Engineering of the complementary mutation site in tobacco mosaic virus p126 to develop a stable attenuated mutant for cross-protection

Abstract Tobacco mosaic virus (TMV; genus Tobamovirus) is one of the most prevailing pathogens that seriously affects the quality and yield of tobacco (Nicotiana tabacum) leaves. Cross-protection using mild strains is a potential strategy for the biological prevention of plant viral diseases. Complementary mutations in attenuated strains may cause attenuated ones to suddenly evolve into virulent strains, which limits the application of cross-protection in practice. To data there has been no study on engineering the complementary mutation sites to generate stable attenuated mutants for cross-protection. In this study, we found that the substitution of the conserved arginine at position 88 (R88) in p126 protein with alanine (A) abolished the cell-to-cell movement and reduced the replication of TMV. However, a spontaneous complementary mutation of serine at position 114 (S114) to lysine (K) in p126 restored TMV virulence. Substitution of S114 with R in p126 restored the systemic infection but not the virulence of TMV, therefore, the mutant TMV-R88A/S114R was an attenuated one. Furthermore, our results showed that TMV-R88A/S114R was a stable attenuated mutant, and could effectively protect tobacco plants against the wild-type TMV infection. This study reports a promising TMV mild mutant for cross-protection in tobacco plants by modifying the complementary mutation site in p126.