Genetic Diversity And Functional Analysis Of RNA Silencing Suppressor Encoded By Sugarcane Yellow Leaf Virus(SCYLV) Genotypes

Sugarcane yellow leaf disease(YLD)caused by Sugarcane yellow leaf virus(SCYLV)is a major disease in sugarcane-producing regions in China and worldwide,leading to significant reductions in cane yield and sucrose content.SCYLV is a member of the Polerovirus genus within Luteoviridae family,which contains six recognized open reading frames(ORFs 0-5).SCYLV genetic population and RNA silencing suppressors encoded by ORFO among different genotypes were investigated in this study.A total of 332 sugarcane leaf samples collected from seven provinces in China were used for SCYLV detection.Besides,we cloned and sequenced the 5' end of SCYLV genomic regions containing a complete P0 sequence among 102 SCYLV-positive samples and two complete genomes of GZ-GZ18 and HN-CP502 isolates.A novel recombinant genotype CHN3 regenerated from the major parent CHN-HN1(BRA genotype)and the minor parent CHN-GD-WY19(PER genotype)was referred for the first time at full genomic and P0 nucleotide levels.Two isolates of CHN3 genotype shared high genetic variability with 86.4%-98.3%of nucleotide sequence identify compared to other isolates in different genotypes.Our results showed that P0 protein contained three significant conserved regions of 1-60,76-125,and 161-210 amino acid residues and eight positive selection sites.The P0 RNA silencing suppressor was proved to be efficient in enhancing reporter gene expression in transient assay using sugarcane young leaf segments.The conserved regions of P0,15-aa in N terminal and 102-aa in C terminal,are necessary for the RNA silencing suppressor activity.Sub-cellular localization of P0 expression was observed in the nucleus through bombarding on onion epidermal cells.The suppressor activity of P0 encoded by four different SCYLV genotypes was analyzed in sugarcane and Nicotiana benthamiana line 16C.The findings revealed that BRA-P0,CHN3-P0 and PER-P0 would be strong suppressors,but not for CHN1-P0.BRA-P0,CHN3-P0,PER-P0 and CHN1-P0 increased more than 3.5,5.3,3.9 and 2.6-fold of EYFP expression level,respectively in sugarcane young leaf segments at 120 h post-bombardment.GFP expression level of mRNA was enhanced by 2.8,1.5,1.7 and 0.6-fold for BRA-P0,CHN3-P0,PER-PO and CHN1-P0,respectively,in 16C line at 3 dpi(days post injection).The interactions of SCYLV P0 protein with Arabidopsis thaliana AGOs and Nicotiana benthamiana S-phase kinase-associated protein 1(NbSKP1)were performed through yeast two hybrid system(Y2HS)and bimolecular fluorescence complementation(BiFC).P0 protein directly interacted with NbSKP1,but not with AtAGO1,AtAG04,AtAG06.Thus,P0 may mediate with AGOs in suppressing RNA silencing by ubiquitination pathway.In this study,highly genetic diversity of SCYLV populations and the differences of P0 suppressing RNA silencing among SCYLV genotypes were investigated.Additionally,we also explored the interaction P0 protein with AGOs and SKP1.The findings contribute to understand SCYLV population genetic structure and the function of P0 suppressor and help of further exploring the molecular mechanisms between SCYLV and plant host.