ORCID Profile
0000-0001-7997-7794
Current Organisation
Washington State University
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Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.VIRUSRES.2015.10.003
Abstract: Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of a wide range of field and horticultural crops. We developed an artificial microRNA (amiRNA) strategy against TSWV, targeting the nucleoprotein (N) and silencing suppressor (NSs) genes. The amiRNA constructs replaced the natural miRNA in a shortened Arabidopsis 173-nucleotide (nt) miR159a precursor backbone (athmiR159a) without the stem base extending beyond the miR/miR* duplex. Further, each amiRNA was modified to contain a mismatch (wobble) sequence at nucleotide position 12 and 13 on the complementary strand amiRNA*, mimicking the endogenous miR159a sequence structure. Transient expression in Nicotiana benthamiana demonstrated that the introduction of a wobble sequence did not alter amiRNA expression levels. Following challenge inoculation with TSWV, plants expressing N-specific amiRNAs with or without the wobble remained asymptomatic and were negative for TSWV by ELISA. In contrast, plants expressing the NSs-specific amiRNAs were symptomatic and accumulated high levels of TSWV. Similar findings were obtained in stably transformed Nicotiana tabacum plants. Our results show that a shortened 173-nt athmiR159a backbone is sufficient to express amiRNAs and that the presence of mismatch at position 12-13 does not influence amiRNA expression or conferring of resistance. We also show that selection of target gene and positional effect are critical in amiRNA-based approach for introducing resistance. These findings open the possibility of employing the amiRNA approach for broad-spectrum resistance to tospoviruses as well as other viruses.
Publisher: MDPI AG
Date: 26-03-2020
DOI: 10.3390/V12040363
Abstract: Tospoviruses cause significant losses to a wide range of agronomic and horticultural crops worldwide. The type member, Tomato spotted wilt tospovirus (TSWV), causes systemic infection in susceptible tomato cultivars, whereas its infection is localized in cultivars carrying the Sw-5 resistance gene. The response to TSWV infection in tomato cultivars with or without Sw-5 was determined at the virus small RNA level in the locally infected leaf. Predicted reads were aligned to TSWV reference sequences. The TSWV genome was found to be differentially processed among each of the three-viral genomic RNAs—Large (L), Medium (M) and Small (S)—in the Sw-5(+) compared to Sw-5(−) genotypes. In the Sw-5(+) cultivar, the L RNA had the highest number of viral small-interfering RNAs (vsiRNAs), whereas in the Sw-5(−) cultivar the number was higher in the S RNA. Among the three-viral genomic RNAs, the distribution of hotspots showed a higher number of reads per million reads of vsiRNAs of 21 and 22 nt class at the 5′ and 3′ ends of the L and the S RNAs, with less coverage in the M RNA. In the Sw-5(−) cultivar, the nature of the 5′ nucleotide-end in the siRNAs varied significantly reads with 5′-adenine-end were most abundant in the mock control, whereas cytosine and uracil were more abundant in the infected plants. No such differences were seen in case of the resistant genotype. Findings provided insights into the response of tomato cultivars to TSWV infection.
Publisher: Springer Science and Business Media LLC
Date: 22-12-2014
DOI: 10.1007/S00705-013-1928-8
Abstract: Tospoviruses cause serious economic losses to a wide range of field and horticultural crops on a global scale. The NSs gene encoded by tospoviruses acts as a suppressor of host plant defense. We identified amino acid motifs that are conserved in all of the NSs proteins of tospoviruses for which the sequence is known. Using tomato spotted wilt virus (TSWV) as a model, the role of these motifs in suppressor activity of NSs was investigated. Using site-directed point mutations in two conserved motifs, glycine, lysine and valine/threonine (GKV/T) at positions 181-183 and tyrosine and leucine (YL) at positions 412-413, and an assay to measure the reversal of gene silencing in Nicotiana benthamiana line 16c, we show that substitutions (K182 to A, and L413 to A) in these motifs abolished suppressor activity of the NSs protein, indicating that these two motifs are essential for the RNAi suppressor function of tospoviruses.
No related grants have been discovered for Ying Zhai.