N~6-methyladenosine Modification Limits Pepino Mosaic Virus(PepMV) Infection By Recruiting The RNA Decay Machinery

Pepino mosaic virus is a positive single-stranded RNA virus,belonging to the genus Potexvirus.Since PepMV was first isolated from pepino in 1974,it has become one of the most important diseases in tomato crops,distributed in most areas of Europe,America,and parts of Africa,Asia,and Oceania.Now,this virus has been reported in Shanghai and Yunnan in China.PepMV mainly infects Solanaceae crops,leading to bubble bumps,chlorosis,yellow leaf spots,and serious mosaic on tomato leaves.In addition,it can also cause marbling,discoloration,and even open fruits,which seriously affects the yield and quality of tomatoes.N~6-methyladenosine(m~6A)modification is the most abundant internal modification of m RNA in eukaryotes,involved in various biological processes.The role of m~6A in antiviral immunity in mammals has been extensively studied.However,the function of m~6A in plant virus infection is obscure.In this paper,the dynamics and function of m~6A in tomato and Nicotiana benthamiana plants were studied during the infection of PepMV.In this study,we analyzed virus-infected tomato and N.benthamiana plants by Me RIP-seq and obtained high-quality m~6A methylome.Meanwhile,it was found that m~6A modifications on the PepMV genome in these two species are quite conserved,and are mainly distributed at the 3’-terminal of PepMV genomic RNA.In order to clarify the role of m~6A modification in PepMV infection,we used the VIGS system to silence m~6A methyltransferase MTA and HAKAI in tomato and N.benthamiana plants,and the results showed that the silencing of MTA and HAKAI promoted PepMV infection.We further obtained the Nb HAKAI knockout mutant.After inoculating with PepMV,the viral RNA level and CP accumulation were significantly increased,and the m~6A modification of viral particle RNA was significantly decreased,indicating that the deletion of HAKAI reduced the m~6A modification level of viral RNA to increase viral RNA loads.On the other hand,we obtained Nb MTA and Nb HAKAI transgene overexpressing plants.After inoculating with PepMV,the accumulation of viral RNA and protein was inhibited,but the m~6A modification of viral RNA was increased.These results suggest that HAKAI and MTA suppress PepMV infection,and m~6A modification levels of viral RNA are negatively correlated with viral pathogenicity.To explore how m~6A-related proteins modify viral RNA.The interactions between m~6A methyltransferases and PepMV encoded proteins by yeast-two hybrid(Y2H)were analyzed.The results showed that Nb HAKAI had a strong interaction with PepMV Rd RP.The interaction was further confirmed by co-localization and bimolecular fluorescence complementation(Bi FC).We further found that the N-terminal RING Ubox domain of Nb HAKAI is required for Nb HAKAI-Rd RP interaction and Nb HAKAI-mediated antiviral activity.These results suggest that the antiviral effect of Nb HAKAI depends on the interaction between Nb HAKAI and Rd RP.We further analyzed the m~6A readers in N.benthamiana and found that several readers were up-regulated during PepMV infection.By VIGS silencing system and transient expression of Nb ECT2A and Nb ECT2B,these two m~6A readers were found to play anti-PepMV roles.Given the negative correlation between viral m~6A level and viral RNA level,we screened Nb ECT2A and Nb ECT2B for interactions with proteins related to the RNA decay-related pathway by Y2H.The results showed that both Nb ECT2A and Nb ECT2B could interact with several RQC key factors.Knocked-out of Nb DCP1,a key gene in P-body,abolished the Nb HAKAI,Nb MTA,Nb ECT2A,and Nb ECT2B-medited anti-PepMV function.These results suggest that the m~6A readers recognize m~6A methylated viral RNAs and recruit them to P-body for degradation.In summary,we reveal a new strategy by which hosts utilize the m~6A modification and RNA degradationto to inhibit PepMV infection,which would provide new insights into the antiviral function of m~6A and RNA degradation in plants.