectors to promote disease. These effectors induce cell death on wheat cultivars carrying dominant susceptibility genes in an inverse gene‐for‐gene manner. However, the molecular mechanisms underpinning these interactions and resulting cell death remain unclear. Here, we used a yeast two‐hybrid library approach to identify wheat proteins that interact with the necrotrophic effector ToxA. Using this strategy, we identified an interaction between ToxA and a wheat transmembrane NDR/HIN1‐like protein (TaNHL10) and confirmed the interaction using in planta co‐immunoprecipitation and confocal microscopy co‐localization analysis. We showed that the C‐terminus of TaNHL10 is extracellular whilst the N‐terminus is localized in the cytoplasm. Further analyses using yeast two‐hybrid and confocal microscopy co‐localization showed that ToxA interacts with the C‐terminal LEA2 extracellular domain of TaNHL10. Random mutagenesis was then used to identify a ToxA mutant, ToxA N109D , which was unable to interact with TaNHL10 in yeast two‐hybrid assays. Subsequent heterologous expression and purification of ToxA N109D in Nicotiania benthamiana revealed that the mutated protein was unable to induce necrosis on Tsn1 ‐dominant wheat cultivars, confirming that the interaction of ToxA with TaNHL10 is required to induce cell death. Collectively, these data advance our understanding on how ToxA induces cell death during infection and further highlight the importance of host cell surface interactions in necrotrophic pathosystems.

Remarkable recent changes in the genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici

Publisher: Wiley

Date: 14-07-2021

DOI: 10.1111/MPP.13101

Abstract: Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici , is one of the most economically important diseases of wheat. Recently, both factors of a gene‐for‐gene interaction between Z . tritici and wheat, the wheat receptor‐like kinase Stb6 and the Z . tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high ersity of AvrStb6 haplotypes present in earlier Z . tritici isolate collections, with up to c.18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic ersity and the incidence of haplotypes allowing evasion of Stb6 ‐mediated resistance in more recent Z . tritici populations. Here we show, using targeted resequencing of AvrStb6 , that this gene is universally present in field isolates s led from major wheat‐growing regions of the world in 2013–2017. However, in contrast to the data from previous AvrStb6 population studies, we report a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z . tritici isolates. Moreover, a remarkably small number of haplotypes, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6‐ containing wheat, were found to predominate among modern Z . tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z . tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence haplotypes tend to be eliminated in subsequent populations.

Development of an in-house protocol for the OFFGEL fractionation of plant proteins

Publisher: Proteomass Scientific Society

Date: 31-12-2011

DOI: 10.5584/JIOMICS.V1I2.59

Emerging Insights into the Functions of Pathogenesis-Related Protein 1

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.TPLANTS.2017.06.013

Abstract: The members of the pathogenesis-related protein 1 (PR-1) family are among the most abundantly produced proteins in plants on pathogen attack, and PR-1 gene expression has long been used as a marker for salicylic acid-mediated disease resistance. However, despite considerable interest over several decades, their requirement and role in plant defence remains poorly understood. Recent reports have emerged demonstrating that PR-1 proteins possess sterol-binding activity, harbour an embedded defence signalling peptide, and are targeted by plant pathogens during host infection. These studies have re-energised the field and provided long-awaited insights into a possible PR-1 function. Here we review the current status of PR-1 proteins and discuss how these recent advances shed light on putative roles for these enigmatic proteins.

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana

Publisher: Wiley

Date: 12-03-2017

DOI: 10.1111/MPP.12535

Victorin, the host-selective cyclic peptide toxin from the oat pathogen Cochliobolus victoriae , is ribosomally encoded

Publisher: Proceedings of the National Academy of Sciences

Date: 14-09-2020

DOI: 10.1073/PNAS.2010573117

Abstract: Studies of the 1940s Victoria blight of oats epidemic discovered that some fungal pathogens secrete HSTs responsible for symptom development and specificity of the associated disease. The causal necrotrophic pathogen of Victoria blight, C. victoriae , secretes the peptide HST victorin, which was, subsequently, shown to constitute a novel class of effectors that exploit host immunity pathways aimed at repelling biotrophic pathogens. Although these discoveries have broadened our mechanistic understanding of plant-pathogen interactions, the genetic and biochemical origins of victorin have remained elusive. Here, we solve this decades-old mystery by demonstrating that victorin is produced ribosomally, not, as assumed, by nonribosomal peptide synthetase. Furthermore, we identify a CAO enzyme responsible for converting victorin to its active form.

Utilizing Gene Tree Variation to Identify Candidate Effector Genes in Zymoseptoria tritici

Publisher: Oxford University Press (OUP)

Date: 04-2016

DOI: 10.1534/G3.115.025197

Abstract: Zymoseptoria tritici is a host-specific, necrotrophic pathogen of wheat. Infection by Z. tritici is characterized by its extended latent period, which typically lasts 2 wks, and is followed by extensive host cell death, and rapid proliferation of fungal biomass. This work characterizes the level of genomic variation in 13 isolates, for which we have measured virulence on 11 wheat cultivars with differential resistance genes. Between the reference isolate, IPO323, and the 13 Australian isolates we identified over 800,000 single nucleotide polymorphisms, of which ∼10% had an effect on the coding regions of the genome. Furthermore, we identified over 1700 probable presence/absence polymorphisms in genes across the Australian isolates using de novo assembly. Finally, we developed a gene tree sorting method that quickly identifies groups of isolates within a single gene alignment whose sequence haplotypes correspond with virulence scores on a single wheat cultivar. Using this method, we have identified & 100 candidate effector genes whose gene sequence correlates with virulence toward a wheat cultivar carrying a major resistance gene.

The utilisation of di/tripeptides by Stagonospora nodorum is dispensable for wheat infection

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1016/J.PMPP.2003.12.003

Interactions between wine volatile compounds and grape and wine matrix components influence aroma compound headspace partitioning

Publisher: American Chemical Society (ACS)

Date: 11-11-2009

DOI: 10.1021/JF902586N

Abstract: A full-factorial design was used to assess the matrix effects of ethanol, glucose, glycerol, catechin, and proline on the volatile partitioning of 20 volatile compounds considered to play a role in wine aroma. Analysis of variance showed that the two-way interactions of ethanol and glucose, ethanol and glycerol, and glycerol and catechin significantly influenced headspace partitioning of volatiles. Experiments were conducted to observe the effect of varied ethanol and glucose concentrations on headspace partitioning of a constant concentration of volatiles. Analysis of variance and linear regression analysis showed that the presence of glucose increased the concentration of volatiles in the headspace, whereas increasing ethanol concentration was negatively correlated with headspace partitioning of volatiles. A subsequent study assessed the effect of diluting white and red wines with water and ethanol. It was again observed that increased ethanol concentration significantly reduced the relative abundance of volatile compounds in the s le headspace. This study investigates some of the complex matrix interactions of the major components of grape and wine that influence volatile compound headspace partitioning. The magnitude of each matrix-volatile interaction was ethanol > glucose > glycerol > catechin, whereas proline showed no apparent interaction. The results clearly identify that increasing ethanol concentrations significantly reduce the headspace concentration of volatile aroma compounds, which may contribute to explaining recent sensory research observations that indicate ethanol can suppress the fruit aroma attributes in wine.

Assessing the mycotoxigenic threat of necrotrophic pathogens of wheat

Publisher: Springer Science and Business Media LLC

Date: 12-08-2011

DOI: 10.1007/S12550-011-0108-5

Abstract: Pathogenic fungi are the causal agents of many significant plant diseases around the world. These diseases often result in significant yield reductions, leading to lower food production rates and economic losses. Several of these pathogenic fungi also produce mycotoxins during infection, which are harmful to human and animal health. Whilst some of these toxins and the fungi that produce them have been studied intensively, the mycotoxigenic potential of many of these pathogens remains largely unknown. Included within these fungi are the necrotrophic pathogens of wheat, Stagonospora nodorum, Pyrenophora tritici-repentis and Alternaria alternata. Recent studies have demonstrated that each of these pathogens is capable of synthesizing an array of mycotoxic compounds during disease development, questioning their status as non-mycotoxin producers. This review summarises recent mycotoxin findings in these necrotrophic wheat pathogens by briefly discussing the mycotoxins identified, their toxicity and their synthesis. Future and emerging threats are also considered.

Characterization of the Interaction of a Novel Stagonospora nodorum Host-Selective Toxin with a Wheat Susceptibility Gene

Publisher: Oxford University Press (OUP)

Date: 07-12-2007

DOI: 10.1104/PP.107.108761

Abstract: Recent work suggests that the Stagonospora nodorum-wheat pathosystem is controlled by host-selective toxins (HSTs SnToxA, SnTox1, and SnTox2) that interact directly or indirectly with dominant host genes (Tsn1, Snn1, and Snn2) to induce disease. Here we describe and characterize a novel HST designated SnTox3, and the corresponding wheat sensitivity/susceptibility gene identified on chromosome arm 5BS, which we designated as Snn3. SnTox3 is a proteinaceous necrosis-inducing toxin between 10 and 30 kD in size. The S. nodorum isolates Sn1501 (SnToxA−, SnTox2+, and SnTox3+), SN15 (SnToxA+, SnTox2+, and SnTox3+), and SN15KO18, a strain of SN15 with a disrupted form of SnToxA, were evaluated on a population of wheat recombinant inbred lines. A compatible Snn3-SnTox3 interaction played a significant role in the development of disease caused by isolates Sn1501 and SN15KO18, with Snn2 being epistatic to Snn3. Snn3 was not significantly associated with disease caused by SN15 presumably due to the major effects observed for Snn2 and Tsn1, which were largely additive. This work introduces a fourth HST produced by S. nodorum and builds on the notion that the S. nodorum-wheat pathosystem is largely based on multiple host-toxin interactions that follow an inverse gene-for-gene scenario.

Editorial: How can secretomics help unravel the secrets of plant-microbe interactions?

Publisher: Frontiers Media SA

Date: 30-11-2016

DOI: 10.3389/FPLS.2016.01777

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Publisher: Research Square Platform LLC

Date: 10-09-2020

DOI: 10.21203/RS.3.RS-40104/V2

Abstract: Background : The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs) – secreted, membrane-bound nanoparticles containing molecular cargo – being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes. Results : Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 – 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs. Conclusion : We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

High burden and frailty: association with poor cognitive performance in older caregivers living in rural areas

Publisher: FapUNIFESP (SciELO)

Date: 04-12-2017

DOI: 10.1590/2237-6089-2016-0085

Abstract: Abstract Introduction: Older caregivers living in rural areas may be exposed to three vulnerable conditions, i.e., those related to care, their own aging, and their residence context. Objective: To analyze the association of burden and frailty with cognition performance in older caregivers in rural communities. Method: In this cross-sectional survey, 85 older caregivers who cared for dependent elders were included in this study. Global cognition (Addenbrooke's Cognitive Examination – Revised Mini Mental State Examination), burden (Zarit Burden Interview) and frailty (Fried's frailty phenotype) were assessed. All ethical principles were observed. Results: Older caregivers were mostly women (76.7%) mean age was 69 years. Cognitive impairment was present in 15.3%, severe burden in 8.2%, frailty in 9.4%, and pre-frailty in 52.9% of the older caregivers. More severely burdened or frail caregivers had worse cognitive performance than those who were not, respectively (ANOVA test). Caregivers presenting a high burden level and some frailty degree (pre-frail or frail) simultaneously were more likely to have a reduced global cognition performance. Conclusion: A significant number of older caregivers had low cognitive performance. Actions and resources to decrease burden and physical frailty may provide better cognition and well-being, leading to an improved quality of life and quality of the care provided by the caregivers.

Metabolite profiling identifies the mycotoxin alternariol in the pathogen Stagonospora nodorum

Publisher: Springer Science and Business Media LLC

Date: 12-03-2009

DOI: 10.1007/S11306-009-0158-2

Extracellular Vesicles from the Apoplastic Fungal Wheat Pathogen Zymoseptoria Tritici

Publisher: Research Square Platform LLC

Date: 07-07-2020

DOI: 10.21203/RS.3.RS-40104/V1

Abstract: Background: The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs) – secreted, membrane-bound nanoparticles containing molecular cargo – being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes. Results: Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 – 100 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs. Conclusion: We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

Pro-domain processing of fungal effector proteins from plant pathogens

Publisher: Public Library of Science (PLoS)

Date: 20-10-2021

DOI: 10.1371/JOURNAL.PPAT.1010000

Influence of geographic origin on the sensory characteristics and wine composition of Vitis vinifera cv. Cabernet Sauvignon wines from Australia

Publisher: American Society for Enology and Viticulture

Date: 25-06-2012

DOI: 10.5344/AJEV.2012.12023

Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum

Publisher: Springer Science and Business Media LLC

Date: 22-09-2009

DOI: 10.1186/1471-2105-10-301

Abstract: Stagonospora nodorum , a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained. In this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellular-associated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome. We conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies.

Quantitative proteomic analysis of G-protein signalling in Stagonospora nodorum using isobaric tags for relative and absolute quantification

Publisher: Wiley

Date: 30-12-2010

DOI: 10.1002/PMIC.200900474

Abstract: The G protein alpha-subunit (Gna1) in the wheat pathogen Stagonospora nodorum has previously been shown to be a critical controlling element in disease ontogeny. In this study, iTRAQ and 2-D LC MALDI-MS/MS have been used to characterise protein expression changes in the S. nodorum gna1 strain versus the SN15 wild-type. A total of 1336 proteins were identified. The abundance of 49 proteins was significantly altered in the gna1 strain compared with the wild-type. Gna1 was identified as having a significant regulatory role on primary metabolic pathways, particularly those concerned with NADPH synthesis or consumption. Mannitol dehydrogenase was up-regulated in the gna1 strain while mannitol 1-phosphate dehydrogenase was down-regulated providing direct evidence of Gna1 regulation over this enigmatic pathway. Enzymatic analysis and growth assays confirmed this regulatory role. Several novel hypothetical proteins previously associated with stress and pathogen responses were identified as positively regulated by Gna1. A short-chain dehydrogenase (Sch3) was also significantly less abundant in the gna1 strains. Sch3 was further characterised by gene disruption in S. nodorum by homologous recombination. Functional characterisation of the sch3 strains revealed their inability to sporulate in planta providing a further link to Gna1 signalling and asexual reproduction. These data add significantly to the identification of the regulatory targets of Gna1 signalling in S. nodorum and have demonstrated the utility of iTRAQ in dissecting signal transduction pathways.

Production of small cysteine‐rich effector proteins in Escherichia coli for structural and functional studies

Publisher: Wiley

Date: 12-04-2016

DOI: 10.1111/MPP.12385

Multi-stage resistance to Zymoseptoria tritici revealed by GWAS in an Australian bread wheat (Triticum aestivum L.) diversity panel

Publisher: Cold Spring Harbor Laboratory

Date: 30-06-2022

DOI: 10.1101/2022.06.29.498182

Abstract: Septoria tritici blotch (STB) has been ranked the third most important wheat disease in the world, threatening a large area of wheat production. Although major genes play an important role in the protection against Zymoseptoria tritici infection, the lifespan of their resistance unfortunately is very short in modern agriculture systems. Combinations of quantitative resistance with minor effects, therefore, are believed to have prolonged and more durable resistance to Z. tritici . In this study new quantitative trait loci (QTLs) were identified that are responsible for seedling-stage resistance and adult-plant stage resistance (APR). More importantly was the characterisation of a previously unidentified QTL that can provide resistance during different stages of plant growth or multi-stage resistance (MSR). At the seedling stage, we discovered a new isolate-specific QTL, QSt.wai.1A.1. At the adult-plant stage, the new QTL QStb.wai.6A.2 provided stable and consistent APR in multiple sites and years, while the QTL QStb.wai.7A.2 was highlighted to have MSR. The stacking of multiple favourable MSR alleles was found to improve resistance to Z. tritici by up to 40%. An Australian GWAS panel discovered three new QTLs associated with seedling-stage resistance, adult-plant stage resistance, and multi-stage resistance, respectively.

Stagonospora nodorum: Cause of stagonospora nodorum blotch of wheat

Publisher: Wiley

Date: 05-2006

DOI: 10.1111/J.1364-3703.2006.00326.X

Abstract: SUMMARY Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf and glume blotch. This review summarizes recent advances in our understanding of taxonomy, control and pathogenicity of this species. Stagonospora (syn. Septoria) nodorum (Berk.) Castell. and Germano [teleomorph: Phaeosphaeria (syn. Leptosphaeria) nodorum (Müll.) Hedjar.], kingdom Fungi, phylum Ascomycota, subphylum Euascomycota, class Dothideomycetes, order Pleosporales, family Phaeosphaeriaceae, genus Phaeosphaeria, species nodorum. Wheat, Triticum aestivum, T. durum, Triticale, are the main hosts but other cereals and wild grasses have been reported to harbour S. nodorum. Disease symptoms are lens-shaped necrotic lesions on leaves, girdling necrosis on stems (especially the nodes, hence 'nodorum') and lesions on glumes. Mature lesions produce pycnidia scattered throughout the lesions, especially as tissue senesces. esearch/deephyphae/htmls/asco_taxlist_spat.html (taxonomic information), c-fact/0002.html (disease information), wwwacnfp.murdoch.edu.au/ (ACNFP homepage), nnotation/fungi/stagonospora_nodorum/index.html (genome sequence homepage), cogeme.ex.ac.uk/efungi/ (genome sequence annotation and analysis).

The Cysteine Rich Necrotrophic Effector SnTox1 Produced by Stagonospora nodorum Triggers Susceptibility of Wheat Lines Harboring Snn1

Publisher: Public Library of Science (PLoS)

Date: 05-01-2012

DOI: 10.1371/JOURNAL.PPAT.1002467

Decoding the mannitol enigma in filamentous fungi

Publisher: Elsevier BV

Date: 06-2007

DOI: 10.1016/J.TIM.2007.04.002

Abstract: Mannitol is a 6-carbon polyol that is among the most abundant biochemical compounds in the biosphere. Mannitol has been ascribed a multitude of roles in filamentous fungi including carbohydrate storage, reservoir of reducing power, stress tolerance and spore dislodgement and/or dispersal. The advancement of genetic manipulation techniques in filamentous fungi has rapidly accelerated our understanding of the roles and metabolism of mannitol. The targeted deletion of genes encoding proteins of mannitol metabolism in several fungi, including phytopathogens, has proven that the metabolism of mannitol does not exist as a cycle and that many of the postulated roles are unsupported. These recent studies have provided a much needed focus on this mysterious metabolite and make this a fitting time to review the roles and metabolism of mannitol in filamentous fungi.

Chemical Ecogenomics-Guided Discovery of Phytotoxic α-Pyrones from the Fungal Wheat Pathogen Parastagonospora nodorum

Publisher: American Chemical Society (ACS)

Date: 18-09-2018

DOI: 10.1021/ACS.ORGLETT.8B02617

Abstract: A biosynthetic gene cluster that is significantly upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection was reconstructed heterologously in Aspergillus nidulans. This led to the discovery of five new α-pyrone polyketides, alternapyrones B-F (2-6). Compounds 5 and 6, which contain a highly substituted dihydrofuran, exhibited phytotoxicity on wheat seed germination. It is demonstrated that only three enzymes, one highly reducing polyketide synthase and two multifunctional P450 oxygenases, are needed to synthesize the structurally complex products.

Remarkable recent changes in genetic diversity of the avirulence geneAvrStb6in global populations of the wheat pathogenZymoseptoria tritici

Publisher: Cold Spring Harbor Laboratory

Date: 18-09-2020

DOI: 10.1101/2020.09.18.303370

Abstract: Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici , is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high ersity of AvrStb6 alleles present in historic Z. tritici isolate collections, with up to ~ 18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic ersity and the incidence of alleles allowing evasion of Stb6 -mediated resistance in more recent Z. tritici populations. Here we show, using targeted re-sequencing of AvrStb6, that this gene is universally present in field isolates s led from major wheat-growing regions of the world between 2013–2017. However, in contrast to the data from studies of historic isolates, our study revealed a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of alleles, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6- containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence alleles tend to be eliminated in subsequent populations.

Investigating the role of calcium/calmodulin-dependent protein kinases inStagonospora nodorum

Publisher: Wiley

Date: 14-09-2006

DOI: 10.1111/J.1365-2958.2006.05380.X

Abstract: Three genes encoding different Ca2+/calmodulin-dependent protein kinases have been characterized in the wheat phytopathogenic fungus Stagonospora nodorum. The kinases were identified from the S. nodorum genome sequence on the basis of sequence homology to known Ca2+/calmodulin-dependent protein kinases. Expression analysis determined that each of the kinases was expressed during growth in vitro and also during infection. The onset of sporulation triggered increased transcript levels of each of the kinases, particularly CpkA where an 11-fold increase in expression was observed during sporulation in planta. The role of the kinases was further determined via a reverse genetics approach. The disruption of CpkA affected vegetative growth in vitro and also sporulation. The cpkA strains produced 20-fold less spores on complex media and were unable to sporulate on defined minimal media. Infection assays showed that CpkA was not required for lesion development but was essential for sporulation at the completion of the infection cycle. Microscopic analysis revealed that the disruption of CpkA resulted in Stagonospora nodorum being unable to differentiate the mycelial knot into immature pycnidia during sporulation. A metabolite analysis of infected leaves during sporulation excluded the possible involvement of mannitol, a compound previously shown to be involved in the sporulation of Stagonospora nodorum. The disruption of CpkB did not effect growth in vitro or pathogenicity. Stagonospora nodorum strains lacking CpkC appeared unaffected during growth in planta but showed delayed lesion development and sporulation during infection.

Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

Publisher: American Society for Microbiology

Date: 09-2010

DOI: 10.1128/EC.00363-09

Abstract: The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger . In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger . Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA , respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger .

Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch)

Publisher: Portland Press Ltd.

Date: 27-09-2006

DOI: 10.1042/BJ20060891

Abstract: The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant–Microbe Interact. 18, 110–115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.

Re-classification of the causal agent of white grain disorder on wheat as three separate species of Eutiarosporella

Publisher: Springer Science and Business Media LLC

Date: 29-07-2015

DOI: 10.1007/S13313-015-0367-2

Proteomic Techniques for Plant–Fungal Interactions

Publisher: Humana Press

Date: 23-11-2011

DOI: 10.1007/978-1-61779-501-5_5

Abstract: Proteomics is a key technique that is helping elucidate many complex biological processes. The analysis of plant-pathogen interactions using proteomics is complicated by the presence of the proteomes of two species, but is benefiting from the developing maturity and power of these techniques. More and more pathogen genomes are being sequenced, so fungal proteomics is reaching its full potential and remains the chosen technology to unravel the molecular pathways of pathogenicity and resistance. In this chapter, we suggest proteomic strategies that have proved successful on various plant-interacting fungal species. Several protein extraction methods are described. For adequate quantitative analyses of protein abundances, we recommend either separation using two-dimensional gel electrophoresis or labelling with isobaric tags followed by two-dimensional HPLC separation. Proteins of interest are then identified using mass spectrometry. Identified proteins can assist in refining genome annotations, otherwise known as proteogenomics.

Volatile Molecules Secreted by the Wheat Pathogen Parastagonospora nodorum Are Involved in Development and Phytotoxicity

Publisher: Frontiers Media SA

Date: 25-03-2020

DOI: 10.3389/FMICB.2020.00466

Recent Fungal Diseases of Crop Plants: Is Lateral Gene Transfer a Common Theme?

Publisher: Scientific Societies

Date: 03-2008

DOI: 10.1094/MPMI-21-3-0287

Abstract: A cursory glance at old textbooks of plant pathology reveals that the diseases which are the current scourge of agriculture in many parts of the world are a different set from those that were prominent 50 or 100 years ago. Why have these new diseases arisen? The traditional explanations subscribe to the “nature abhors a vacuum” principle—that control of one disease creates the condition for the emergence of a replacement—but does little to explain why the new pathogen succeeds. The emergence of a new disease requires a series of conditions and steps, including the enhanced fecundity of the new pathogen, enhanced survival from season to season, and spread around the world. Recently, evidence was obtained that wheat tan spot emerged through a lateral gene transfer event some time prior to 1941. Although there have been sporadic and persistent reports of lateral gene transfer between and into fungal plant pathogens, most ex les have been dismissed through incomplete evidence. The completion of whole genome sequences of an increasing number of fungal pathogens no longer allows such proposed cases of lateral gene transfer to be dismissed so easily. How frequent are lateral gene transfers involving fungal plant pathogens, and can this process explain the emergence of many of the new diseases of the recent past? Many of the apparently new diseases are dependant on the expression of host-specific toxins. These are enigmatic molecules whose action requires the presence of plant genes with products that specifically encode sensitivity to the toxin and susceptibility to the disease. It is also notable that many new diseases belong to the fungal taxon dothideomycetes. This review explores the coincidence of new diseases, interspecific gene transfer, host-specific toxins, and the dothideomycete class.

A thousand-genome panel retraces the global spread and climatic adaptation of a major crop pathogen

Publisher: Cold Spring Harbor Laboratory

Date: 27-08-2022

DOI: 10.1101/2022.08.26.505378

Abstract: Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici , a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

PR1‐mediated defence via C‐terminal peptide release is targeted by a fungal pathogen effector

Publisher: Wiley

Date: 03-01-2021

DOI: 10.1111/NPH.17128

Effects of fighting decisions on formation and structure of dominance hierarchies

Publisher: Informa UK Limited

Date: 03-2007

DOI: 10.1080/10236240701237932

Acquisition and loss of secondary metabolite clusters shaped the evolutionary path of three recently emerged phytopathogens of wheat

Publisher: Cold Spring Harbor Laboratory

Date: 18-03-2018

DOI: 10.1101/283416

Abstract: White grain disorder is a recently emerged wheat disease in Australia, caused by three Botryosphaeriaceae spp. Eutiarosporella darliae, E. pseudodarliae , and E. tritici-australis . The disease cycle of these pathogens and the molecular basis of their interaction with wheat are poorly understood. To address this, we undertook a comparative genomics approach to identify potential pathogenicity factors. Subsequent genome analysis revealed that each of the white grain disorder species harbour modular polyketide synthase genes. To our knowledge, this is the first report of fungi harbouring such genes. Further comparative analysis using the modular polyketide synthase genes discovered their presence in the closely related Macrophomina phaseolina . Phylogenetic analysis implicates horizontal acquisition of these genes from a bacterial or a protist species. Both E. darliae and E. pseudodarliae possess a secondary metabolite cluster with multiple polyketide/non-ribosomal peptide synthase genes ( Hybrid-1, -2, and -3 ). In contrast, only remnant and partial genes homologous to this cluster were identified at a syntenic locus in E. tritici-australis suggesting loss of this cluster. Homologues of Hybrid-2 in other fungi have been proposed to facilitate disease induction in woody plants. Subsequent assays confirmed that E. darliae and E. pseudodarliae were both pathogenic on woody plant hosts, but E. tritici-australis was not, implicating woody plants as potential host reservoirs for the fungi. We hypothesise that loss of the cluster in E. tritici-australis represents a committed lifestyle jump to grasses. Combined, our observations relating to the secondary metabolite potential of the WGD Eutiarosporella spp. have contributed novel data to the field by expanding the range of known fungal secondary metabolite genes, and helped develop our understanding of the lifestyle and potential host-range of a recently emerged pathogen of wheat.

A thousand-genome panel retraces the global spread and adaptation of a major fungal crop pathogen

Publisher: Springer Science and Business Media LLC

Date: 24-02-2023

DOI: 10.1038/S41467-023-36674-Y

Abstract: Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici , a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

Biosynthesis of a Tricyclo[6.2.2.0^2,7]dodecane System by a Berberine Bridge Enzyme-Like Aldolase

Publisher: Wiley

Date: 28-10-2019

DOI: 10.1002/CHEM.201904360

Abstract: The aldol reaction is one of the most fundamental stereocontrolled carbon-carbon bond-forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell-free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)-like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.0

Correction to “Combining Protein Ratio p-Values as a Pragmatic Approach to the Analysis of Multirun iTRAQ Experiments”

Publisher: American Chemical Society (ACS)

Date: 08-10-2015

DOI: 10.1021/ACS.JPROTEOME.5B00897

The necrotrophic effector SnToxA induces the synthesis of a novel phytoalexin in wheat

Publisher: Wiley

Date: 19-06-2013

DOI: 10.1111/NPH.12356

Abstract: S tagonospora nodorum and P yrenophora tritici‐repentis produce the effector T ox A that interacts with the dominant susceptibility gene in wheat, Tsn1 . However, the way in which T ox A induces cell death and causes disease is unclear. Here, we performed comprehensive metabolite profiling of T ox A ‐infiltrated wheat ( T riticum aestivum ) to observe the secondary metabolite response to this effector. A strong induction of secondary metabolism subsequent to S n T ox A infiltration was observed, including the monoamine serotonin. We established a novel role for serotonin as a phytoalexin in wheat and demonstrated that serotonin strongly inhibited sporulation of S . nodorum . Microscopy revealed that serotonin interferes with spore formation and maturation within pycnidial structures of the fungus. Subsequent analysis of S . nodorum exposed to serotonin revealed metabolites changes previously associated with sporulation, including trehalose and alternariol. Furthermore, we identified significantly lower concentrations of serotonin during infection compared with infiltration with T ox A , providing evidence that S . nodorum may suppress plant defence. This is the first study demonstrating induction of plant secondary metabolites in response to a necrotrophic effector that have significant antifungal potential against the pathogen. While it is generally accepted that necrotrophs exploit host cell responses, the current research strengthens the notion that necrotrophs require mechanisms to overcome plant defence to survive initial stages of infection.

Simple and efficient heterologous expression of necrosis-inducing effectors using the model plant Nicotiana benthamiana

Publisher: Cold Spring Harbor Laboratory

Date: 03-05-2021

DOI: 10.1101/2021.05.02.442377

Abstract: Plant fungal pathogens cause devastating diseases on cereal plants and threaten global food security. During infection, these pathogens secrete proteinaceous effectors that promote disease. Some of these effectors from necrotrophic plant pathogens induce a cell death response (necrosis), which facilitates pathogen growth in planta . Characterisation of these effectors typically requires heterologous expression and microbial expression systems such as bacteria and yeast are the predominantly used. However, microbial expression systems often require optimization for any given effector and are, in general, not suitable for effectors involving cysteine bridges and posttranslational modifications for activity. Here, we describe a simple and efficient method for expressing such effectors in the model plant Nicotiana benthamiana . Briefly, an effector protein is transiently expressed and secreted into the apoplast of N. benthamiana by Agrobacterium-mediated infiltration. Two-to-three days subsequent to agroinfiltration, the apoplast from the infiltrated leaves is extracted and can be directly used for phenotyping on host plants. The efficacy of this approach was demonstrated by expressing the ToxA, Tox3 and Tox1 necrosis-inducing effectors from Parastagonospora nodorum . All three effectors produced in N. benthamiana were capable of inducing necrosis in wheat lines, and two of three showed visible bands on Coomassie-stained gel. These data suggest that N. benthamiana- agroinfiltration system is a feasible tool to obtain fungal effectors, especially those that require disulfide bonds and posttranslational modifications. Furthermore, due to the low number of proteins typically observed in the apoplast (compared to intracellular), this simple and high-throughput approach circumvents the requirement to lyse cells and further purify the target proteins that is required in other heterologous systems. Because of its simplicity and potential for high-throughput, this method is highly amenable to the phenotyping of candidate protein effectors on host plants.

Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast

Publisher: Springer Science and Business Media LLC

Date: 12-06-2017

DOI: 10.1186/S12934-017-0713-X

Malayamycin, a new streptomycete antifungal compound, specifically inhibits sporulation ofStagonospora nodorum(Berk) Castell and Germano, the cause of wheat glume blotch disease

Publisher: Wiley

Date: 2008

DOI: 10.1002/PS.1632

Abstract: Malayamycin is a novel perhydrofuropyran C-nucleoside isolated from Streptomyces malaysiensis that shows promising antifungal activity, fully controlling a range of diseases when applied to plants at 100 microg mL(-1). The goal of this study was to determine the mode of action. Malayamycin exhibited in vitro antifungal activity against Stagonospora nodorum (Berk) Castell & Germano, the cause of stagonospora nodorum blotch of wheat. Growth in liquid minimum medium was merely delayed at 50 microg mL(-1), but sporulation was suppressed by more than 50% by 10 microg mL(-1) of malayamycin. When applied to wheat seedlings 36 h prior to infection, 10 microg mL(-1) of malayamycin reduced lesion size and significantly reduced pycnidiation to only 5% of the non-treated level. A transcription factor gene, Mrg1 (malayamycin response gene) whose expression was upregulated by application of malayamycin, was identified. Both Mrg1 knockout and overexpression strains were created. These strains were fully pathogenic, suggesting that the expression of Mrg1 did not affect pathogenicity. Interestingly, a strain that expressed Mrg1 50 times more than wild type showed a significant reduction in sporulation. However, all the tested knockout and overexpression strains retained sensitivity to malayamycin. Malayamycin is a new type of antifungal compound that acts primarily by inhibiting sporulation. Although Mrg1 may be involved in the sporulation process, it is not the major contributor for sporulation inhibition caused by malayamycin treatment.

Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

Publisher: Springer Science and Business Media LLC

Date: 31-03-2020

DOI: 10.1186/S40694-020-00094-0

Abstract: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. These data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

The nutrient supply of pathogenic fungi; a fertile field for study

Publisher: Wiley

Date: 26-03-2003

DOI: 10.1046/J.1364-3703.2003.00161.X

Abstract: SUMMARY Phytopathogenic fungi must feed on their hosts to propagate and cause disease. Their ability to access the rich nutrient supply offered by living plants is one of the most obvious properties that distinguish pathogens from saprophytes. Successful invasion by pathogens depends as much on their ability to utilize the available nutrient sources offered by plants as on their ability to penetrate plants and evade defensive mechanisms. Here, we review current knowledge on the nature of the nutrient supplies utilized by pathogens during infection. The available evidence is rudimentary in most cases. There is much evidence to suggest that fungal metabolism can be ided into at least two phases. The first is based on lipolysis and occurs during germination and penetration of the host. The second phase uses glycolysis and predominates during the invasion of host tissue. We also propose, mainly on theoretical grounds, that a third phase of nutrition occurs late in infection during which new spores are produced. Contrary to early assumptions, the nitrogen sources available to some pathogens appear to be abundant. The idea that nitrogen starvation is a cue that controls fungal gene expression during infection may need to be reassessed. Very little is known about the micronutrient (Fe, S, P) or vitamin supply. The knowledge gained from this research may enable the design of new antifungal strategies targeting potential weaknesses in fungal metabolism and will also impact on agronomic practices.

Analysis and evaluation of the top- $$k$$ k most influential location selection query

Publisher: Springer Science and Business Media LLC

Date: 16-01-2014

DOI: 10.1007/S10115-013-0720-0

SnTox3 Acts in Effector Triggered Susceptibility to Induce Disease on Wheat Carrying the Snn3 Gene

Publisher: Public Library of Science (PLoS)

Date: 18-09-2009

DOI: 10.1371/JOURNAL.PPAT.1000581

New developments in pathogenicity and virulence of necrotrophs

Publisher: Elsevier BV

Date: 08-2010

DOI: 10.1016/J.PBI.2010.05.003

Abstract: It was generally considered that necrotrophic plant pathogenic fungi possessed simplistic pathogenic mechanisms being typically reliant on 'blasting' their way through host tissue with a battery of lytic and degradative enzymes. However recent studies have suggested that this is not true and that necrotrophic fungal pathogens can subtly manipulate the host during infection in a manner similar to biotrophic pathogens. For ex le, it has been demonstrated that the wheat pathogens Stagonospora nodorum and Pyrenophora tritici-repentis secrete small unique proteins (effectors) that are internalised by host cells and interact with the host in a gene-for-gene relationship to initiate disease, albeit in an inverse manner compared to biotrophs. This paper reviews recent developments in necrotrophic fungal pathogenicity throughout a critical period that arguably saw this field come of age.

Role of Cereal Secondary Metabolites Involved in Mediating the Outcome of Plant-Pathogen Interactions

Publisher: MDPI AG

Date: 15-12-2011

DOI: 10.3390/METABO1010064

Characterising the role of GABA and its metabolism in the wheat pathogen Stagonospora nodorum

Publisher: Public Library of Science (PLoS)

Date: 12-11-2013

DOI: 10.1371/JOURNAL.PONE.0078368

Functional genomics-guided discovery of a light-activated phytotoxin in the wheat pathogen Parastagonospora nodorum via pathway activation

Publisher: Wiley

Date: 10-04-2017

DOI: 10.1111/1462-2920.13711

Abstract: Parastagonospora nodorum is an important pathogen of wheat. The contribution of secondary metabolites to this pathosystem is poorly understood. A biosynthetic gene cluster (SNOG_08608-08616) has been shown to be upregulated during the late stage of P. nodorum wheat leaf infection. The gene cluster shares several homologues with the Cercospora nicotianae CTB gene cluster encoding the biosynthesis of cercosporin. Activation of the gene cluster by overexpression (OE) of the transcription factor gene (SNOG_08609) in P. nodorum resulted in the production of elsinochrome C, a perelyenequinone phytotoxin structurally similar to cercosporin. Heterologous expression of the polyketide synthase gene elcA from the gene cluster in Aspergillus nidulans resulted in the production of the polyketide precursor nortoralactone common to the cercosporin pathway. Elsinochrome C could be detected on wheat leaves infected with P. nodorum, but not in the elcA disruption mutant. The compound was shown to exhibit necrotic activity on wheat leaves in a light-dependent manner. Wheat seedling infection assays showed that ΔelcA exhibited reduced virulence compared with wild type, while infection by an OE strain overproducing elsinochrome C resulted in larger lesions on leaves. These data provided evidence that elsinochrome C contributes to the virulence of P. nodorum against wheat.

Dissecting the role of G-protein signalling in primary metabolism in the wheat pathogen Stagonospora nodorum

Publisher: Microbiology Society

Date: 09-2013

DOI: 10.1099/MIC.0.067009-0

Abstract: Mutants of the wheat pathogenic fungus Stagonospora nodorum lacking G-protein subunits display a variety of phenotypes including melanization defects, primary metabolic changes and a decreased ability to sporulate. To better understand the causes of these phenotypes, Stagonospora nodorum strains lacking a Gα, Gβ or Gγ subunit were compared to a wild-type strain using metabolomics. Agar plate growth at 22 °C revealed a number of fundamental metabolic changes and highlighted the influential role of these proteins in glucose utilization. A further characterization of the mutants was undertaken during prolonged storage at 4 °C, conditions known to induce sporulation in these sporulation-deficient signalling mutants. The abundance of several compounds positively correlated with the onset of sporulation including the dissacharide trehalose, the tryptophan degradation product tryptamine and the secondary metabolite alternariol metabolites all previously associated with sporulation. Several other compounds decreased or were absent during sporulation. The levels of one such compound (Unknown_35.27_2194_319) decreased from being one of the more abundant compounds to absence during pycnidial maturation. This study has shed light on the role of G-protein subunits in primary metabolism during vegetative growth and exploited the cold-induced sporulation phenomenon in these mutants to identify some key metabolic changes that occur during asexual reproduction.

A Revised Nomenclature for ToxA Haplotypes Across Multiple Fungal Species

Publisher: Scientific Societies

Date: 28-08-2023

DOI: 10.1094/PHYTO-01-23-0017-SC

Abstract: ToxA is one of the most studied proteinaceous necrotrophic effectors produced by plant pathogens. It has been identified in four pathogens ( Pyrenophora tritici-repentis, Parastagonospora nodorum, Parastagonospora pseudonodorum [formerly Parastagonospora avenaria f. sp. tritici], and Bipolaris sorokiniana) causing leaf spot diseases on cereals worldwide. To date, 24 different ToxA haplotypes have been identified. Some P. tritici-repentis and related species also express ToxB, another small protein necrotrophic effector. We present here a revised and standardized nomenclature for these effectors, which could be extended to other poly-haplotypic genes found across multiple species.

The rise of necrotrophic effectors.

Publisher: Wiley

Date: 11-2022

DOI: 10.1111/NPH.17811

Abstract: This article is a Commentary on Kariyawasam et al . (2022), 233 : 409–426 and Richards et al . (2022), 233 : 427–442 .

Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum

Publisher: Elsevier BV

Date: 05-2009

DOI: 10.1016/J.FGB.2009.02.002

Abstract: Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2-3weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S.nodorum.

Fungal endophyte infection of ryegrass reprograms host metabolism and alters development

Publisher: Wiley

Date: 25-08-2015

DOI: 10.1111/NPH.13614

Abstract: Beneficial associations between plants and microbes play an important role in both natural and agricultural ecosystems. For ex le, associations between fungi of the genus Epichloë , and cool‐season grasses are known for their ability to increase resistance to insect pests, fungal pathogens and drought. However, little is known about the molecular changes induced by endophyte infection. To study the impact of endophyte infection, we compared the expression profiles, based on RNA sequencing, of perennial ryegrass infected with Epichloë festucae with noninfected plants. We show that infection causes dramatic changes in the expression of over one third of host genes. This is in stark contrast to mycorrhizal associations, where substantially fewer changes in host gene expression are observed, and is more similar to pathogenic interactions. We reveal that endophyte infection triggers reprogramming of host metabolism, favouring secondary metabolism at a cost to primary metabolism. Infection also induces changes in host development, particularly trichome formation and cell wall biogenesis. Importantly, this work sheds light on the mechanisms underlying enhanced resistance to drought and super‐infection by fungal pathogens provided by fungal endophyte infection. Finally, our study reveals that not all beneficial plant–microbe associations behave the same in terms of their effects on the host.

A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat.

Publisher: Springer Science and Business Media LLC

Date: 04-11-2019

DOI: 10.1038/S41598-019-52444-7

Abstract: The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The Zn 2 Cys 6 transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P . nodorum wildtype and a strain deleted in PnPf2 ( pf2-69 ) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation.

Reprogramming of the apoplast metabolome ofLolium perenneupon infection with the mutualistic symbiontEpichloë festucae

Publisher: Cold Spring Harbor Laboratory

Date: 08-12-2019

DOI: 10.1101/861450

Abstract: Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with Lolium perenne . Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae . A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infected L. perenne . Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloë metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë -derived metabolites, we deleted a gene encoding a NRPS ( lgsA ) that is highly expressed in planta . Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

SnPKS19 encodes the polyketide synthase for alternariol mycotoxin biosynthesis in the wheat pathogen Parastagonospora nodorum

Publisher: American Society for Microbiology

Date: 15-08-2015

DOI: 10.1128/AEM.00278-15

Abstract: Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat pathogen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 ( SnPKS19 ), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA , is required for AOH production. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus nidulans . The expression profile of SnPKS19 based on previous P. nodorum microarray data correlated with the presence of AOH in vitro and its absence in planta . Subsequent characterization of the Δ SnPKS19 mutants showed that SnPKS19 and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of the P. nodorum SnPKS19 cast light on a possible alternative AOH synthase gene in Alternaria alternata and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.

Assessing the impact of transcriptomics, proteomics and metabolomics on fungal phytopathology

Publisher: Wiley

Date: 09-2009

DOI: 10.1111/J.1364-3703.2009.00565.X

Both Mating types of Phaeosphaeria (anamorph Stagonospora) nodorum are Present in Western Australia

Publisher: Springer Science and Business Media LLC

Date: 08-2004

DOI: 10.1023/B:EJPP.0000041565.42836.C1

A chemical ecogenomics approach to understand the roles of secondary metabolites in fungal cereal pathogens

Publisher: Frontiers Media SA

Date: 19-11-2014

DOI: 10.3389/FMICB.2014.00640

Does the oxidative stress used by plants for defence provide a source of nutrients for pathogenic fungi?

Publisher: Elsevier BV

Date: 10-2004

DOI: 10.1016/J.TPLANTS.2004.08.006

Genetic mapping of Stb19, a new resistance gene to Zymoseptoria tritici in wheat

Publisher: Springer Science and Business Media LLC

Date: 20-09-2018

DOI: 10.1007/S00122-018-3189-0

Abstract: A new and dominant R gene Stb19 is identified from a soft wheat cultivar 'Lorikeet' and was mapped on the distal region of chromosome 1DS. Two tightly linked KASP markers were also discovered and validated for molecular-assisted breeding programs. A new R gene, designated as Stb19, provides resistance to Zymoseptoria tritici in wheat. This new dominant gene resides on the short arm of chromosome 1D, exhibiting complete resistance to three Z. tritici isolates, WAI332, WAI251, and WAI161, at the seedling stage. A genetic linkage map, based on an F

Pathogenicity of Stagonospora nodorum requires malate synthase

Publisher: Wiley

Date: 10-06-2004

DOI: 10.1111/J.1365-2958.2004.04178.X

Genomics-Driven Discovery of Phytotoxic Cytochalasans Involved in the Virulence of the Wheat Pathogen Parastagonospora nodorum

Publisher: American Chemical Society (ACS)

Date: 09-12-2020

DOI: 10.1021/ACSCHEMBIO.9B00791

Abstract: The etiology of fungal pathogenesis of grains is critical to global food security. The large number of orphan biosynthetic gene clusters uncovered in fungal plant pathogen genome sequencing projects suggests that we have a significant knowledge gap about the secondary metabolite repertoires of these pathogens and their roles in plant pathogenesis. Cytochalasans are a family of natural products of significant interest due to their ability to bind to actin and interfere with cellular processes that involved actin polymerization however, our understanding of their biosynthesis and biological roles remains incomplete. Here, we identified a putative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene cluster (

Molecular plant immunity against biotrophic, hemibiotrophic, and necrotrophic fungi

Publisher: Portland Press Ltd.

Date: 09-2022

DOI: 10.1042/EBC20210073

Abstract: Pathogenic fungi use erse infection strategies to obtain nutrients from plants. Biotrophic fungi feed only on living plant tissue, whereas necrotrophic fungi kill host cells to extract nutrients. To prevent disease, plants need to distinguish between pathogens with different life cycles, as a successful defense against a biotroph, which often involves programmed cell-death around the site of infection, is not an appropriate response to some necrotrophs. Plants utilize a vast collection of extracellular and intracellular receptors to detect the signatures of pathogen attack. In turn, pathogens are under strong selection to mask or avoid certain receptor responses while enhancing or manipulating other receptor responses to promote virulence. In this review, we focus on the plant receptors involved in resistance responses to fungal pathogens and highlight, with ex les, how the infection strategy of fungal pathogens can determine if recognition responses are effective at preventing disease.

A review of wheat diseases—a field perspective

Publisher: Wiley

Date: 26-12-2017

DOI: 10.1111/MPP.12618

Emergence of a new disease as a result of interspecific virulence gene transfer

Publisher: Springer Science and Business Media LLC

Date: 09-07-2006

DOI: 10.1038/NG1839

Abstract: New diseases of humans, animals and plants emerge regularly. Enhanced virulence on a new host can be facilitated by the acquisition of novel virulence factors. Interspecific gene transfer is known to be a source of such virulence factors in bacterial pathogens (often manifested as pathogenicity islands in the recipient organism) and it has been speculated that interspecific transfer of virulence factors may occur in fungal pathogens. Until now, no direct support has been available for this hypothesis. Here we present evidence that a gene encoding a critical virulence factor was transferred from one species of fungal pathogen to another. This gene transfer probably occurred just before 1941, creating a pathogen population with significantly enhanced virulence and leading to the emergence of a new damaging disease of wheat.

The necrotrophic effector ToxA from Parastagonospora nodorum interacts with wheat NHL proteins to facilitate Tsn1-mediated necrosis

Publisher: Cold Spring Harbor Laboratory

Date: 23-09-2021

DOI: 10.1101/2021.09.22.461440

Abstract: The plant pathogen Parastagonospora nodorum secretes necrotrophic effectors to promote disease. These effectors induce cell death on wheat cultivars carrying dominant susceptibility genes in an inverse gene-for-gene manner. However, the molecular mechanisms underpinning these interactions and resulting cell death remain unclear. Here, we used a yeast-two-hybrid library approach to identify wheat proteins that interact with the necrotrophic effector ToxA. Using this strategy, we identified an interaction between ToxA and a wheat transmembrane NDR/HIN1-like protein (TaNHL10) and confirmed the interaction using in-planta co-immunoprecipitation and confocal microscopy co-localization analysis. We showed that the C-terminus of TaNHL10 is extracellular whilst the N-terminus was localized in the cytoplasm. Further analyses using yeast-two-hybrid and confocal microscopy co-localization showed that ToxA interacts with the C-terminal LEA2 extracellular domain of TaNHL10. Random mutagenesis was then used to identify a ToxA mutant, ToxA N109D , which was unable to interact with TaNHL10 in yeast-two-hybrid assays. Subsequent heterologous expression and purification of ToxA N109D in Nicotiania benthamiana revealed that the mutated protein was unable to induce necrosis on Tsn1 -dominant wheat cultivars confirming that the interaction of ToxA with TaNHL10 is required to induce cell death. Collectively, these data advance our understanding on how ToxA induces cell death during infection and further highlights the importance of host cell surface interactions in necrotrophic pathosystems.

Transcriptome analysis of Stagonospora nodorum: gene models, effectors, metabolism and pantothenate dispensability

Publisher: Wiley

Date: 06-12-2011

DOI: 10.1111/J.1364-3703.2011.00770.X

Comparative Pathogenomics Reveals Horizontally Acquired Novel Virulence Genes in Fungi Infecting Cereal Hosts

Publisher: Public Library of Science (PLoS)

Date: 27-09-2012

DOI: 10.1371/JOURNAL.PPAT.1002952

Quantitative disease resistance assessment by real-time PCR using the Stagonospora nodorum-wheat pathosystem as a model

Publisher: Wiley

Date: 06-2008

DOI: 10.1111/J.1365-3059.2007.01787.X

Phytopathogen emergence in the genomics era

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1016/J.TPLANTS.2015.01.009

Abstract: Phytopathogens are a global threat to plant agriculture and bio ersity. The genomics era has lead to an exponential rise in comparative gene and genome studies of both economically significant and insignificant microorganisms. In this review we highlight some recent comparisons and discuss how they identify shared genes or genomic regions associated with host virulence. The two major mechanisms of rapid genome adaptation - horizontal gene transfer and hybridisation - are reviewed and we consider how intra-specific pan-genome sequences encode alternative host specificity. We also discuss the power that access to expansive gene databases provides in aiding the study of phytopathogen emergence. These databases can rapidly enable the identification of an unknown pathogen and its origin, as well as genomic adaptations required for emergence.

Mannitol 1-phosphate metabolism is required for sporulation in planta of the wheat pathogen Stagonospora nodorum

Publisher: Scientific Societies

Date: 02-2005

DOI: 10.1094/MPMI-18-0110

Abstract: An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wildtype strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.

Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

Publisher: Research Square Platform LLC

Date: 11-02-2020

DOI: 10.21203/RS.2.23248/V1

Abstract: Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (gRNA) which interact to form a Cas9-gRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and gRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting gene transgene-free knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

Publisher: Research Square Platform LLC

Date: 23-03-2020

DOI: 10.21203/RS.2.23248/V2

Abstract: Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

Coverage and consistency

Publisher: American Chemical Society (ACS)

Date: 20-09-2013

DOI: 10.1021/PR400531Y

Abstract: The hexaploid genome of bread wheat (Triticum aestivum) is large (17 Gb) and repetitive, and this has delayed full sequencing and annotation of the genome, which is a prerequisite for effective quantitative proteomics analysis. Aware of these constraints we investigated the most effective approaches for shotgun proteomic analyses of bread wheat that would support large-scale quantitative comparisons using iTRAQ reagents. We used a data set that was generated by two-dimensional LC-MS of iTRAQ labeled peptides from wheat leaves. The main items considered in this study were the choice of sequence database for matching LC-MS data, the consistency of identification when multiple LC-MS runs were acquired, and the options for downstream functional analysis to generate useful insight. For peptide identification we examined the extensive NCBInr plant database, a smaller composite cereals database, the Brachypodium distachyon model plant genome, the EST-based SuperWheat database, as well as the genome sequence from the recently sequenced D-genome progenitor Aegilops tauschii. While the most spectra were assigned by using the SuperWheat database, this extremely large database could not be readily manipulated for the robust protein grouping that is required for large-scale, multirun quantitative experiments. We demonstrated a pragmatic alternative of using the composite cereals database for peptide spectra matching. The stochastic aspect of protein grouping across LC-MS runs was investigated using the smaller composite cereals database where we found that attaching the Brachypodium best BLAST hit reduced this problem. Further, assigning quantitation to the best Brachypodium locus yielded promising results enabling integration with existing downstream data mining and functional analysis tools. Our study demonstrated viable approaches for quantitative proteomics analysis of bread wheat s les and shows how these approaches could be similarly adopted for analysis of other organisms with unsequenced or incompletely sequenced genomes.

Autoimmunity and effector recognition in Arabidopsis thaliana can be uncoupled by mutations in the RRS1-R immune receptor.

Publisher: Wiley

Date: 03-02-2019

DOI: 10.1111/NPH.15617

Abstract: Plant nucleotide-binding leucine-rich repeat (NLR) disease resistance proteins recognize specific pathogen effectors and activate a cellular defense program. In Arabidopsis thaliana (Arabidopsis), Resistance to Ralstonia solanacearum 1 (RRS1-R) and Resistance to Pseudomonas syringae 4 (RPS4) function together to recognize the unrelated bacterial effectors PopP2 and AvrRps4. In the plant cell nucleus, the RRS1-R/RPS4 complex binds to and signals the presence of AvrRps4 or PopP2. The exact mechanism underlying NLR signaling and immunity activation remains to be elucidated. Using genetic and biochemical approaches, we characterized the intragenic suppressors of sensitive to low humidity 1 (slh1), a temperature-sensitive autoimmune allele of RRS1-R. Our analyses identified five amino acid residues that contribute to RRS1-R

Metabolomics Protocols for Filamentous Fungi

Publisher: Humana Press

Date: 23-11-2011

DOI: 10.1007/978-1-61779-501-5_15

Abstract: Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi.

A Core Gene Set Describes the Molecular Basis of Mutualism and Antagonism in Epichloë spp.

Publisher: Scientific Societies

Date: 03-2015

DOI: 10.1094/MPMI-09-14-0293-FI

Abstract: Beneficial plant–fungal interactions play an important role in the ability of plants to survive changing environmental conditions. In contrast, phytopathogenic fungi fall at the opposite end of the symbiotic spectrum, causing reduced host growth or even death. In order to exploit beneficial interactions and prevent pathogenic ones, it is essential to understand the molecular differences underlying these alternative states. The association between the endophyte Epichloë festucae and Lolium perenne (perennial ryegrass) is an excellent system for studying these molecular patterns due to the existence of several fungal mutants that have an antagonistic rather than a mutualistic interaction with the host plant. By comparing gene expression in a wild-type beneficial association with three mutant antagonistic associations disrupted in key signaling genes, we identified a core set of 182 genes that show common differential expression patterns between these two states. These gene expression changes are indicative of a nutrient-starvation response, as supported by the upregulation of genes encoding degradative enzymes, transporters, and primary metabolism, and downregulation of genes encoding putative small-secreted proteins and secondary metabolism. These results suggest that disruption of a mutualistic symbiotic interaction may lead to an elevated uptake and degradation of host-derived nutrients and cell-wall components, reminiscent of phytopathogenic interactions.

The Mak2 MAP kinase signal transduction pathway is required for pathogenicity in Stagonospora nodorum

Publisher: Springer Science and Business Media LLC

Date: 14-06-2005

DOI: 10.1007/S00294-005-0588-Y

Abstract: A gene encoding a mitogen-activated protein kinase (MAPK) putatively orthologous to Pmk1 from Magnaporthe grisea was cloned and characterised from the wheat glume blotch pathogen Stagonospora nodorum. Protein sequence alignments showed the cloned gene, Mak2, is closely related to homologues from other dothideomycete fungi. Expression studies revealed Mak2 is up-regulated during in vitro growth upon nitrogen starvation but is not sensitive to carbon starvation or osmotic stress. Transcript analysis in planta showed Mak2 to be expressed throughout infection and up-regulated during the sporulation phase of the infection cycle. Fungal strains harbouring a disrupted Mak2 gene were created by homologous gene recombination. The mutant strains had a severely altered phenotype in vitro with reduced growth rate and failure to sporulate. Further phenotypic analysis revealed that the mutants had near-normal levels of secreted protease activity, were not hypersensitive to osmotic stress and appeared to have melanin synthesis intact. The mak2 strains were essentially non-pathogenic to wheat leaves. No penetration structures formed and although entry was observed through stomates, the infection rarely continued. The results within this study are discussed within the context of the differences in downstream regulation of the Mak2 MAPK pathway and the cAMP signal transduction pathway in S. nodorum and differences are compared to mak2 mutant strains in other pathogenic fungi.

Influence of yeast strain, canopy management, and site on the volatile composition and sensory attributes of cabernet sauvignon wines from Western Australia

Publisher: American Chemical Society (ACS)

Date: 03-03-2011

DOI: 10.1021/JF104324D

Abstract: Understanding what factors are the major influences on wine composition will assist in the successful management of grape composition in the vineyard and/or variables in the winery to produce wines with specific sensory attributes. A recently developed analytical method [headspace solid-phase microextraction comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry] was employed to analyze over 350 volatile compounds in research scale wines and was combined with descriptive sensory analysis. Both compositional and sensory results showed significant differences among the wines, and in many cases, multiple factors influenced the abundance of wine volatile compounds. Site had the most significant influence on sensory scores and wine composition, followed by canopy management. Unexpectedly, yeast strain had no significant sensory effect despite the fact that a number of volatile compounds were significantly different in the wines made from different strains. PLS analysis, combining the sensory and chemical analyses, also supports the concept of volatile compound interactions contributing to the aroma characteristics of Cabernet Sauvignon wine.

Photochemical characterization of a novel fungal rhodopsin from Phaeosphaeria nodorum

Publisher: Elsevier BV

Date: 11-2011

DOI: 10.1016/J.BBABIO.2011.07.005

Abstract: Eukaryotic microbial rhodopsins are widespread bacteriorhodopsin-like proteins found in many lower eukaryotic groups including fungi. Many fungi contain multiple rhodopsins, some significantly erged from the original bacteriorhodopsin template. Although few fungal rhodopsins have been studied biophysically, both fast-cycling light-driven proton pumps and slow-cycling photosensors have been found. The purpose of this study was to characterize photochemically a new subgroup of fungal rhodopsins, the so-called auxiliary group. The study used the two known rhodopsin genes from the fungal wheat pathogen, Phaeosphaeria nodorum. One of the genes is a member of the auxiliary group while the other is highly similar to previously characterized proton-pumping Leptosphaeria rhodopsin. Auxiliary rhodopsin genes from a range of species form a distinct group with a unique primary structure and are located in carotenoid biosynthesis gene cluster. Amino acid conservation pattern suggests that auxiliary rhodopsins retain the transmembrane core of bacteriorhodopsins, including all residues important for proton transport, but have unique polar intramembrane residues. Spectroscopic characterization of the two yeast-expressed Phaeosphaeria rhodopsins showed many similarities: absorption spectra, conformation of the retinal chromophore, fast photocycling, and carboxylic acid protonation changes. It is likely that both Phaeosphaeria rhodopsins are proton-pumping, at least in vitro. We suggest that auxiliary rhodopsins have separated from their ancestors fairly recently and have acquired the ability to interact with as yet unidentified transducers, performing a photosensory function without changing their spectral properties and basic photochemistry.

Differential effector gene expression underpins epistasis in a plant fungal disease

Publisher: Wiley

Date: 07-07-2016

DOI: 10.1111/TPJ.13203

Simple and efficient heterologous expression of necrosis‐inducing effectors using the model plant Nicotiana benthamiana

Publisher: Wiley

Date: 08-2021

DOI: 10.1002/PLD3.341

Abstract: Plant fungal pathogens cause devastating diseases on cereal plants and threaten global food security. During infection, these pathogens secrete proteinaceous effectors that promote disease. Some of these effectors from necrotrophic plant pathogens induce a cell death response (necrosis), which facilitates pathogen growth in planta . Characterization of these effectors typically requires heterologous expression, and microbial expression systems such as bacteria and yeast are the predominantly used. However, microbial expression systems often require optimization for any given effector and are, in general, not suitable for effectors involving cysteine bridges and posttranslational modifications for activity. Here, we describe a simple and efficient method for expressing such effectors in the model plant Nicotiana benthamiana . Briefly, an effector protein is transiently expressed and secreted into the apoplast of N. benthamiana by Agrobacterium‐mediated infiltration. Two to three days subsequent to agroinfiltration, the apoplast from the infiltrated leaves is extracted and can be directly used for phenotyping on host plants. The efficacy of this approach was demonstrated by expressing the ToxA, Tox3, and Tox1 necrosis‐inducing effectors from Parastagonospora nodorum . All three effectors produced in N. benthamiana were capable of inducing necrosis in wheat lines, and two of three showed visible bands on Coomassie‐stained gel. These data suggest that N. benthamiana –agroinfiltration system is a feasible tool to obtain fungal effectors, especially those that require disulfide bonds and posttranslational modifications. Furthermore, due to the low number of proteins typically observed in the apoplast (compared with intracellular), this simple and high‐throughput approach circumvents the requirement to lyse cells and further purifies the target proteins that are required in other heterologous systems. Because of its simplicity and potential for high‐throughput, this method is highly amenable to the phenotyping of candidate protein effectors on host plants.

The transcription factor stua regulates central carbon metabolism, mycotoxin production, and effector gene expression in the wheat pathogen Stagonospora nodorum

Publisher: American Society for Microbiology

Date: 07-2010

DOI: 10.1128/EC.00064-10

Abstract: The Stagonospora nodorum StuA transcription factor gene SnStuA was identified by homology searching in the genome of the wheat pathogen Stagonospora nodorum . Gene expression analysis revealed that SnStuA transcript abundance increased throughout infection and in vitro growth to peak during sporulation. To investigate its role, the gene was deleted by homologous recombination. The growth of the resulting mutants was retarded on glucose compared to the wild-type growth, and the mutants also failed to sporulate. Glutamate as a sole carbon source restored the growth rate defect observed on glucose, although sporulation remained impaired. The SnstuA strains were essentially nonpathogenic, with only minor growth observed around the point of inoculation. The role of SnstuA was investigated using metabolomics, which revealed that this gene's product played a key role in regulating central carbon metabolism, with glycolysis, the TCA cycle, and amino acid synthesis all affected in the mutants. SnStuA was also found to positively regulate the synthesis of the mycotoxin alternariol. Gene expression studies on the recently identified effectors in Stagonospora nodorum found that SnStuA was a positive regulator of SnTox3 but was not required for the expression of ToxA . This study has uncovered a multitude of novel regulatory targets of SnStuA and has highlighted the critical role of this gene product in the pathogenicity of Stagonospora nodorum .

A signaling-regulated, short-chain dehydrogenase of Stagonospora nodorum regulates asexual development

Publisher: American Society for Microbiology

Date: 11-2008

DOI: 10.1128/EC.00237-08

Abstract: The fungus Stagonospora nodorum is a causal agent of leaf and glume blotch disease of wheat. It has been previously shown that inactivation of heterotrimeric G protein signaling in Stagonospora nodorum caused development defects and reduced pathogenicity [P. S. Solomon et al., Mol. Plant-Microbe Interact. 17:456-466, 2004]. In this study, we sought to identify targets of the signaling pathway that may have contributed to phenotypic defects of the signaling mutants. A comparative analysis of Stagonospora nodorum wild-type and Gα-defective mutant ( gna1 ) intracellular proteomes was performed via two-dimensional polyacrylamide gel electrophoresis. Several proteins showed significantly altered abundances when comparing the two strains. One such protein, the short-chain dehydrogenase Sch1, was 18-fold less abundant in the gna1 strain, implying that it is positively regulated by Gα signaling. Gene expression and transcriptional enhanced green fluorescent protein fusion analyses of Sch1 indicates strong expression during asexual development. Mutant strains of Stagonospora nodorum lacking Sch1 demonstrated poor growth on minimal media and exhibited a significant reduction in asexual sporulation on all growth media examined. Detailed histological experiments on sch1 pycnidia revealed that the gene is required for the differentiation of the subparietal layers of asexual pycnidia resulting in a significant reduction in both pycnidiospore size and numbers.

Impaired nutrient signaling and body weight control in a Na ⁺ neutral amino acid cotransporter (Slc6a19)-deficient mouse

Publisher: Elsevier BV

Date: 07-2011

DOI: 10.1074/JBC.M111.241323

Normalization of metabolites in heterogenous systems using genomics

Publisher: Elsevier BV

Date: 03-2006

DOI: 10.1016/J.AB.2005.11.044

The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro‐domain protease processin

Publisher: Wiley

Date: 24-06-2021

DOI: 10.1111/NPH.17516

Abstract: Plant pathogens cause disease through secreted effector proteins, which act to promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilized a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum , which causes cell death in wheat‐lines carrying the sensitivity gene Snn3 . We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination and functional studies. We show this approach can be successfully applied to study effectors from other pathogenic fungi. The β‐barrel fold of SnTox3 is a novel fold among fungal effectors. Structure‐guided mutagenesis enabled the identification of residues required for Snn3 recognition. SnTox3 is a pre‐pro‐protein, and the pro‐domain of SnTox3 can be cleaved in vitro by the protease Kex2. Complementing this, an in silico study uncovered the prevalence of a conserved motif (LxxR) in an expanded set of putative pro‐domain‐containing fungal effectors, some of which can be cleaved by Kex2 in vitro . Our in vitro and in silico study suggests that Kex2‐processed pro‐domain (designated here as K2PP) effectors are common in fungi and this may have broad implications for the approaches used to study their functions.

A functional genomics approach to dissect the mode of action of the Stagonospora nodorum effector protein SnToxA in wheat

Publisher: Wiley

Date: 24-11-2012

DOI: 10.1111/J.1364-3703.2011.00763.X

Proteinaceous necrotrophic effectors in fungal virulence

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/FP10067

Abstract: The host–pathogen interface can be considered as a biological battlefront. Molecules produced by both the pathogen and the host are critical factors determining the outcome of the interaction. Recent studies have revealed that an increasing number of necrotrophic fungal pathogens produce small proteinaceous effectors that are able to function as virulence factors. These molecules can cause tissue death in host plants that possess dominant sensitivity genes, leading to subsequent pathogen colonisation. Such effectors are only found in necrotrophic fungi, yet their roles in virulence are poorly understood. However, several recent key studies of necrotrophic effectors from two wheat (Triticum aestivum L.) pathogens, Pyrenophora tritici-repentis (Died.) Drechs. and Stagonospora nodorum (Berk.) Castell. & Germano, have shed light upon how these effector proteins serve to disable the host from the inside out.

University Of Queensland

Location: Australia

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Australian National University

Location: Australia

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The Identification Of Mycosphaerella Graminicola Effectors That Promote Pathogenicity On Wheat

Start Date: 2011

End Date: 2015

Funder: Australian Research Council

Proteome Mapping Of The Model Fungal Plant Pathogen Stagonospora Nodorum Using LC-LC-MS/MS

Start Date: 2008

End Date: 2011

Funder: Australian Research Council

Investigating A New Paradigm For Plant-pathogen Interactions; Identification Of Host-selective Toxin Proteins In The Wheat Pathogen Stagonospora Nodorum

Start Date: 2009

End Date: 2011

Funder: Australian Research Council

Phytosphere: New Facilities For Controlled Manipulation Of Effects Of Climate Change & Airborne Pollutants On Disease Epidemiology & Plant Performance

Start Date: 2009

End Date: 2009

Funder: Australian Research Council

The Functional Characterisation Of A Novel Immune Response In Plants

Start Date: 2018

End Date: 2020

Funder: Australian Research Council

Functional Characterisation Of The Necrotrophic Effector Proteins Tox1 And Tox3 From The Wheat Pathogen Stagonospora Nodorum

Start Date: 2012

End Date: 2014

Funder: Australian Research Council

The Development Of Mass Spectrometry Techniques For Mapping Post-translational Modifications In The Wheat Pathogen Stagonospora Nodorum

Start Date: 2011

End Date: 2013

Funder: Australian Research Council

Biosynthesis And Functions Of Two Phytotoxins In Septoria Nodorum Blotch

Start Date: 2017

End Date: 2019

Funder: Australian Research Council

Multiphoton Confocal Microscope

Start Date: 2010

End Date: 2010

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120103558

Start Date: 2012

End Date: 06-2015

Amount: $350,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989071

Start Date: 01-2009

End Date: 07-2012

Amount: $600,000.00

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT110100698

Start Date: 05-2012

End Date: 12-2016

Amount: $803,186.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180102355

Start Date: 02-2018

End Date: 07-2022

Amount: $363,164.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP110100690

Start Date: 12-2011

End Date: 12-2017

Amount: $90,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0986139

Start Date: 2009

End Date: 12-2011

Amount: $230,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200101880

Start Date: 04-2020

End Date: 04-2023

Amount: $569,499.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100078

Start Date: 2010

End Date: 12-2010

Amount: $600,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0883556

Start Date: 03-2009

End Date: 03-2012

Amount: $76,881.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP170100228

Start Date: 2017

End Date: 12-2019

Amount: $428,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC230100027

Start Date: 2023

End Date: 12-2027

Amount: $5,000,000.00

Funder: Australian Research Council