Screening Of Ptoato Virus Y Mild Mutants And Evaluation Of Their Effects On Cross Protection

Potato virus Y (PVY) is the type species of the genus Potyvirus (family Potyviridae) and causes huge economic losses to many crops of the family Solanaceae including potato and tobacco. PVY isolates can be divided into two strains:O and N. Isolates of strain O induce mosaic in tobacco leaves, while those of strain N induce veinal necrosis on tobacco leaves, which bring more severe damage to tobacco leaf production, In the present study, we obtained mild mutants with mutations at multiple sites by site-directed mutagenesis, tested their effects on cross protection, and introduce mutation on the site involved in synergy with Potato virus X (PVX). The main results read as follows:1. Four single site mutants A (D205G), B (N339D), C (K400R) and D (E419D) were obtained from the full-length cDNA infectious clone of PVYN-605. Compared with the wild type, the symptoms of those mutants in tobacco were milder. Then we introduced three or four mutations to one molecule and obtained two mutants with triple mutations (ABC, ABD), and one with four mutations (ABCD). All of them could not induce vein necrosis in tobacco leaves. The accumulation levels of these mild strains are reduced by25%,38%,47%.2. The cross protection effects of mild mutants with multiple amino acid mutations against wild type were evaluated. Plants of N. tabacum cv. NC89were inoculated by the mild mutants as the protective inoculation. The second fully expanded leaves of the plants were mechanically challenged with wild type PVYN-60515or20days later. The results indicated that the symptoms were significantly reduced in protected N. tabacum cv. NC89plants, and virus accumulation reduced to30%, indicating that the mutants ABC, ABD, and ABCD provided good cross protection effects.3. The amino acid responsible for synergy of PVY HC-Pro with PVX were elucidated. The mutations of motif FINK to FRNK and NRT to ART in PVY HC-Pro abolished its RNA silencing suppression activity. Inserting PVY HC-Pro with one of these mutations to the infectious clone of pgR106abolished the synergism between PVY HC-Pro and PVX in that no systemic necrosis in Nicotiana benthamiana was observed.4. The synergsim-abolishing mutation was introduced to the PVY mutants with multiple mutations by site-directed mutatagenesis. In the end we obtained the mild mutants which abolished the ability of synergism. This mutant confer cross protection effect against wild type PVY but has no synergism with PVX.