Development and Application of Attenuated Plant Viruses as Biological Control Agents in Japan.

In 1929, it was reported that yellowing symptoms caused by a tobacco mosaic virus (TMV) yellow mosaic isolate were suppressed in tobacco plants that were systemically infected with a TMV light green isolate. Similar to vaccination, the phenomenon of cross-protection involves a whole plant being infected with an attenuated virus and involves the same or a closely related virus species. Therefore, attenuated viruses function as biological control agents. In Japan, many studies have been performed on cross-protection. For example, the tomato mosaic virus (ToMV)-L11A strain is an attenuated isolate developed by researchers and shows high control efficiency against wild-type ToMV in commercial tomato crops. Recently, an attenuated isolate of zucchini yellow mosaic virus (ZYMV)-2002 was developed and registered as a biological pesticide to control cucumber mosaic disease. In addition, attenuated isolates of pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tobacco mild green mosaic virus (TMGMV), melon yellow spot virus (MYSV), and watermelon mosaic virus (WMV) have been developed in Japan. These attenuated viruses, sometimes called plant vaccines, can be used not only as single vaccines but also as multiple vaccines. In this review, we provide an overview of studies on attenuated plant viruses developed in Japan. We also discuss the application of the attenuated strains, including the production of vaccinated seedlings.

Antiviral Mechanisms of Anisomycin Produced by Streptomyces albulus SN40 on Potato Virus Y.

Microbial secondary metabolites produced by Streptomyces have diverse application prospects in the control of plant diseases. Herein, the fermentation filtrate of Streptomyces SN40 effectively inhibited the infection of tobacco mosaic virus (TMV) in Nicotiana glutinosa and systemic infection of potato virus Y (PVY) in Nicotiana benthamiana. Additionally, metabolomic analysis indicated that anisomycin (C14H19NO4) and trans-3-indoleacrylic acid (C11H9NO2) were highly abundant in the crude extract and that anisomycin effectively suppressed the infection of TMV as well as PVY. Subsequently, transcriptomic analysis was conducted to elucidate its mechanisms on the induction of host defense responses. Furthermore, the results of molecular docking suggested that anisomycin can potentially bind with the helicase domain (Hel) of TMV replicase, TMV coat protein (CP), and PVY helper component proteinase (HC-Pro). This study demonstrates new functions of anisomycin in virus inhibition and provides important theoretical significance for the development of new biological pesticides to control diverse plant viruses.

TOM1 family conservation within the plant kingdom for tobacco mosaic virus accumulation.

The susceptibility factor TOBAMOVIRUS MULTIPLICATION 1 (TOM1) is required for efficient multiplication of tobacco mosaic virus (TMV). Although some phylogenetic and functional analyses of the TOM1 family members have been conducted, a comprehensive analysis of the TOM1 homologues based on phylogeny from the most ancient to the youngest representatives within the plant kingdom, analysis of support for tobamovirus accumulation and interaction with other host and viral proteins has not been reported. In this study, using Nicotiana benthamiana and TMV as a model system, we functionally characterized the TOM1 homologues from N. benthamiana and other plant species from different plant lineages. We modified a multiplex genome editing tool and generated a sextuple mutant in which TMV multiplication was dramatically inhibited. We showed that TOM1 homologues from N. benthamiana exhibited variable capacities to support TMV multiplication. Evolutionary analysis revealed that the TOM1 family is restricted to the plant kingdom and probably originated in the Chlorophyta division, suggesting an ancient origin of the TOM1 family. We found that the TOM1 family acquired the ability to promote TMV multiplication after the divergence of moss and spikemoss. Moreover, the capacity of TOM1 orthologues from different plant species to promote TMV multiplication and the interactions between TOM1 and TOM2A and between TOM1 and TMV-encoded replication proteins are highly conserved, suggesting a conserved nature of the TOM2A-TOM1-TMV Hel module in promoting TMV multiplication. Our study not only revealed a conserved nature of a gene module to promote tobamovirus multiplication, but also provides a valuable strategy for TMV-resistant crop development.

Nanoparticle-dsRNA Treatment of Pollen and Root Systems of Diseased Plants Effectively Reduces the Rate of Tobacco Mosaic Virus in Contemporary Seeds.

Most crop viruses are carried and spread by seeds. Virus-infected seeds are seed-borne viral disease infections, and thus, reducing the rate of seed infection is an urgent problem in the seed-production industry. The objective of this study was to use nanoparticles (NPs) to directly deliver dsRNA into plants or pollen to initiate RNA interference (RNAi) to reduce viral carryover in seeds. Chitosan quaternary ammonium salt (HACC), complexed with dsRNAs, was selected for targeting the genes for the tobacco mosaic virus (TMV) coat protein (CP) and TMV RNA-dependent RNA polymerase (RdRP) to form HACC-dsRNA NPs. These NP-based dsRNAs were delivered to the plants using four different methods, including infiltration, spraying, root soaking, and pollen internalization. All four methods were able to reduce the seed-carrying rate of offspring seeds of the TMV-infected plants, with pollen internalization being the most effective in reducing the TMV-carrying rate from 95.1 to 61.1% in the control group. By measuring the plant uptake of fluorescence-labeled NPs and dsRNAs, the transportation of the HACC-dsRNA NPs into the plants was observed, and the uptake of dsRNA in combination with small RNA sequencing was further confirmed, resulting in the silencing of homologous RNA molecules during the topical application. The results demonstrated that the incidence of TMV infection was reduced by various degrees via RNAi induction without the need to develop transgenic plants. These results demonstrate the advantages of NP-based RNAi technology in breeding for disease resistance and developing a new strategy for virus-resistant breeding in plants.

Plant virus movement proteins originated from jelly-roll capsid proteins.

Numerous, diverse plant viruses encode movement proteins (MPs) that aid the virus movement through plasmodesmata, the plant intercellular channels. MPs are essential for virus spread and propagation in distal tissues, and several unrelated MPs have been identified. The 30K superfamily of MPs (named after the molecular mass of tobacco mosaic virus MP, the classical model of plant virology) is the largest and most diverse MP variety, represented in 16 virus families, but its evolutionary origin remained obscure. Here, we show that the core structural domain of the 30K MPs is homologous to the jelly-roll domain of the capsid proteins (CPs) of small RNA and DNA viruses, in particular, those infecting plants. The closest similarity was observed between the 30K MPs and the CPs of the viruses in the families Bromoviridae and Geminiviridae. We hypothesize that the MPs evolved via duplication or horizontal acquisition of the CP gene in a virus that infected an ancestor of vascular plants, followed by neofunctionalization of one of the paralogous CPs, potentially through the acquisition of unique N- and C-terminal regions. During the subsequent coevolution of viruses with diversifying vascular plants, the 30K MP genes underwent explosive horizontal spread among emergent RNA and DNA viruses, likely permitting viruses of insects and fungi that coinfected plants to expand their host ranges, molding the contemporary plant virome.

Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus.

BACKGROUND: Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. METHODS AND RESULTS: The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CONCLUSIONS: CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

The coat protein of tobacco mosaic virus as an anti-tobacco mosaic virus: a molecular dynamics simulation.

The Coat Protein (CP) of the Tobacco Mosaic Virus (TMV) executes an important duty in the protection of virus RNA. The interaction between the virus CP and host plant proteins induces infection in the host and creates dark and light green mosaics on crops, which disturb the growth and function of the plant. The interaction between the virus CP and the modified CP, expressed in transgenic plants, causes Coat Protein-Mediated Resistance (CP-MR), which reduces virus infection in transgenic plants. In this study, a model is suggested for resistance as "stop assembly of CP" in the virus. It is based on the fact that the CP, when mutated, acts as a dead-end in virus assembly. For evaluation of the model, we investigated the effect of four mutants including CBT28I, ABT42W, ABD77R, and ABT89W complexes on plant resistance against TMV infection by molecular dynamics simulation. Previous studies had shown the influence of such mutations on the CP-MR. The MD results of in the present study further confirmed the mentioned effect and demonstrated how the mutations could be the cause of CP-MR. The results are calculated by the RMSD, Rg, H-bond, and g-MMPBSA scripts. The change in binding energy between two chains is consistent with CP-MR such that with increase in binding energy, the affinity between two chains was reduced and the CP-MR increased. Based on this model, it is possible to design mutants with a high level of efficiency.Communicated by Ramaswamy H. Sarma.

Tobamoviruses Show Broad Host Ranges and Little Genetic Diversity Among Four Habitat Types of a Heterogeneous Ecosystem.

Host ranges of plant viruses are poorly known, as studies have focused on pathogenic viruses in crops and adjacent wild plants. High-throughput sequencing (HTS) avoids the bias toward plant-virus interactions that result in disease. Here we study the host ranges of tobamoviruses, important pathogens of crops, using HTS analyses of an extensive sample of plant communities in four habitats of a heterogeneous ecosystem. Sequences of 17 virus operational taxonomic units (OTUs) matched references in the Tobamovirus genus, eight had narrow host ranges, and five had wide host ranges. Regardless of host range, the OTU hosts belonged to taxonomically distant families, suggesting no phylogenetic constraints in host use associated with virus adaptation, and that tobamoviruses may be host generalists. The OTUs identified as tobacco mild green mosaic virus (TMGMV), tobacco mosaic virus (TMV), pepper mild mottle virus, and Youcai mosaic virus had the largest realized host ranges that occurred across habitats and exhibited host use unrelated to the degree of human intervention. This result is at odds with assumptions that contact-transmitted viruses would be more abundant in crops than in wild plant communities and could be explained by effective seed-, contact-, or pollinator-mediated transmission or by survival in the soil. TMGMV and TMV had low genetic diversity that was not structured according to habitat or host plant taxonomy, which indicated that phenotypic plasticity allows virus genotypes to infect new hosts with no need for adaptive evolution. Our results underscore the relevance of ecological factors in host range evolution, in addition to the more often studied genetic factors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Potential risk of plant viruses entering disease cycle in surface water in protected vegetable growing areas of Eastern China.

With the expansion of protected vegetable growing areas (PVGAs), viral plant diseases have become more prevalent, causing severe economic losses to the vegetable production industry in China. At present, researches on plant viruses mainly focus on plants, but there is only a few reports on the species of viruses in surface water from PVGAs. The surface water samples in PVGAs are representative to a certain extent, which has an important reference value for studying the characteristics of plant viruses in surface water. The purpose of this study was to identify the diversity and the possibility of entering disease infection cycle of plant viruses in water samples collected from PVGAs in eastern China. A total of 144 water samples were collected, and eight plant viruses including tobacco mosaic virus (TMV, 8.33%), cucumber green mottle mosaic virus (CGMMV, 33.33%), pepper mild mottle virus (PMMoV, 6.94%), cucumber mosaic virus (CMV, 0.69%), tomato masaic virus (ToMV, 3.47%), tomato mottle mosaic virus (ToMMV, 0.69%), tomato chlorosis virus (ToCV, 4.17%), and tomato yellow leaf curl virus (TYLCV, 5.56%) were examined using RT-PCR and PCR. The species of viruses in surface water varied greatly by location. CGMMV, TMV, ToCV, ToMV, ToMMV, and TYLCV were identified in Shandong, a northern part of Eastern China, whereas only PMMoV was found in Shanghai, a southern part of Eastern China. After healthy tobacco plants were inoculated with the concentrated solutions of TMV, ToMV, CGMMV, and PMMoV, could cause disease in healthy tobacco, indicating that the plant viruses in the concentrated solution have the infectivity, and the plant viruses in surface water have the possibility of entering the infection cycle of disease. The results will improve the understanding of the potential risks of waterborne disease transmission.

Facilitating viral vector movement enhances heterologous protein production in an established plant system.

Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system-wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant-based expression system that utilizes viral-based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.

Convenient site-selective protein coupling from bacterial raw lysates to coenzyme A-modified tobacco mosaic virus (TMV) by Bacillus subtilis Sfp phosphopantetheinyl transferase.

A facile enzyme-mediated strategy enables site-specific covalent one-step coupling of genetically tagged luciferase molecules to coenzyme A-modified tobacco mosaic virus (TMV-CoA) both in solution and on solid supports. Bacillus subtilis surfactin phosphopantetheinyl transferase Sfp produced in E. coli mediated the conjugation of firefly luciferase N-terminally extended by eleven amino acids forming a 'ybbR tag' as Sfp-selective substrate, which even worked in bacterial raw lysates. The enzymes displayed on the protein coat of the TMV nanocarriers exhibited high activity. As TMV has proven a beneficial high surface-area adapter template stabilizing enzymes in different biosensing layouts in recent years, the use of TMV-CoA for fishing ybbR-tagged proteins from complex mixtures might become an advantageous concept for the versatile equipment of miniaturized devices with biologically active proteins. It comes along with new opportunities for immobilizing multiple functionalities on TMV adapter coatings, as desired, e.g., in handheld systems for point-of-care detection.

The Tobacco Mosaic Virus Origin of Assembly Sequence is Dispensable for Specific Viral RNA Encapsidation but Necessary for Initiating Assembly at a Single Site.

We have investigated whether the presence of the origin of assembly sequence (OAS) of tobacco mosaic virus (TMV) is necessary for the specific encapsidation of replicating viral RNA. To this end TMV coat protein was expressed from replicating RNA constructs with or without the OAS in planta. In both cases the replicating RNA was specifically encapsidated to give nucleoprotein nanorods, though the yield in the absence of the OAS was reduced to about 60% of that in its presence. Moreover, the nanorods generated in the absence of the OAS were more heterogeneous in length and contained frequent structural discontinuities. These results strongly suggest that the function of the OAS is to provide a unique site for the initiation of viral assembly, leading to a one-start helix, rather than the selection of virus RNA for packaging.

Inhibition of Cell-Free Translation and Replication of Tobacco Mosaic Virus RNA by Exogenously Added 5'-Proximal Fragments of the Genomic RNA.

Replication proteins of tobacco mosaic virus (TMV), a positive-sense RNA virus, co-translationally bind to a 5'-proximal ~70-nucleotide (nt) region of the genomic RNA, referred to as the nuclease-resistant (NR) region for replication template selection. Therefore, disruption of the interaction between the viral replication proteins and viral genomic RNA is expected to inhibit the replication of TMV. In this study, we demonstrate that the addition of small RNA fragments (18-33 nts in length) derived from different regions within the NR region inhibit the binding of TMV replication proteins to viral RNA and TMV RNA replication in a cell-free system. Intriguingly, some of the small RNA fragments also inhibited the translation of mRNA in a sequence-nonspecific manner. These results highlight the pleiotropic roles of the 5'-proximal region of the TMV genome.

Construction of electrochemiluminescence biosensor via click chemistry and ARGET-ATRP for detecting tobacco mosaic virus RNA.

Herein, an electroluminescence (ECL) biosensor was constructed by combining click chemistry with activators regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP) to sensitively assay tobacco mosaic virus (TMV) RNA for the first time. First, hairpin DNA (hDNA) was self-assembled on the gold electrode surface through Au-S bonding. The hDNA hybridized with the tDNA to form tRNA/hDNA hybrids in the presence of TMV RNA (tRNA), so that the azide group labelled at the end of the hDNA was kept away from the electrode surface. Subsequently, the initiator for the ARGET-ATRP reaction was modified on the electrode surface by chemical bonds via click chemistry. Then, N-acryloxysuccinimide (NAS)-labelled polymer chains were successfully formed on the electrode surface by ARGET-ATRP. Under the optimized conditions, a good linear relationship existed with the ECL signal and the logarithm of tRNA concentration in the range of 0.1 pM-10 nM, and the limit of detection was 2.61 fM. In addition, this strategy can identify mismatched bases and performs well in recovery assays in real samples. For its high sensitivity, selectivity, simplicity and economy, the ECL biosensor shows great potential for practical applications.

Induction of Systemic Resistance to Tobacco mosaic virus in Tomato through Foliar Application of Bacillus amyloliquefaciens Strain TBorg1 Culture Filtrate.

The application of microbe-derived products as natural biocontrol agents to boost systemic disease resistance to virus infections in plants is a prospective strategy to make agriculture more sustainable and environmentally friendly. In the current study, the rhizobacterium Bacillus amyloliquefaciens strain TBorg1 was identified based on 16S rRNA, rpoB, and gyrA gene sequences, and evaluated for its efficiency in conferring protection of tomato from infection by Tobacco mosaic virus (TMV). Under greenhouse circumstances, foliar sprays of TBorg1 culture filtrate (TBorg1-CF) promoted tomato growth, lowered disease severity, and significantly decreased TMV accumulation in systemically infected leaves of treated plants relative to untreated controls. TMV accumulation was reduced by 90% following the dual treatment, applied 24 h before and after TMV infection. Significant increases in levels of total soluble carbohydrates, proteins, and ascorbic acid were also found. In addition, a significant rise in activities of enzymes capable of scavenging reactive oxygen species (PPO and POX), as well as decreased levels of non-enzymatic oxidative stress markers (H2O2 and MDA) were observed, compared to untreated plants. Enhanced systemic resistance to TMV was indicated by significantly increased transcript accumulation of polyphenolic pathway (C4H, HCT, and CHI) and pathogenesis-related (PR-1 and PR-5) genes. Out of the 15 compounds identified in the GC-MS analysis, 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester and phenol, 2,4-bis(1,1-dimethylethyl), as well as L-proline, N-valeryl-, and heptadecyl ester were present in the highest concentrations in the ethyl acetate extract of TBorg1-CF. In addition, significant amounts of n-hexadecanoic acid, pyrrolo [1,2-a] pyrazine-1,4-dione hexahydro-3-(2-methylpropyl)-, nonane, 5-butyl-, and eicosane were also detected. These compounds may act as inducers of systemic resistance to viral infection. Our findings indicate that the newly isolated B. amyloliquefaciens strain TBorg1 could be a potentially useful rhizobacterium for promoting plant growth and a possible source of biocontrol agents for combating plant virus infections.

Bioconjugation Strategies for Tobacco Mild Green Mosaic Virus.

Tobacco mild green mosaic virus (TMGMV) is a plant virus closely related to Tobacco mosaic virus (TMV), sharing many of its structural and chemical features. These rod-shaped viruses, comprised of 2130 identical coat protein subunits, have been utilized as nanotechnological platforms for a myriad of applications, ranging from drug delivery to precision agriculture. This versatility for functionalization is due to their chemically active external and internal surfaces. While both viruses are similar, they do exhibit some key differences in their surface chemistry, suggesting the reactive residue distribution on TMGMV should not overlap with TMV. In this work, we focused on the establishment and refinement of chemical bioconjugation strategies to load molecules into or onto TMGMV for targeted delivery. A combination of NHS, EDC, and diazo coupling reactions in combination with click chemistry were used to modify the N-terminus, glutamic/aspartic acid residues, and tyrosines in TMGMV. We report loading with over 600 moieties per TMGMV via diazo-coupling, which is a >3-fold increase compared to previous studies. We also report that cargo can be loaded to the solvent-exposed N-terminus and carboxylates on the exterior/interior surfaces. Mass spectrometry revealed the most reactive sites to be Y12 and Y72, both tyrosine side chains are located on the exterior surface. For the carboxylates, interior E106 (66.53 %) was the most reactive for EDC-propargylamine coupled reactions, with the exterior E145 accounting for >15 % reactivity, overturning previous assumptions that only interior glutamic acid residues are accessible. A deeper understanding of the chemical properties of TMGMV further enables its functionalization and use as a multifunctional nanocarrier platform for applications in medicine and precision farming.

Tobacco Mosaic Virus and the History of Molecular Biology.

The history of tobacco mosaic virus (TMV) includes many firsts in science, beginning with its being the first virus identified. This review offers an overview of a history of research on TMV, with an emphasis on its close connections to the emergence and development of molecular biology.

Isolation of Tobacco Mosaic Virus-Binding Peptides for Biotechnology Applications.

Tobacco mosaic virus (TMV) was the first virus to be discovered and it is now widely used as a tool for biological research and biotechnology applications. TMV particles can be decorated with functional molecules by genetic engineering or bioconjugation. However, this can destabilize the nanoparticles, and/or multiple rounds of modification may be necessary, reducing product yields and preventing the display of certain cargo molecules. To overcome these challenges, we used phage display technology and biopanning to isolate a TMV-binding peptide (TBPT25 ) with strong binding properties (IC50 =0.73 µM, KD =0.16 µM), allowing the display of model cargos via a single mixing step. The TMV-binding peptide is specific for TMV but does not recognize free coat proteins and can therefore be used to decorate intact TMV or detect intact TMV particles in crude plant sap.

Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

BACKGROUND: Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. METHODS AND RESULTS: In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720 nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. CONCLUSIONS: These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

Identification of genetic determinants of tomato brown rugose fruit virus that enable infection of plants harbouring the Tm-22 resistance gene.

Tomato cultivars containing the Tm-22 resistance gene have been widely known to resist tobacco mosaic virus (TMV) and tomato mosaic virus. Tomato brown rugose fruit virus (ToBRFV), a new emerging tobamovirus, can infect tomato plants carrying the Tm-22 gene. However, the virulence determinant of ToBRFV that overcomes the resistance conferred by the Tm-22 gene remains unclear. In this study, we substituted the movement protein (MP) encoding sequences between ToBRFV and TMV infectious clones and conducted infectivity assays. The results showed that MP was the virulence determinant for ToBRFV to infect Tm-22 transgenic Nicotiana benthamiana plants and Tm-22 -carrying tomato plants. A TMV MP chimera with amino acid residues 60-186 of ToBRFV MP failed to induce hypersensitive cell death in the leaves of Tm-22 transgenic N. benthamiana plants. Chimeric TMV containing residues 60-186 of ToBRFV MP could, but chimeric ToBRFV containing 61-187 residues of TMV MP failed to infect Tm-22 transgenic N. benthamiana plants, indicating that 60-186 residues of MP were important for ToBRFV to overcome Tm-22 gene-mediated resistance. Further analysis showed that six amino acid residues, H67 , N125 , K129 , A134 , I147 , and I168 of ToBRFV MP, were critical in overcoming Tm-22 -mediated resistance in transgenic N. benthamiana plants and tomato plants. These results increase our understanding of the mechanism by which ToBRFV overcomes Tm-22 -mediated resistance.