The tomato Tm-1 gene confers resistance to tomato mosaic virus (ToMV). Here, we report that the extracts of Tm-1 tomato cells (GCR237) have properties that inhibit the in vitro RNA replication of WT ToMV more strongly than that of the Tm-1-resistancebreaking mutant of ToMV, LT1. We purified this inhibitory activity and identified a polypeptide of Ϸ80 kDa (p80 GCR237 ) using LCtandem MS. The amino acid sequence of p80 GCR237 had no similarity to any characterized proteins. The p80 GCR237 gene cosegregated with Tm-1; transgenic expression of p80 GCR237 conferred resistance to ToMV within tomato plants; and the knockdown of p80 GCR237 sensitized Tm-1 tomato plants to ToMV, indicating that Tm-1 encodes p80 GCR237 itself. We further show that in vitro-synthesized Tm-1 (p80 GCR237 ) protein binds to the replication proteins of WT ToMV and inhibits their function at a step before, but not after, the viral replication complex is formed on the membrane surfaces. Such binding was not observed for the replication proteins of LT1. These results suggest that Tm-1 (p80 GCR237 ) inhibits the replication of WT ToMV RNA through binding to the replication proteins.Tm-1 ͉ Tomato mosaic virus ͉ inhibitory interaction

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through expression of Cas9 and single guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base-editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus-vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.