Genome-Wide Identification and Functions against Tomato Spotted Wilt Tospovirus of PR-10 in Solanum lycopersicum.

Tomato spotted wilt virus impacts negatively on a wide range of economically important plants, especially tomatoes. When plants facing any pathogen attack or infection, increase the transcription level of plant genes that are produced pathogenesis-related (PR) proteins. The aim of this study is a genome-wide identification of PR-10 superfamily and comparative analysis of PR-10 and Sw-5b gene functions against tomato responses to biotic stress (TSWV) to systemic resistance in tomato. Forty-five candidate genes were identified, with a length of 64-210 amino acid residues and a molecular weight of 7.6-24.4 kDa. The PR-10 gene was found on ten of the twelve chromosomes, and it was determined through a genetic ontology that they were involved in six biological processes and molecular activities, and nine cellular components. Analysis of the transcription level of PR-10 family members showed that the PR-10 gene (Solyc09g090980) has high expression levels in some parts of the tomato plant. PR-10 and Sw-5b gene transcription and activity in tomato leaves were strongly induced by TSWV infection, whereas H8 plants having the highest significantly upregulated expression of PR-10 and Sw-5b gene after the inoculation of TSWV, and TSWV inoculated in M82 plants showed significantly upregulated expression of PR-10 gene comparatively lower than H8 plants. There was no significant expression of Sw-5b gene of TSWV inoculated in M82 plants and then showed highly significant correlations between PR-10 and Sw-5b genes at different time points in H8 plants showed significant correlations compared to M82 plants after the inoculation of TSWV; a heat map showed that these two genes may also participate in regulating the defense response after the inoculation of TSWV in tomato.

Tomato zonate spot virus induced hypersensitive resistance via an auxin-related pathway in pepper.

Tomato zonate spotvirus (TZSV) often incurs significant losses in many food and ornamental crops in Yunnan province, China, and the surrounding areas. The pepper (Capsicum chinensePI152225)can develop hypersensitive resistance following infection with TZSV, through an as yet unknown mechanism. The transcriptome dataset showed a total of 45.81 GB of clean data were obtained from six libraries, and the average percentage of the reads mapped to the pepper genome was over 90.00 %. A total of 1403 differentially expressed genes (DEGs) were obtained after TZSV infection, including 825significantly up-regulated genes and 578 down-regulated genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that most up-regulated DEGs were involved in basal defenses. RT-qPCR, and virus induced gene silencing (VIGS) were used preliminarily to identifyBBC_22506 and BBC_18917, among total of 71 differentially expressed genes (DEGs), that play a key role in mediating the auxin-induced signaling pathway that might take part in hypersensitive response (HR) conferred resistance to viral infection in pepper (PI152225) byTZSV. This is the first study on the mechanism of auxin resistance, involved in defense responses of pepper against viral diseases, which lay the foundation for further study on the pathogenic mechanism of TZSV, as well as the mechanism of resistance to TZSV, in peppers.

Defense-Related Gene Expression Following an Orthotospovirus Infection Is Influenced by Host Resistance in Arachis hypogaea.

Planting resistant cultivars is the most effective tactic to manage the thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) in peanut plants. However, molecular mechanisms conferring resistance to TSWV in resistant cultivars are unknown. In this study, transcriptomes of TSWV-susceptible (SunOleic 97R) and field-resistant (Tifguard) peanut cultivars with and without TSWV infection were assembled and differentially expressed genes (DEGs) were compared. There were 4605 and 2579 significant DEGs in SunOleic 97R and Tifguard, respectively. Despite the lower number of DEGs in Tifguard, an increased proportion of defense-related genes were upregulated in Tifguard than in the susceptible cultivar. Examples included disease resistance (R) proteins, leucine-rich repeats, stilbene synthase, dicer, and calmodulin. Pathway analysis revealed the increased downregulation of genes associated with defense and photosynthesis in the susceptible cultivar rather than in the resistant cultivar. These results suggest that essential physiological functions were less perturbed in the resistant cultivar than in the susceptible cultivar and that the defense response following TSWV infection was more robust in the resistant cultivar than in the susceptible cultivar.

Auxin drives tomato spotted wilt virus (TSWV) resistance through epigenetic regulation of auxin response factor ARF8 expression in tomato.

Tomato spotted wilt virus (TSWV) causes severe losses of tomato crops worldwide. To cope dynamically with such a threat, plants deploy strategies acting at the molecular and the epigenetic levels. We found that tomato symptoms progress in a specific-genotype-manner upon TSWV infection. Susceptible genotypes showed within the Auxin Response Factor (ARF8) promoter coupled to enhanced expression of miRNA167a, reduced ARF8 gene and decreased levels of the hormone auxin. This constitutes a deliberate attempt of TSWV to disrupt plant growth to promote spread in sensitive cultivars. Epigenetic regulation through the level of cytosine methylation and the miR167a-ARF8 module are part of a complex network modulating auxin-triggered synthesis and shaping tomato responses to TSWV. Furthermore, modulation of miR167a-ARF8 regulatory module could be applied in tomato-resistance breeding programs.

NbTMP14 Is Involved in Tomato Spotted Wilt Virus Infection and Symptom Development by Interaction with the Viral NSm Protein.

Tomato spotted wilt virus (TSWV) is one of the most destructive plant viruses, causing severe losses in many important crops worldwide. The non-structural protein NSm of TSWV is a viral movement protein that induces viral symptoms. However, the molecular mechanisms by which NSm contributes to symptom development are unclear. Here, we present evidence that NSm directly interacts with Nicotiana benthamiana chloroplast thylakoid membrane protein TMP14 (NbTMP14) by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The interaction between NSm and NbTMP14 led to the translocation of the NbTMP14 protein from the chloroplast to the cytoplasm in TSWV-infected plants, and overexpressing NSm decreased NbTMP14 mRNA accumulation. In addition, abnormal chloroplasts and starch accumulation were observed in TSWV-infected plants. Silencing of NbTMP14 by TRV VIGS also showed similar results to those of TSWV-infected plants. Overexpressing NbTMP14 in transgenic N. benthamiana plants impeded TSWV infection, and silencing NbTMP14 in N. benthamiana plants increased disease symptom severity and virus accumulation. To our knowledge, this is the first report showing that the plant chloroplast TMP14 protein is involved in viral infection. Knowledge of the interaction between NSm and NbTMP14 advances our understanding of the molecular mechanisms underlying TSWV symptom development and infection.

Co-existence of chlorosis inducing strain of Cucumber mosaic virus with tospoviruses on hot pepper (Capsicum annuum) in India.

Cucumo- and tospoviruses are the most destructive viruses infecting hot pepper (chilli). A diagnostic survey was conducted to assess the prevalence of cucumo and tospoviruses in chilli growing tracts of Tamil Nadu. Infected plants showing mosaic with chlorotic and necrotic rings, veinal necrosis, mosaic mottling, leaf filiformity and malformation were collected. Molecular indexing carried out through reverse transcription polymerase chain reaction (RT-PCR) with coat protein gene specific primer of Cucumber mosaic virus (CMV) and tospovirus degenerate primer corresponding to the L segment (RdRp). Ostensibly, amplifications were observed for both CMV and tospoviruses as sole as well for mixed infections. The sequence analysis indicated that the Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) to be involved with CMV in causing combined infections. The co-infection of CMV with CaCV was detected in 10.41% of the symptomatic plant samples and combined infection of CMV with GBNV was recorded in around 6.25% of the symptomatic plants surveyed. The amino acid substitution of Ser129 over conserved Pro129 in coat protein of CMV implies that CMV strain involved in mixed infection as chlorosis inducing strain. Further, the electron microscopy of symptomatic plant samples explicated the presence of isometric particles of CMV and quasi spherical particles of tospoviruses. This is the first molecular evidence for the natural co-existence of chlorosis inducing CMV strain with CaCV and GBNV on hot pepper in India.

Induction of Plant Resistance in Tobacco (Nicotiana tabacum) against Tomato Spotted Wilt Orthotospovirus through Foliar Application of dsRNA.

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) continues to be a constraint to peanut, pepper, tobacco, and tomato production in Georgia and elsewhere. TSWV is being managed by an integrated disease management strategy that includes a combination of cultural practices, vector management, and growing virus-resistant varieties where available. We used a non-transgenic strategy to induce RNA interference (RNAi)-mediated resistance in tobacco (Nicotiana tabacum) plants against TSWV. Double-stranded RNA (dsRNA) molecules for the NSs (silencing suppressor) and N (nucleoprotein) genes were produced by a two-step PCR approach followed by in vitro transcription. When topically applied to tobacco leaves, both molecules elicited a resistance response. Host response to the treatments was measured by determining the time to symptom expression, and the level of resistance by absolute quantification of the virus. We also show the systemic movement of dsRNA_N from the inoculated leaves to younger, non-inoculated leaves. Post-application, viral siRNAs were detected for up to nine days in inoculated leaves and up to six days in non-inoculated leaves. The topical application of dsRNAs to induce RNAi represents an environmentally safe and efficient way to manage TSWV in tobacco crops and could be applicable to other TSWV-susceptible crops.

A step towards Balkan Capsicum annuum L. core collection: Phenotypic and biochemical characterization of 180 accessions for agronomic, fruit quality, and virus resistance traits.

Region-specific local landraces represent a germplasm diversity adapted and acclimatized to local conditions, and are ideal to breed for targeted market niches while maintaining the variability of heirloom traits. A collection of 180 pepper accessions, collected from 62 diverse locations across six Balkan countries, were characterized and evaluated for phenotypic and biochemical variation during a multi-year environment. An assortment of 32 agro-morphological, fruit quality, and virus resistance traits were evaluated, and the top 10% accessions were identified. A wide range of trait variation concerning plant architecture, inflorescence and fruit traits, yield and fruit quality was observed, and appreciable variation was noticed. According to hierarchical clustering, six distinct clusters were established based on pre-defined varietal groups. Divergence among accessions for phenotypic and fruit compositional variability was analyzed, and eight principal components were identified that contributed ~71% of the variation, with fruit shape, width, wall thickness, weight, and fruit quality traits being the most discriminant. Evaluation of the response to tobacco mosaic virus (TMV) and pepper mild mottle mosaic virus (PMMoV) showed that 24 and 1 accession were resistant, respectively while no tomato spotted wilt virus (TSWV) resistance was found. Considerable diversity for agro-bio-morphological traits indicates the Balkan pepper collection as good gene sources for pre-breeding and cultivar development that are locally adapted.

Grafting alters tomato transcriptome and enhances tolerance to an airborne virus infection.

Grafting of commercial tomato varieties and hybrids on the tomato ecotype Manduria resulted in high levels of tolerance to the infection of Sw5 resistance-breaking strains of tomato spotted wilt virus and of severe cucumber mosaic virus strains supporting hypervirulent satellite RNAs that co-determine stunting and necrotic phenotypes in tomato. To decipher the basis of such tolerance, here we used a RNAseq analysis to study the transcriptome profiles of the Manduria ecotype and of the susceptible variety UC82, and of their graft combinations, exposed or not to infection of the potato virus Y recombinant strain PVYC-to. The analysis identified graft- and virus-responsive mRNAs differentially expressed in UC82 and Manduria, which led to an overall suitable level of tolerance to viral infection confirmed by the appearance of a recovery phenotype in Manduria and in all graft combinations. The transcriptome analysis suggested that graft wounding and viral infection had diverging effects on tomato transcriptome and that the Manduria ecotype was less responsive than the UC82 to both graft wounding and potyviral infection. We propose that the differential response to the two types of stress could account for the tolerance to viral infection observed in the Manduria ecotype as well as in the susceptible tomato variety UC82 self-grafted or grafted on the Manduria ecotype.

Changes in the GN/GCof the M segment show positive selection and recombination of one aggressive isolate and two mild isolates of tomato spotted wilt virus.

We isolated and compared three tomato spotted wilt virus (TSWV) isolates from lettuce (TSWV-Let), pepper (TSWV-Pep), and tomato (TSWV-Tom) from central Mexico to determine their ability to infect a set of eighteen differential plant species from seven families. TWSV-Let was an aggressive isolate with the ability to infect up to 52% of the differential plants, including maize, under greenhouse conditions. The nucleotide (nt) sequences of the three isolates are more than 90% similar in the M and S RNA segments. In the M segment of the TSWV-Let isolate, we detected nt changes in their intergenic region (IGR) and, in the Gc gene, a region containing a recombination site, as well as a synapomorphy associated with one of three sites under positive selection with a change in one aa residue (a cysteine-to-valine mutation). We speculate on the association of these features in the Gc gene with host selection, adaptation, aggressiveness, and ability to infect maize plants.

Rescue of tomato spotted wilt virus entirely from complementary DNA clones.

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.

A thrips vector of tomato spotted wilt virus responds to tomato acylsugar chemical diversity with reduced oviposition and virus inoculation.

There is increasing evidence that acylsugars deter insect pests and plant virus vectors, including the western flower thrips (WFT), Frankliniella occidentalis (Pergande), vector of tomato spotted wilt virus (TSWV). Acylsugars are sugar-polyesters composed of saturated, un-saturated, and variously branched short and long chain fatty acids (FAs) esterified to a glucose (acylglucose) or sucrose (acylsucrose) moiety. We sought to understand how acylsucrose amount and composition of associated FA profiles interacted to mediate resistance to WFT oviposition and TSWV inoculation on tomato leaves. Towards this goal, we examined WFT oviposition and TSWV inoculation behavior on tomato lines bred to exude varying amounts of acylsucrose in association with diverse FA profiles. Our data show that as acylsucrose amounts increased, WFT egg-laying (oviposition) decreased and TSWV inoculation was suppressed. Western flower thrips also responded to FA profiles that included iC4, iC11, nC12 and nC10 FA. These findings support improving acylsugar-mediated resistance against WFT by breeding tomatoes exuding greater amounts of acylsucrose associated with specific FA profiles. We show that increasing acylsucrose amount output by type IV trichomes and selecting for particular FA profiles through advanced breeding profoundly affects WFT behavior in ways that benefit management of WFT as direct pests and as TSWV vectors.

The XIth International Symposium on Thysanoptera and Tospoviruses Co-sponsored by Yunnan Academy of Agricultural Sciences and Nanjing Agricultural University in Kunming, China 2019.

The XIth International Symposium on Thysanoptera and Tospoviruses co-hosted by the Yunnan Academy of Agricultural Sciences, and Nanjing Agricultural University was held from September 21-25 in Kunming, China (Figure 1) [...].

Tomato glycosyltransferase Twi1 plays a role in flavonoid glycosylation and defence against virus.

BACKGROUND: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. RESULTS: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1-silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. CONCLUSIONS: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection.

Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance.

RNA interference (RNAi)-based tools are used in multiple organisms to induce antiviral resistance through the sequence-specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi-based tools include artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). syn-tasiRNAs have emerged as a promising antiviral tool allowing for the multi-targeting of viral RNAs through the simultaneous expression of several syn-tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn-tasiRNA construct expressing four different syn-tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn-tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn-tasiRNA lines was not exclusive of lines with high syn-tasiRNA accumulation. Collectively, these results suggest that syn-tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn-tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn-tasiRNA.

Comprehensive transcriptome analysis and functional characterization of PR-5 for its involvement in tomato Sw-7 resistance to tomato spotted wilt tospovirus.

Tomato spotted wilt tospovirus (TSWV), one of the most important plant viruses, causes yield losses to many crops including tomato. The current disease management for TSWV is based mainly on breeding tomato cultivars containing the Sw-5 locus. Unfortunately, several Sw-5 resistance-breaking strains of TSWV have been identified. Sw-7 is an alternative locus conferring resistance to a broad range of TSWV strains. In an effort to uncover gene networks that are associated with the Sw-7 resistance, we performed a comparative transcriptome profiling and gene expression analysis between a nearly-isogenic Sw-7 line and its susceptible recurrent parent (Fla. 8059) upon infection by TSWV. A total of 1,244 differentially expressed genes were identified throughout a disease progression process involving networks of host resistance genes, RNA silencing/antiviral defense genes, and crucial transcriptional and translational regulators. Notable induced genes in Sw-7 include those involved in callose accumulation, lignin deposition, proteolysis process, transcriptional activation/repression, and phosphorylation. Finally, we investigated potential involvement of PR-5 in the Sw-7 resistance. Interestingly, PR-5 overexpressed plants conferred enhanced resistance, resulting in delay in virus accumulation and symptom expression. These findings will facilitate breeding and genetic engineering efforts to incorporate this new source of resistance in tomato for protection against TSWV.

Iris yellow spot virus-induced chloroplast malformation results in male sterility.

Iris yellow spot virus (IYSV) is one of the most devastating viral pathogens, which causes high economic losses in the onion yield. Physiological and genetic changes are associated with the appearance of chlorotic symptom in the infected plants. IYSV-N gene sequence analysis revealed that it shared sequence identity of 99% with other Egyptian isolates, at both genomic and proteomic levels. In addition, N protein sequence with computational examination indicated many motifs involved and played different roles in the virus activity and its regulation and stability were detected. In the Differential Display-Polymerase Chain Reaction (DD-PCR) study, a highly up-regulated gene at 15 days post-biological IYSV inoculation (dpi) was selected for sequencing. Based on the sequencing results that showed the identified gene was coding for a chloroplast-related gene, degenerate specific primers were designed for Real-Time PCR analysis. A significant change in the transcription level of the chloroplast-related gene after 15 dpi suggested that some IYSV proteins interact and/or regulate with chloroplast proteins and this finding supports the DD-PCR results. At 20 dpi, the ultrathin sections showed that IYSV infection caused many dramatic chloroplasts malformations. The malformation appeared as chloroplast broken envelope with the presence of numerous spherical particles inside it and chloroplasts with long stromule. Our findings indicated that IYSV interrupts normal chloroplast functions, as a part of the onion defence response, however many crucial factors remain to be elucidated and further studies are needed at both biological and molecular levels.

Integrating transcriptome and microRNA analysis identifies genes and microRNAs for AHO-induced systemic acquired resistance in N. tabacum.

3-Acetonyl-3-hydroxyoxindole (AHO) induces systemic acquired resistance (SAR) in Nicotiana. However, the underlying molecular mechanism is not well understood. To understand the molecular regulation during SAR induction, we examined mRNA levels, microRNA (miRNA) expression, and their regulatory mechanisms in control and AHO-treated tobacco leaves. Using RNA-seq analysis, we identified 1,445 significantly differentially expressed genes (DEGs) at least 2 folds with AHO treatment. The DEGs significantly enriched in six metabolism pathways including phenylpropanoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis for protective cuticle and wax. Key DEGs including PALs and PR-10 in salicylic acid pathway involved in SAR were significantly regulated. In addition, we identified 403 miRNAs belonging to 200 miRNA families by miRNA sequencing. In total, AHO treatment led to 17 up- and 6 down-regulated at least 2 folds (Wald test, P < 0.05) miRNAs (DEMs), respectively. Targeting analysis implicated four DEMs regulating three DEGs involved in disease resistance, including miR156, miR172f, miR172g, miR408a, SPL6 and AP2. We concluded that both mRNA and miRNA regulation enhances AHO-induced SAR. These data regarding DEGs, miRNAs, and their regulatory mechanisms provide molecular evidence for the mechanisms involved in tobacco SAR, which are likely to be present in other plants.

A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast.

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus of the order Bunyavirales, family Tospoviridae, genus Orthotospovirus. TSWV infects a broad range of plant species, causing serious economic losses. Despite its agronomic importance, molecular biological understanding of TSWV has been limited, partly due to the lack of a reverse genetics system, which would enable genetic manipulation of the virus. Here, we report that RNA synthesis by TSWV RNA polymerase occurs in the yeast Saccharomyces cerevisiae using a segment of the TSWV genome, S RNA expressed from cloned cDNA, as a template. Viral nucleocapsid protein was required for RNA synthesis. Replacement of the protein-coding and intergenic regions of TSWV S RNA by a yellow fluorescent protein (YFP)-coding sequence drastically increased the accumulation of both sense and antisense strands of the RNA, showing that this RNA was replicated. Using this system, we revealed that efficient RNA synthesis by TSWV RNA polymerase in yeast requires the 5'-terminal 17-nt and 3'-terminal ~50-nt regions of the TSWV S cRNA (complementary RNA to the genomic RNA) template.

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper.

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.