Development of a stable attenuated double-mutant of tobacco mosaic virus for cross-protection1

Tobacco () and tomato () are two major economic crops in China. Tobacco mosaic virus (TMV; genus ) is the most prevalent virus infecting both crops. Currently, some widely cultivated tobacco and tomato cultivars are susceptible to TMV and there is no effective strategy to control this virus. Cross-protection can be a safe and environmentally friendly strategy to prevent viral diseases. However, stable attenuated TMV mutants are scarce. In this study, we found that the substitutions in the replicase p126, arginine at position 196 (R) with aspartic acid (D), glutamic acid at position 614 (E) with glycine (G), serine at position 643 (S) with phenylalanine (F), or D at position 730 (D) with S, significantly reduced the virulence and replication of TMV. However, only the mutation of S to F reduced the RNA silencing suppression activity of TMV p126. A double-mutant TMV-E614G-S643F induced no visible symptom and was genetically stable through six successive passages in tobacco plants. Furthermore, our results showed that TMV-E614G-S643F double-mutant could provide effective protection against the wild-type TMV infection in tobacco and tomato plants. This study reports a promising mild mutant for cross-protection to control TMV in tobacco and tomato plants.