Role Of Conserved Amino Acids And Acetylated Amino Acids Of Potato Virus Y Coat Protein In Tobacco Veinal Necrosis

Potato virus Y（PVY）is a representative species of the genus Potyvirus and has a wide host range,and frequent recombination in genome.PVY infects many crops including potato and tobacco and causes huge economic losses.The coat protein（CP）of PVY is a multifunctional protein and involved in the virus particle assembly,aphid transmission,cell-to-cell and long distance movement,genome replication,and symptom formation.Conserved amino acids and acetylated amino acids are involved in the regulation of protein function,but the function of CP conserved amino acids and acetylated amino acids on CP and their roles in PVY tobacco veinal necrosis are not clear.The results of this study,I showed that recombinant strain became the PVY dominant strain in seed potatoes,could cause veinal necrosis of tobacco leaves.CP-conserved aspartic acid at position 6 and glycine at position 9and 9 acetylated lysine in CP of necrosis strain play a key role in tobacco veinal necrosis.The detailed results are read as follows:1.Seven PVY isolates detected in seed potatoes could cause veinal necrosis of tobacco leaves.In this study,we tested 362 seed potatoes of 7 cultivars using PVY specific antibody,and found that the incidence of PVY was up to 12.0%,and varied in different seed potato cultivars.Complete sequences of 7 PVY isolates were obtained by RT-PCR amplification,and the results of recombination analysis showed that all the genomes of 7 isolates were recombinant;phylogenetic analysis showed that 6 isolates were PVY^N-Wistrain and 1 isolate was PVY^NTN-NW（SYR-II）strain.These 7 isolates can cause veinal necrosis symptoms on Nicotiana tabacum cv.Xanthi after mechanical inoculation.2.Mutations of CP conserved amino acids D~6and G~9decreased the interaction between CP and HC-Pro,and affected the occurrence of tobacco veinal necrosis caused by PVY.Using site-directed mutagenesis technique,p Cam PVY-CP^D6Nand p Cam PVY-CP^G9Rwere obtained by mutating D~6and G~9of CP into natural asparagine（N）and arginine（R）,respectively,in p Cam PVY^N605 infectious clone.Nicotiana tabacum cv.Xanthi was inoculated by agrobacterium infiltration,at 10 days post agro-infiltration（dpi）,PVY^N605 and all mutants could systemic infection,and there was no significant difference in virus accumulation.PVY^N605 induced veinal necrosis in the systematic leaves of Nicotiana tabacum cv.Xanthi,but the mutants PVY-CP^D6Nand PVY-CP^G9Rdid not cause veinal necrosis.The interaction between CP,CP^D6N,CP^G9Rand HC-Pro was observed by luciferase complementation assay（LCA）.The results show that the fluorescence intensity of CP^D6Nand HC-Pro was significantly lower than that of CP and HC-Pro,and the fluorescence intensity of CP^G9Rand HC-Pro was slightly weaker than that of CP and HC-Pro,indicating that D6N and G9R mutations affected the interaction between CP and HC-Pro.These results suggest that D6N and G9R mutations may affect veinal necrosis in PVY by decreasing the interaction between CP and HC-Pro.3.The mutation of CP acetylated amino acids K¹⁶,K²²¹and K²⁶⁵affected the infection of PVY.PVY-infected potatoes were analyzed by acetylation modification omics analysis and 9lysine sites of acetylation modification were identified.These 9 K are K¹²,K¹³,K¹⁶,K²⁸and K³²in the N-terminus,K¹⁷⁷,K²²¹and K²²⁶in the central region and K²⁶⁵in the C-terminus.These sites were mutated into glutamine（Q）as a mimic of acetyl lysine and arginine（R）as a mimic of the unmodified state,respectively,using site-directed mutagenesis.After 10 days of inoculation of PVY^N605 and 18 mutants in Nicotiana tabacum cv.Xanthi,it was found that except for reverse mutation at K²²¹,PVY^N605 and all mutants could systemic infection,and there was no significant difference in virus accumulation.Veinal necrosis was induced by PVY^N605,but only when K¹⁶and K²⁶⁵were mutated to mimic non-acetylation R,the virus mutant did not cause veinal necrosis.Both mimic of acetyl mutation and mimic of the unmodified mutation at K²²¹site affect the replication and movement of PVY,but the mutation at K²²¹site is unstable and will reverse to wild type.In conclusion,The necrosis strain has become the dominant strain of PVY in seed potatoes.CP conserved amino acids D~6and G~9and acetylated modified amino acids K¹⁶and K²⁶⁵are involved in the formation of veinal necrosis symptoms of PVY,and acetylated amino acids K²²¹affected the infection of PVY.