Identification Of Amino Acid Residues And Domains Of Sugarcane Yellow Leaf Virus CHN3 Genotype Essential For Suppressing RNA Silencing

Sugarcane yellow Leaf disease(YLD)caused by Sugarcane yellow leaf virus(SCYLV)is a major disease in sugarcane-producing regions in China and worldwide,causing significant reductions in cane yield and sucrose content.SCYLV is a member of the genus Polerovirus and family Luteoviridae.The virus genome contains six recognized open reading frames(ORFs 0-5)and the ORF0 encodes the P0 protein that is an RNA silencing suppressor.POs of poleroviruses possessed high divergence in the sequences and constructs contribute to different mechnisams of P0 suppressing the RNA silencing,whereas it is not clearly known for the molecular mechnisam of SCYLV P0 RNA silencing suppressor.Thus,here we indetified the crucial amino acids(aa)residues and domains for SCYLV-CHN3 P0(P0CHN3)suppressing RNA silencing and investigated the interaction between P0CHN3 and Nicotiana benthamiana SKP1 and 14-3-3 proteins.Firstly,six conservative regions,1-50,51-71,76-125,129-157,161-210,and 211-251 aa in P0CHN3 protein were predicted.We deleted these six conservative domains,the first 15 aa of each N and C terminal,and the F-box motif and then cloned into the plant-expressiong vectors,respectively.The activity of RNA silencing suppressors of these mutants of P0 proteins were verified through GFP transient expression system in N.benthamiana.All of above-mentioned deletions of P0CHN3 resulted to significant decarese in the acitivty of RNA silencing suppressors.Secondly,12 conservative amino acids sites were observed aomng the P0 protein of poleroviruses and 11 amino acids sites subjected to positive-selection were found in the SCYLV P0.Then the amino acids being essential for P0CHN3 suppressing the RNA silencing was identified by single point mutagenesis.The deletions of each positive-selection amino acids did not affect on the activity of RNA silencing suppressor,whearas the two mutants of L85A in F-box motif and R138A in conservative sites depressed the activity of RNA silencing suppressor.Hence,we confered the L85 and R138 would be essential for P0CHN3 RNA silencing suppressor.Lastly,we investigated that the interaction in vivo between P0CHN3 and NbSKP1 as well as Nb14-3-3 using the yeast two-hybrid system.A slight interaction between P0CHN3 with NbSKP1 was observed,whereas a significant signal was found between P0CHN3 with Nb 14-3-3.The results demonstrated that the close relationship between P0CHN3 and Nb 14-3-3.In the present work,we found two key amino acids and domains in P0CHN3 RNA silencing suppressor by mutagenesis and the strong interaction signal between P0CHN3 and Nb 14-3-3.Our findings provided an important foundation to further studies on the molecular mechanism between SCYLV and host plants.