Mechanism Of A Novel Ubiquitin-like Protein-mediated 26S Proteasome Pathway To Degrade The Rice Streak Virus Silencing Suppressor P3

Rice stripe blight,caused by rice stripe virus and transmitted by Laodelphax striatellus eggs in a sustained circulation,is a kind of vital viral disease on the agricultural industry,which engenders rice a large area production shortfall and brings enormous financial loss.RSV is a type of negative single-strand RNA virus,consisting of four single-strand RNAs that encoding seven proteins.RNA3 strand adopts negative-encoding strategy to encode two proteins,which positive encodes a nonstructural protein p3 with a protein size 23.9 kDa,has been identified as a suppressor of gene silencing.p3 may specifically recognize and bind to double-strand or single-strand siRNA,which prevents siRNA enter into RNA-induced silencing complex to counteract host gene silencing.To date,there is less report about the effect on p3 function through host protein.In the previous research work,the rice ubiquitin-like protein 5(named OsUBL5),interacting with p3,was screened from rice cDNA library through yeast two-hybrid system.Furthermore,interaction betweenUBL5 and p3 were tested by Y2 H,BiFC and Co-IP experiments.In order to determine the function of OsUBL5 during RSV infection,RSV was mechanically inoculated onto N.benthamiana leaves basing on its experimental condition property,so the following work was done in N.benthamiana plants.Three OsbUBL5 homologous sequences were annotated on the solgenomic database by BLAST search and they shared98% amino acid similarity,so the interaction between NbUBL5 s and p3 were also confirmed by Y2 H,BiFC and Co-IP technology.Next,conservatism ofUBL5 in different species was analyzed by DNAMAN,result showingUBL5 had nine conserved amino acid sites in open reading frame.Based on those conserved sites mutant,N7 or D64 was confirmed as the key interacting site with p3 via Y2 H and BiFC.To determine the biological significance of interaction betweenUBL5 and p3 during RSV infection,partial NbUBL5 segment was constructed into TRV vector and NbUBL5 s was systematically silenced basing on virus induced gene silencing system.During the expression of NbUBL5 s down-regulated to 15%,plant showed slight chlorosis and stunting symptom on the systemic leaves.After RSV was inoculated onto leaves,accumulation of RSV was analyzed by northern blot.Result showed NbUBL5s-silenced plants has more RSV-infected yellow spot and RSV RNAs accumulation on the inoculated leaves,indicating the silencing NbUBL5 s facilitate RSV infection.In addition,qRT-PCR result showed RSV infection induces OsUBL5 and NbUBL5 s up-regulated expression,overexpressing of NbUBL5 plants were also obtained by agrobacterium-mediated genetic transformation method and three overexpressed transgenic lines were identified by qRT-PCR.Transgenic plants had no obvious development phenotype comparing with WT.After RSV was inoculated onto plants,RSV symptom was milder on transgenic plants than WT and the accumulation of RSV RNAs was also more less,indicating overexpressing of NbUBL5 confer resistant to RSV.Meanwhile,overexpressing of OsUBL5 rice plants were obtained based on the same way,which also had no obvious difference in growth and development than wild type.After Laodelphax striatellus of carrying RSV fed the overexpressed plant and wild type,morbidity and accumulation of RSV RNAs on transgenic plants were less than wild type plants,indicating overexpressing of OsUBL5 plants also conferred the resistant to RSV.In a word,UBL5 involves in host antiviral response to RSV infection.RSV p3 is known as RSV suppressor,which plays essential role in RSV successful infection,so NbUBL5 or OsUBL5 having an effect on p3 suppressor function was further done by transient expression way.Result showed the expressing of NbUBL5 or OsUBL5 inhibit p3 suppressor function on 16 c plants and the accumulation of p3 protein was strikingly reduced.Meanwhile,the expression of NbUBL5 and OsUBL5 on N.benthamiana plants also reduced p3 protein accumulation,suggesting the expressing of NbUBL5 and OsUBL5 affect p3 suppressor function.The interacted defect of D64 mutant results confirmed that NbUBL5-mediated p3 degradation depend on interaction between p3 andUBL5.Next,to clarity the mechanism of p3 degradation,according toUBL5 belong to ubiquitin-like protein family member,having similar structure with ubiquitin,so co-expressing p3 and NbUBL5 or OsUBL5 leaves were treated by using inhibitor MG132.Western blot showed accumulation of p3 protein was more than control,which indicatedUBL5 degrades p3 through 26 S proteasome.Ub and UBLs could be recognized by the receptor subunits of 26 S proteasome,which was a necessary process against canonical protein degradation via ubiquitin-proteasome pathway.We further confirmed NbUBL5 and NbRPN10 or NbRPN13 interaction,and also OsUBL5 and OsRPN10 or OsRPN13 interaction through Y2 H,BiFC and Co-IP.Moreover,the degradation of p3 caused byUBL5 had an obvious recovery on the NbRPN10-silenced or NbRPN13-silenced plants.These results further indicate thatUBL5 mediated p3 degradation depends on interaction with ubiquitin receptorRPN10 and RPN13.In a word,this study identified a novel ubiquitin-like proteinUBL5,which interacted with p3,and determinedUBL5-mediated p3 degradation through 26 S proteasome pathway,and also the interaction between p3 andUBL5 was necessary to affect p3 suppressor function,which had a resistant to RSV.Those help us well understand the relationship of RSV-host interaction,plant antiviral and viral pathogenicity,and also give the theoretical basis for extending pathway of plant antiviral.