Functional Analysis Of The Multimer Structure And Interacting Protein Of Cucumber Mosaic Virus 2b Protein

The Cucumber mosaic virus(CMV)2b protein is one of the early identified RNA silencing suppressor and exhibits inhibitory roles in multiple steps of RNA silencing pathway.First,2b interacts with AGO1 and AG04 proteins to compromise their cleavage activities.In addition,2b can bind long double-stranded RNA and siRNA of different lengths,which impedes siRNA amplification and assembling of functional RNA-induced silencing complex(RISC).Among these functions,the siRNA binding activity of 2b plays an essential role in suppressing post-transcriptional gene silencing(PTGS).However,the functional structure of 2b in viral pathogenicity and suppressor activities remains elusive.In the current studies,we investigated the multimeric forms of 2b protein of CMV Fny strain in the suppressor function and viral pathogenicity.Besides,we identified the interaction of the Arabidopsis DAYSLEEPER gene with 2b protein,and explored the effect of DAYSLEEPER gene on 2b's suppressor activities and viral pathogenicity.Our previous studies have found that the leucine at position 15(L15)and the methionine at position 18(M18)of the N-terminal region of CMV 2b protein played a key role in viral pathogenicity.The virus mutant harboring double alanine-substitution of L15 and M18 of 2b protein causes a attenuated infectivity in wild-type plants,but retains the ability of inducing severe symptoms in mutant plants deficient in RNA-dependent RNA polymerase 6(RDR6).Here,we constructed single mutant viruses CMV-2bl and CMV-2bm,and challenged the null mutants of DICER-LIKE(DCL)with single and double viral mutants.The results showed that the accumulation of mutant viruses were significantly increased in dcl4 and dcl2/4 mutant plants,indicating that DCL4 plays a vital role against CMV-2bl,CMV-2bm,and CMV-2blm.The 2b and 2blm transgenic plants were obstained and showed that the 2b transgenic Arabidopsis showed abnormal development,such as serrated leaves,dwarf,delayed flowering time,whereas the 2blm transgenic plants exhibited similar phenotypes with empty vector transgenic and non-transgenic plants.We further found that the transgenic 2b protein,but 2blm,rescued the pathogenicity and accumulation of CMV-m2b,a viral mutant defect in 2b protein expression.Agrobacterium-mediated transient expression of green fluorescent protein system demonstrated that the suppressor abilities of single mutants 2bl,2bm and double mutant 2blm were significantly compromised than 2b protein,although still had weak suppressor activities compared with the empty vector.Collectively,these results indicated that the L15 and M18 are required for 2b to suppress RNA silencing.To explore the implication of L15 and M18 in the structure and biochemical properties of 2b,we expressed and purified N terminus(amino acid 1-61)of 2b and mutant proteins in prokaryotic expression system.Through electrophoretic mobility shift assay(EMSA),we proved that the binding ability of 2blm with siRNA were largely decreased,but 2blm maintained the high affinity activity with long double-stranded RNA.In vitro cross-linking and in vivo experiments showed that CMV 2b protein could form dimer and tetramer structures,in contrast with that 2blm only formed dimer but not tetramer.These results show that N-terminal L15 and M18 of CMV 2b protein are key sites for siRNA binding activity and tetramer formation of 2b protein,which is critical for viral pathogenicity and 2b suppressor function.Our findings provide an insightful understanding in the function of CMV 2b protein multimerization in its suppressor activity and viral pathogenicity.During the interaction with their hosts,many plant viruses establish successful infection through encoding RNA silencing suppressor to defense against host antiviral RNA silencing pathway.To limit viral pathogenicity,host plants may evolve some endogenous factors to restrain suppressor activities.Here,2b protein was used as a bait to screen its interacting proteins in Arabidopsis plants.We found a transposase DAYSLEEPER in Arabidopsis interacted with CMV 2b protein in the cytoplasm and nucleus of host plants through bimolecular fluorescence complementation(BiFC)and co-immunoprecipitation(Co-IP)assays.Pull-down experiments further confirmed that CMV 2b directly interacted with DAYSLEEPER in vitro.Through the analysis of DAYSLEEPER and 2b truncated mutants,we found that the N-terminal 1-149 amino acid of DAYSLEEPER containing a nuclear localization signal(NLS)and BED zinc finger domain could interact with 2b 1-61 amino acids in its N-terminal region,a siRNA binding domain.Consequently,DAYSLEEPER weakens suppressor activity of 2b protein in the Agrobacterium-mediated transient expression system.We further verified that DAYSLEEPER could negatively regulate 2b binding activity with siRNA through EMSA assays.Over-expression of DAYSLEEPER significantly reduces accumulation of wild-type CMV but did not affect that of CMV-m2b in Nicotiana benthamiana plants.Taken altogether,the transposase DAYSLEEPER improves antiviral immunity of host plants through interacting with and inactivating 2b in the suppression of RNA silencing.Our findings uncover a new evidence to show the long-term arm race during the evolution of plant viruses and their hosts.