Studies On Molecular Basis Of Purple Leaf Symptom And Pathogenicity Of Brassica Yellows Virus

Brassica yellows virus(BrYV)is a polerovirus isolated from crucifers and reported by our lab.It is a spherical viron with positive single strained RNA genome,and sharing all the common features of Polerovirus,including phloem limited and transmitted by aphids.So far,we found that there are three different genotypes of BrYV,named BrYV-A,BrYV-B,and BrYV-C respectively,and the infectious cDNA clone of these three genotypes have been constructed successfully.By detecting the samples collected from all over China we found that BrYV is widely distributed in mainland China.By sequence homology analysis of the viruses in Polerovirus,we found that Turnip yellows virus(TuYV)is the closest relative of BrYV.Poleroviruses have been well known as a group of important plant pathogens which cause severe damage on crops.For example,TuYV is widely distributed in European countries and was thought to be the principal reason why commercial Oilseed rape crops do not reach their potential genetic yield.China is one of the world's largest producers of Oilseed rape,and taking the close relationship between BrYV and TuYV and the widely distribution of BrYV in our country into consideration,we highly suspect that BrYV might be a big threat to Oilseed rape production in our country,and that is why we start this study.To confirm the pathogenicity of BrYV,we constructed sets of transgenic Arabidopsis plants which contain the full length cDNA of BrYV(BrYV amplicon).Molecular analysis showed that the sub-genomic RNAs(sgRNAs)of BrYV and the coat protein(CP)translated by the subgenomic RNAs all can be detected in the BrYV amplicon transformed Arabidopsis plants.Many abnormal phenotypes exhibited on the BrYV amplicon transformed Arabidopsis plants,including loss of apical domination,dwarf,shorter silique,increased rosette leaves,and leaves turn into purple.These abnormal phenotypes verified the strong pathogenicity of BrYV on crucifers.For the aim of revealing the molecular mechanism of pathogenesis on cruciferous plants,we sequenced the transcriptome profile of two BrYV amplicon transformed Arabidopsis lines(line 111 and line 412,T5 generation).Next generation sequencing(NGS)results showed that there are plenty of differentially expressed genes(DEGs)between Col-0 and BrYV amplicon transformed Arabidopsis plants.These DEGs are distributed in plenty of biological and chemical processes,and they might be the reason for the appearance of those abnormal phenotypes mentioned above.Bioinformatics analysis of the NGS data showed that most genes involved in anthocyanin biosynthesis pathway were up-regulated in line 111 and line 412.And this might be the reason for the purple leaf phenotype of BrYV amplicon transformed Arabidopsis and the purple leaf symptom caused by BrYV on oilseed rape in the field.Take the published papers into consideration we doubt that the movement protein(MP)of BrYV might be the viral agent which cause this symptom.We constructed a binary vector which contains the MP and transformed it into the Arabidopsis plants.By hygromycin selection and molecular analysis we finally got the transgenic Arabidopsis plants which can express the MP of BrYV.We tested the RNA expression level of genes involved in anthocyanin in these MP expressing transgenic plants and found that these genes are indeed up-regulated,and they also exhibit a purple leaf phenotype.All these results showed that MP of BrYV is the very viral agent,which cause the purple leaf symptom on BrYV infected crucifers.Former study had confirmed that the P0 protein of BrYV is a viral suppressor of RNA silencing(VSR)and it can cause necrosis on Agro-infiltrated Nicotiana benthamiana leaves.By alanine mutation scanning,several P0 mutants,which can either affect the VSR or necrosis function or both of these two functions,were obtained.For better understanding the molecular mechanism of how P0 carry on these functions,we transformed P0 and three mutants[Q2A(a mutant loss both VSR and necrosis functions),LP63AA(a mutant loss local VSR but still have the function of systemic VSR and necrosis),L184A(a mutant loss systemic VSR and necrosis functions but still have local VSR function)]into Arabidopsis plants using XVE system,and finally got the transgenic Arabidopsis plants which can express these proteins.We sent the total RNA of these transgenic Arabidopsis for small RNA sequencing in order to find out the different expression pattern of P0 and its mutants.We further detected the RNA expression profile of P0WT by NGS,and found some genes which are differentially expressed in P0WT transformed Arabidopsis plants compared with Col-0.All in all,by transgenic approach,we confirmed the high pathogenicity of BrYV on crucifers,and revealed that the movement protein of BrYV might be the very viral agent which led to the purple leaf symptom on cruciferous plants.The NGS data of different transgenic plants which we got in this study are useful resources on revealing the molecular mechanism of BrYV pathogenicity.