Screening Of Potato Virus Y And Tomato Brown Rugose Fruit Virus Mild Mutants And Evaluation Of Their Cross Protection Efficiency

Viral disease is one of the most important constraints to agricultural production.Potato virus Y(PVY)infects many crops such as Solanaceae plants.Strain N of PVY induces veinal necrosis on tobacco leaves,which damages tobacco leaves thereby affecting production.Also,tomato brown rugose fruit virus(To BRFV)infects tomatoes and causes brown rugose or yellow spots on fruits,which seriously affects the yield and quality of tomatoes.The PVY-resistant tobacco cultivar or To BRFV-resistant tomato cultivar is unavailable now.Cross protection is an effective method to control viral diseases for lack of effective antiviral agent in practice.In this study,we conducted site-directed mutations of the full-length c DNA infectious clone of PVY-GZ and To BRFV-SD to obtain mild mutants,and evaluated their effects on cross protection.The main findings are as follows:1.The coat protein(CP)is required for PVY to induce veinal necrosis on tobacco leaves,and multiple-site mild mutants of PVY which were constructed had good cross protection effects against wild type PVY.Seven single-site mutants were generated from the full-length c DNA infectious clone of PVY-GZ.Compared with the wild type,the mutant HC(K50E)and HC(K124R)had no difference in symptoms,the mutant HC(K124L),HC(K182R)and CP(D6N)had milder symptoms and no veinal necrosis,and the mutant HC(N166A)and CP(G9R)lost pathogenicity.Mutants which showed no veinal necrosis were selected and aggregated to form four mutants containing multiple mutations:HC(K124L?K182R),HC(K124L)-CP(D6N),HC(K182R)-CP(D6N)and HC(K124L?K182R)-CP(D6N).Compared with the wild type PVY,all of those mutants had milder symptoms and no veinal necrosis,accumulation of viral CP and CP RNA were decreased by 50%,53%,40%,57% and 98%,99%,60%,98%,respectively.The cross protection effects of the mild mutants without veinal necrosis against the wild type were evaluated.Plants of Nicotiana tabacum cv.Xanthi inoculated with the mild mutants were selected for cross protection.The fully expanded leaves of the plants were mechanically challenged with the wild type PVY SYR-I 15 days after.The results indicated that the symptoms were significantly reduced in the protected N.tabacum cv.Xanthi plants.GFP accumulation of wild type PVY could not be detected,and the relative protection efficiencys were 63%,54%,22% and 63%,respectively,indicating that the multiple-site mild mutants provided good cross protection effects.2.The amino acids Y697 and D711 of To BRFV replicase p126 are the determinants of viral pathogenicity in Nicotiana benthamiana.A mild mutant Y697 A was obtained and had good cross protection effects against the wild type To BRFV.Fourteen single-site mutants were generated from the full-length c DNA infectious clone of To BRFV-SD.Compared with the wild type,the mutant Y697 A and D711 A had no symptoms on N.benthamiana.The cross protection effects of mild mutant Y697 A and D711 A against the wild type were evaluated.Plants of N.benthamiana inoculated with the mild mutant Y697 A and D711 A were selected for cross protection.The fully expanded leaves of the plants were mechanically challenged with the wild type To BRFV 15 days after.The results indicated that the symptoms were significantly reduced in the protected N.benthamiana by the Y697 A mutant as compared to the unprotected N.benthamiana,and accumulation of GFP and GFP RNA of the wild type To BRFV were significantly reduced by 79% and 99.9%,respectively.The relative protection efficiencys of Y697 A on the tomato cultivars Qidali and Shanghaihuihe 405 that were challenged 15 days after the protective inoculation were43% and 32%,respectively,indicating that the mutant Y697 A provided good cross protection effects.This findings can help in managing the incidence of PVY and To BRFV in the field to enhance production of tobacco and tomatoes.