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.

Engineered Resistance to Tobamoviruses

“…However, inserting a single specific mutation in a viral coding sequence may put selective pressure on the protective strain, resulting in acquisition of a compensatory mutation elsewhere in its genome. For example, a mild protective variant of TMV was produced by replacing arginine with alanine at residue 88 in the 126 kDa protein sequence, but a spontaneous mutant that caused severe symptoms (with replacement of serine with lysine at residue 114 of the 126 kDa protein) arose independently in 3 out of 14 plants inoculated with the protective strain [75]. Thus, engineered protective strains need to be investigated extensively and may need further mutations to be introduced to make reversion to a virulent form less likely.…”

Engineered Resistance to Tobamoviruses

“…However, inserting a single specific mutation in a viral coding sequence may put selective pressure on the protective strain, resulting in the acquisition of a compensatory mutation elsewhere in its genome. For example, a mild protective variant of TMV was produced by replacing arginine with alanine at residue 88 in the 126 kDa protein sequence, but a spontaneous mutant that caused severe symptoms (with replacement of serine with lysine at residue 114 of the 126 kDa protein) arose independently in 3 out of 14 plants inoculated with the protective strain [75]. Thus, engineered protective strains need to be investigated extensively and may need further mutations to be introduced to make reversion to a virulent form less likely.…”