Studies On The PR-10 And Symptoms Expression Induced By Beet Necrotic Yellow Vein Virus P31 In Nicotiana Benthamiana

The rhizomania disease causes serious losses in sugar beet yield and sugar industry in China and other countries.Beet necrotic yellow vein virus(BNYVV)which causes beet rhizomania in sugarbeet is representative species of the genus Benyvirus.The viral genome of BNYVVV consists of 4-5 linear positive sense ssRNAs,and BNYVV is transmitted by the soil-borne plasmodiophorid Polymyxa betae.On the basis of previous researches,this dissertation investigated the translation initiation strategy of P31 ORF,PR-10 expression and the pathogenicity to Nicotiana benthamiana induced by P31 protein,and explored the possibility of BNYVV replication system in yeast.These results help improve our understanding of the molecular mechanisms involved in BNYVV infection.Previous studies of our lab have shown that the P31 protein cannot be detected from BNYVV-infected N.benthamiana by Western blotting using P31 polyclonal antibody raised from rabbit.The P31 C-ter-Flag,-HA or-Myc tag fusion proteins expressed based on RNA4 cDNA clone pUOFl-6 during virus infection could not be detected either.Putative P31 open reading frame(ORP)harbored 5 initiation codons within 200 nt of the 5' terminal sequence in RNA4.Several frame-shift mutants for 2nd-5th AUG were constructed and fused to the GUS report gene to investigate which AUG is crucial for translation.After inoculated to Tetragonia expansa and N.benthamiana,the result showed that the first AUG was the main translation initiation site,if it was inactivated,the second and third AUG can initiate translation with more lower efficiency.And then,a series of deletion mutants were constructed,and revealed that the corresponding nucleic acid sequence encoding 15-21 amino acid of P31 specifically regulated P31 translation efficiency.RNA4 of BNYVV is not essential for virus propagation in N.benthamiana,but it has a major effect on symptom expression.Early reports showed that RNA4-encoded P31 was associated with severe symptoms such as curling and dwarfing in N.benthamiana.The real-time PCR results indicated that pathogenesis-related protein 10(PR-10)gene was up-regulated in BNYVV-infected N.benthamiana in the presence of RNA4,and it had a close link with symptom development.The PR-10 gene upregulted after inoculation,and reached a peak of the parabola at 7 dpi,then reduced to nomal level.The results of frame-shift,deletion and substitution mutation analysis showed that only intact P31 could induce the up-regulation of PR-10 during BNYVV infection.Meanwhile,two tryptophans(W259 and W276)and six cysteines(C174,C183,C186,C190,C197 and C199)in the cysteine-rich P31 had been proved to be required for the upregulation of PR-10 expression.The PR-10 gene was cloned from N.benthamiana(NbPR-10),and expressed NbPR-10 in E.coli exhibited RNase activity,which could digested both single strand RNAs and double strand RNAs.However,P31 failed to interact directly with NbPR-10 in yeast-two hybridization assay.RNA1 and RNA2 of BNYVV Hu3 isolate were cloned and sequenced(GenBank accession numbers KM434313 and KM434314,respectively).Sequence alignments with other isolates from all parts of world showed that nucleic acid and amino acid identities are more than 98%(RNA1)and 94%(RNA2),and genetic distance is relatively far.BNYVV replication system in yeast was investigated.Primary result indicated that the subgenomic RNA of RNA2 could be detected by Northern blotting,but some details still need exploration for stable expression.