| Potato virus Y（PVY）is the most widespread and economically important virus in potatoes,causeing serious losses to potato production.Accurate identification of the tupe of PVY strain is of great significance for prevention and control of potato virus disease.PVY can be classified into PVYC,PVYO,PVYN,PVYZ and PVYE strains according to their recognition of disease resistance gens in potatoes and their symptoms in tobacco.Further,PVYN can be subdivided into PVYNTN,PVYN:O:O and PVYNTN-NWTN-NW strains.In this study,the isolate A12 from Heilongjiang was identified using biology,serology and molecular biology,and the reason why A12 did not cause vein necrosis in Nicotiana tabacum was analyzed.The main results are as follow.1.PVY isolate A12 was identified to belong to SYR-II type of NTN-NW strainELISA results showed that A12 could be specifically recognized by the monoclonal antibody specific for PVYN.The open reading frame of A12 contained 9186 nt,and coded3061 amino acids.Consistency analysis presented that nucleotide and amino acid sequences of A12 had the highest consistency with isolate SYR-II-Be1 from PVYNTN-NW strain,98.3%and 99.2%respectively.Recombination analysis demonstrated that A12 was the recombinant of N-605 and Oz,and the recombinant type was the same as SYR-II-2-8.Phylogenetic analysis revealed that A12 was aggregated into a cluster of SYR-II of PVYNTN-NW strain.The above results suggest that A12 belongs to SYR-II type of PVYNTN-NW strain.2.Elucidate the reason why isolate A12 from PVYNTN-NWTN-NW strain could not cause vein necrosis in Nicotiana tabacumIsolate A12 belongs to SYR-II type of PVYNTN-NW strain,but common isolates from PVYNTN-NW strain show vein necrosis and mosaic in Nicotiana tabacum.A12 only caused mosaic.Sequence alignment showed that the amino acids at positions 182 and 245 in helper component-proteinase（HC-Pro）of A12 were both Arginine（R）,instead of Lysine（K）in other PVYNTN-NW isolates.Therefore,it is speculated that the reason why A12 does not cause veinal necrosis was due to mutation of amino acids at 182 and/or 245 of HC-Pro.Site-directed mutation was introduced into pCamPVYN605,an infectious clone of PVY N605,to produce plasmids pCamPVY N605-HCK182R182R and pCambiaPVY N605-HCK245R.Amino acid K at positions 182 and 245 in the HC-Pro of progeny viruses derived from these two plasmids was mutated to R,respectively.After introduced Nicotiana tabacum cv.Xanthi,it was found that pCamPVY N605-HCK245R and pCamPVYN605 inoculated plants caused the same symptoms,and all showed vein necrosis and accumulated similar levels of virus.However,the plants inoculated with pCamPVY N605-HCK182R182R showed only mosaic,and accumulated significantly lower than that of wild type.These suggest that mutation at position 182 of HC-Pro from K to R not causing vein necrosis.It is speculated that A12 does not cause vein necrosis in that the 182 amino acid of HC-Pro is mutated to arginine（R）.Compared with wild-type HC-Pro,the mutation of 182 amino acid to arginine weakened the RNA silencing suppressor activity of HC-Pro.3.Dissected the role of Arg18080 in the FRNK motif of PVY HC-Pro on symptom expression and RNA silencing suppressionSite-directed mutation was used to introduce into pCamPVYN605 to produce plasmid pCambiaPVY-HCR180I.The 180 amino acid of HC-Pro encoded by plasimid was was mutated from R to I.Plants of Nicotiana tabacum cv.Xanthi inoculated with pCambiaPVY-HCR180I180I only produced symptom of mosaic and could not accumulate virus in the systemic leaves of inoculated Xanthi plants.Compared with the wild-type HC-Pro,the expression level of GFP mRNA in the infiltrated regions of Nicotiana benthamiana leaves was significantly reduced.These results indicated that that mutation of R to I at position 180 of PVY HC-Pro abolished its RNA silencing suppression acitivity,by which affected the virulence of PVY. |
