Molecular Mechanisms Of Cucumber Mosaic Virus Coat Protein In Negatively Regulating Viral Infection Of The Shoot Apical Meristem

Cucumber mosaic virus(CMV)could infect monocots and dicots,including crops,ornamentals,vegetables,and caused serious economic loss worldwide.The coat protein(CP)and suppressor 2b are both multifunctional factors in systemic movement and virulence determination processes.In our previous studies,the wild-type CMV(CMVWT)transiently invades SAM and then disappears in the late infection of SAM,which causes symptom "recovery" in infected N.benthamiana plants.In contrast,a mutant of CMV,named CMVRV harboring alanine substitution in the N-terminal R-rich region(R13RRRPRR19)of CP,persistently invades SAM with high accumulation and induces the reduction in apical dominance.Additionally,CMVWT induces more potent antiviral RNA silencing than CMVRA in SAM regions,suggesting that RNA silencing is involved in the negative regulation of CP on the SAM invasion of CMV.In co-infiltration assays,2b could suppress GFP-induced local silencing,which was attenuated by co-expression of CPWT but not CPRA.Moreover,the analysis for accumulations of GFP mRNA and siRNA indicated that CPWT,rather than CPRA,significantly induced siRNAs amplification in co-infiltrated patches.We further inoculated the wild-type Arabidopsis thaliana,rdr6,and sgs3 mutant plants with CMVWT and CMVRA,showing that CMVWT induced host RDR6/SGS3-dependent antiviral silencing and negatively regulated the viral virulence in SAM.Since the N-terminal R-rich region was highly enriched in basic amino acid residues,CMV CP bind RNA nonspecificly and inhibited translation,which might recruit SGS3/RDR6 complexes to induce siRNA amplification.The R-rich region is highly conserved in the CPs of cucumoviuses and mediates negative effect on 2b-mediated silencing suppression.In addition,CPWT could also compromise the suppression of other silencing suppressors,implying that CMV CP might modulate synergistic infections naturally.Previous studies have shown that transgenic Arabidopsis plants expressing the 2b protein of the CMV Fny strain exhibited developmental abnormalities,which mainly attribute to the interaction of 2b and AGO1 in endogenous miRNA pathways.Here,the 2b-induced abnormal developments were markedly compromised in the transgenic plants co-expressing CPWT and 2b.In addition,co-expression of CPRA could also rescue the 2b-induced abnormal phenotype,indicating the siRNA amplification and translation inhibition caused by CPWT but not CPRA are not responsible for the negative effects of CMV CP on the regulation of 2b in endogenous miRNA pathways.The in vivo and in vitro interaction assays showed that CP could bind to 2b and interfere the 2b-AGO1 interaction,leading to rescue of AGO 1 in miRNA pathways.Collectively,the CP-2b interaction plays important roles in viral pathogenesis in host plants.Firstly,CP could induce more potent antiviral silencing by facilitating the amplification of siRNA,and modulate the accumulation of 2b protein by translation inhibition,which cause symptom recovery in plants.Secondly,CP could bind 2b competitively and attenuate the 2b-AGO1 interaction to release AGO1 for miRNA pathway.They are both new evolutionary strategies to mediate viral self-attenuation to ensure the relatively healthy state of hosts for the long-term infection of virus.