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.

Summary: Hypersensitive resistance ( HR ) to strains O and C of Potato virus Y ( PVY , genus Potyvirus ) is conferred by potato genes Ny _tbr and Nc _tbr , respectively; however, PVY N strains overcome these resistance genes. The viral helper component proteinases ( HC pro, 456 amino acids) from PVY ^N and PVY ^O are distinguished by an eight‐amino‐acid signature sequence, causing HC pro to fold into alternative conformations. Substitution of only two residues ( K269R and R270K ) of the eight‐amino‐acid signature in PVY ^N HC pro was needed to convert the three‐dimensional ( 3D ) model of PVY ^N HC pro to a PVY ^O ‐like conformation and render PVY ^N avirulent in the presence of Ny _tbr , whereas four amino acid substitutions were necessary to change PVY ^O HC pro to a PVY ^N ‐like conformation. Hence, the HCpro conformation rather than other features ascribed to the sequence were essential for recognition by Ny _tbr . The 3D model of PVY ^C HC pro closely resembled PVY ^O , but differed from PVY ^N HC pro. HC pro of all strains was structurally similar to β‐catenin. Sixteen PVY ^N 605 ‐based chimeras were inoculated to potato cv. Pentland Crown ( Ny _tbr ), King Edward ( Nc _tbr ) and Pentland Ivory ( Ny _tbr / Nc _tbr ). Eleven chimeras induced necrotic local lesions and caused no systemic infection, and thus differed from both parental viruses that infected K ing Edward systemically, and from PVY ^N 605 that infected P entland Crown and P entland Ivory systemically. These 11 chimeras triggered both Ny _tbr and Nc _tbr and, in addition, six induced veinal necrosis in tobacco. Further, specific amino acid residues were found to have an additive impact on necrosis. These results shed new light on the causes of PVY ‐related necrotic symptoms in potato.

14-3-3 proteins play vital roles in plant defense against various pathogen invasions. To date, how 14-3-3 affects virus infections in plants remains largely unclear. In this study, we found that Nicotiana benthamiana 14-3-3h interacts with TRANSLATIONALLY CONTROLLED TUMOR PROTEIN (TCTP), a susceptibility factor of potato virus Y (PVY). Silencing of Nb14-3-3h facilitates PVY accumulation, whereas overexpression of Nb14-3-3h inhibits PVY replication. The antiviral activities of 3 Nb14-3-3h dimerization defective mutants are significantly decreased, indicating that dimerization of Nb14-3-3h is indispensable for restricting PVY infection. Our results also showed that the mutant Nb14-3-3h^E16A, which is capable of dimerizing but not interacting with NbTCTP, has reduced anti-PVY activity; the mutant NbTCTP^I65A, which is unable to interact with Nb14-3-3h, facilitates PVY replication compared with the wild-type NbTCTP, indicating that dimeric Nb14-3-3h restricts PVY infection by interacting with NbTCTP and preventing its proviral function. As a counter-defense, PVY 6K1 interferes with the interaction between Nb14-3-3h and NbTCTP by competitively binding to Nb14-3-3h and rescues NbTCTP to promote PVY infection. Our results provide insights into the arms race between plants and potyviruses.

Cross-protection is a promising measure to control plant viral diseases. Reverse genetics had been recently adopted to generate attenuated mutants that have potential in cross-protection. But studies on the variability of the progeny viruses of the attenuated mutants are scarce. Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is the prevalent virus inducing maize dwarf mosaic disease in China. Here, we showed that the substitution of arginine with isoleucine in the FRNK motif at position 184 of helper component-proteinase (HC-Pro) abolished its RNA silencing suppression (RSS) activity, drastically reduced the virulence and accumulation level of SCMV, and impaired the synergism between SCMV and maize chlorotic mottle virus. The attenuated mutant could protect maize plants from a severe infection of SCMV. However, a spontaneous mutation of glycine at position 440 to arginine in HC-Pro rescued the virulence and synergism with maize chlorotic mottle virus of SCMV and the RSS activity of HC-Pro. Similar results were obtained with tobacco vein banding mosaic virus and watermelon mosaic virus. These results provide novel evidence for the complementary mutation of potyviruses in maintaining the HC-Pro RSS activity and potyviral virulence and remind us of evaluating the potential risk of attenuated mutants thoroughly before applying for the control of plant viral diseases via cross-protection.

Cross protection is a promising alternative to control plant viral diseases. One critical factor limiting the application of cross protection is the availability of attenuated mutants or mild strains. Potato virus X (PVX) infects many crops and induces huge economic losses to agricultural production. However, researches on the variability and mechanism of PVX virulence are scarce. The mutants were obtained by introducing mutations into the RNA dependent RNA polymerase (RdRp) gene of PVX via site-directed mutagenesis. Attenuated mutants were screen according to their symptoms in Nicotiana benthamiana plants. The protection efficacy against severe infection were evaluated with interval of 5, 10 and 15 days. Among the 40 mutants obtained, four mutants carrying substitutions of either Glu46, Asn863, Asn968 or Glu1001 to Ala in PVX RdRp showed drastically attenuated symptom, accompanying with reduced accumulation levels of coat protein, plus- and minus-sense RNAs. When the interval between protective and challenging inoculations was 15 days, mutant E1001A (with substitution of Glu1001 to Ala in RdRp) provided complete protection against severe infection in both Nicotiana benthamiana and tomato, while E46A (Glu46 mutated to Ala) provided incomplete protection. To reduce the risk of reverse mutation, we constructed mutant dM which carries double mutations of both Glu46 and Glu1001 to Ala in RdRp. The mutant dM could provide effective protection against severe PVX infection. Mutations of Glu46, Asn863, Asn968 or Glu1001 to Ala in PVX RdRp significantly reduced the viral symptoms. Mutants E1001A and E46A could provide effective protection against wild type PVX in both Nicotiana benthamiana and tomato. These results provide theoretical and practical bases for the control of PVX via cross protection.