Zhi-Yong Yan

Tomato brown rugose fruit virus (ToBRFV) overcomes Tm‐2 ² ‐mediated resistance, posing a serious threat to global tomato production. We previously identified that key residues in the movement protein (MP)‐H67, N125, K129, A134, I147 and I168 are essential for ToBRFV evasion of Tm‐2 ² resistance. However, the underlying mechanisms remain unclear. In Tm‐2 ² ‐transgenic Nicotiana benthamiana plants at later infection stages, MP mutations H67C, N125A, K129Q and A134N abolished ToBRFV‐GFP cell‐to‐cell and systemic movement, whereas I168N abolished ToBRFV‐GFP systemic but not cell‐to‐cell movement, and I147M partially impaired both. Correspondingly, the MP ^H67C , MP ^N125A , MP ^K129Q and MP ^A134N mutants elicited strong Tm‐2 ² ‐mediated hypersensitive response (HR), MP ^I168N elicited mild HR and MP ^I147M elicited none. Confocal microscopy revealed I147M reduced MP localisation at plasmodesmata in Tm‐2 ² ‐transgenic N. benthamiana leaves. The interaction with Tm‐2 ² was weak for wild‐type MP and I147M (slightly enhanced), but moderately or strongly enhanced for I168N and the other four mutants. Wild‐type MP and MP ^I147M failed to induce Tm‐2 ² self‐association, MP ^I168N only weakly induced it, whereas the other four markedly induced it. Collectively, these findings demonstrate that ToBRFV evades Tm‐2 ² ‐mediated resistance by attenuating MP–Tm‐2 ² interaction strength, thereby avoiding Tm‐2 ² self‐association and downstream immune activation.

Cell-intrinsic restriction factors (CIRFs) negatively regulate plant virus infections and represent valuable resources for breeding virus-resistant crops. However, the potential for ribosomal proteins to function as CIRFs and the underlying mechanisms remains elusive. Our previous work demonstrated that the Nicotiana benthamiana chloroplastic ribosomal protein of the large subunit 1 (NbRPL1) promotes the infection of tobacco vein banding mosaic virus (TVBMV) by antagonizing the NbBeclin1-mediated degradation of the viral RNA-dependent RNA polymerase, NIb. Continuing this line of research, we explored the role of another nonchloroplastic ribosomal protein, large ribosomal protein 4 (RPL4), in TVBMV replication. We found that TVBMV NIb interacts with NbRPL4; however, the NIb proteins of 2 other related potyviruses, potato virus Y (PVY) and turnip mosaic virus (TuMV), did not interact with NbRPL4. Overexpression of NbRPL4 inhibited, whereas its downregulation promoted, TVBMV replication. NbRPL4 did not affect PVY or TuMV replication. The nuclear-cytoplasmic distribution of NbRPL4 positively correlated with its antiviral effect on TVBMV replication. NbRPL4 interfered with NbXPO1-mediated nuclear export of the NIb protein, subsequently affecting the translocation of NIb into the viral replication compartment. Our work indicates that NbRPL4 functions as a restriction factor for TVBMV by inhibiting NbXPO1-mediated nuclear export of NIb in a virus-specific manner.

Summary: To establish systemic infection, plant viruses must replicate, and conduct intra‐ and intercellular movement and long‐distance movement, all of which require the participation of host factors. Tobamoviruses move in the form of movement protein (MP)–viral RNA complex and utilize endocytosis for intracellular movement. However, how tobamoviral MPs hijack host factors to reach the plasma membrane (PM) and then plasmodesmata (PD) is still largely unknown. Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that mainly infects tomatoes and peppers. Here, we show that tomato RabE1a, a small Rab GTPase, interacts with ToBRFV MP and participates in the movement of ToBRFV. Knocking out RabE1a in tomatoes could inhibit infection by ToBRFV. RabE1a positively regulates MP transport to the PM, and this transport process is regulated by its nucleotide‐binding state. Furthermore, RabE1a interacts with the exocyst subunit SEC10b to jointly regulate MP transport to the PM and intracellular movement of ToBRFV. The adaptor protein AP2β interacts with MP and transports MP from the PM to the PD for intercellular movement of ToBRFV. We further find that knocking out RabE1a could also inhibit the infection of other tobamoviruses. In summary, MP exocytosis is coregulated by RabE1a and the exocyst subunit SEC10b for transport to the PM, where it then uses AP2β‐regulated endocytosis to PD. These results provide a comprehensive overview of tobamoviral MP intracellular transport and are insightful for breeding tomato plants resistant to ToBRFV and related tobamoviruses.

Paprika mild mottle virus (PaMMV), a tobamovirus from the Virgaviridae family, has been reported to infect pepper (Capsicum annuum L.) in the Netherlands (García-Luque et al., 1993), Japan (Hamada et al., 2003), Bulgaria (Ruíz del Pino et al., 2003), and Israel (Luria et al., 2018). Pepper has become the most widely planted vegetable and the most heavily consumed spicy condiment, with a large planting area (>21,000 km²) in China (Zou et al., 2021). However, the crop's productivity is limited by viral diseases. In August 2021, a survey of viral diseases was conducted across five pepper-growing regions in Linyi, Shandong Province, China, revealed that 5% to 10% of plants in most fields exhibited symptoms such as stunting, leaf narrowing, chlorosis, and crinkling of leaf tissue (Fig. S1-a). Subsequently, seven symptomatic samples (Fig. S1-b) were collected from a plot (118° 29' E, 34° 65' N) where thin-skinned peppers were grown. All samples tested positive in a Western blot assay using a polyclonal antibody for tobacco mosaic virus (TMV), indicating the presence of a tobamovirus (Fig. S1-c). To identify the virus, total RNAs were extracted from 10 different symptomatic pepper plant samples, using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and combined into a single sample in equal amounts (100 ng/μL each). RNA-Seq was performed on this mixture using the Illumina NovaSeq 6000 (Illumina, San Diego, USA). Raw reads (6.92G) in fastq format were processed with in-house perl scripts to yield clean reads (6.68G), which were assembled into 780 contigs (mean length: 2036 bp) using Trinity software (v2.6.6). These contigs were then analysed using the NCBI BLASTX program (http://www.ncbi.nlm.nih.gov/blast) against the viral RefSeq database. The results demonstrated that the contigs exhibited an average genome coverage of 40.16%. Notably, one unique contig (6453 bp) was mapped to the PaMMV genome (reference sequence KX187305.1) with 98.34% identity. To verify the RNA-Seq result, a PaMMV-specific primer pair (forward: 5'-GAGTTCATAGAGGCAGTACC-3'; reverse: 5'-CTTCGATTTAAGTGGAGGGAT-3') was designed for amplifying an 800-nt fragment of the PaMMV coat protein (CP) gene by RT-PCR. The 800-nt fragment was successfully amplified from all the symptomatic samples (Fig. S1-d). Sequenced RT-PCR products (GenBank No. OR365081.1) showed 99.75% nucleotide identity with PaMMV isolates (OQ198318.1, China, goat) according to BLAST analysis. Mechanical transmission of PaMMV from three infected pepper plants collected in Linyi to healthy pepper plants confirmed pathogenicity. Three pepper cultivars (trade name: Qiemen, Tianyu, Haonong11) were tested and showed varying symptoms post-inoculation (Fig. S1-e). All cultivars were confirmed in the lab to be PMMoV-sensitive and exhibited significant viral disease symptoms after PMMoV inoculation (data not shown). Each variety was inoculated with at least 3 seedlings from different field-collected virulence sources. Qiemen (cultivar: sweet pepper) exhibited severe symptoms with necrotic spots, Tianyu (cultivar: Chaotian pepper) had mild leaf wrinkling, and Haonong11 (cultivar: Pickled pepper) showed yellow mottle and leaf shrinking. All tested seedlings were positive for PaMMV by RT-PCR (Fig. S1-f). To our knowledge, this is the first report of PaMMV infecting pepper in China, highlighting the need for vigilant monitoring to protect the chili industry and prevent the virus's spread to other crops.

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.

Tomato mottle mosaic virus (ToMMV) poses a threat to production and quality of tomato fruits. The Tm-2² gene confers resistance to some tobamoviruses by recognizing viral movement proteins. However, Tm-2²-mediated resistance against ToMMV is not well known. Here, we found that ToMMV could infect wild-type but not Tm-2² transgenic Nicotiana benthamiana plants and could also infect tomato cultivar Moneymaker but not resistant cultivar Jili with homozygous Tm-2². Chimeric viral ToMMV^ToBRFV−MP with swapped ToMMV MP to MP of tomato brown rugose fruit virus could systemically infect Tm-2² transgenic N. benthamiana and tomato cultivars Jili plants. Further, transient expression of ToMMV MP in the leaves of Tm-2² transgenic N. benthamiana plants induced hypersensitive response-associated cell death, suggesting that the MP of ToMMV was the avirulent factor for the Tm-2² resistance gene. ToMMV could infect Tm-2²-containing cultivar Jinpeng 1 but not Chaobei. Sequence analysis revealed that cultivars Chaobei and Jinpeng 1 were heterozygous, where Chaobei consists of Tm-2² and Tm-2 genes, while Jinpeng 1 consists of Tm-2² and tm-2 genes. Transient co-expression assays showed that both Tm-2² and Tm-2 but not tm-2 could recognize ToMMV MP and induce hypersensitivity response-associated cell death in N. benthamiana leaves, suggesting that homozygous tomato harboring Tm-2² and heterozygous tomato containing Tm-2² and Tm-2 may exhibit more durable resistance to ToMMV than heterozygous tomato carrying Tm-2² and tm-2. Further, Tm-2² transgenic N. benthamiana and tomato cultivar Jili plants with silenced Tm-2² gene were susceptible to ToMMV. Also, silencing type-I J-domain MIP1 gene compromised Tm-2²-mediated resistance to ToMMV in Tm-2² transgenic N. benthamiana and tomato cultivar Jili. Moreover, we found that viral RNA could accumulate in the systemic leaves of Tm-2² transgenic N. benthamiana plants and tomato cultivar Jili at 35°C, but not at 20, 25, or 30°C. Altogether, our findings reveal that the Tm-2² confers resistance to ToMMV by recognizing MP, and the resistance is regulated by the allele combinations, accumulation levels of Tm-2², MIP1, and the temperature.