| Antiviral RNAi is a broad and conservative epigenetic regulatory process and similar to RNAi-mediated gene silencing.After virus infection,the double-stranded RNA(ds RNAs)generated in replication are recognized by Dicer protein and cleaved into 21-30 nt si RNAs.si RNA and Argonaute(AGO)form RNA-induced silencing complex(RISC)to induce degradation or translation of viral RNA.Arabidopsis(Arabidopsis thaliana)encodes 4 Dicer-like proteins(DCLs)and 5 double-stranded RNA(ds RNA)binding proteins(ds RBPs).And the 21 nt si RNA generated by DCL4 cleavage plays a major role in antiviral immunity.The specific interaction between DCL4 and DRB4 plays a key role in the generation of endogenous si RNA in plants,however,the specific function of DRB4 in the antiviral process is unknown.In this study,the drb4 mutant was obtained through CRISPR/CAS9 gene editing technology.After inoculation with virus,it was found that the virus accumulation was not increased significantly in the drb4 mutant,probably because other DRBs play redundant roles as DRB4.However,our study found that DRB4 interacts with antiviral regulatory factors(GLTP1 and LPEAT1)newly discovered in our laboratory.In addition,we found that the localization of DRB4-YFP fusion protein in antiviral RNAi defective ala1/ala2 mutant was changed.These results lay the foundation for revealing the specific function of DRB4 in antiviral RNAi.Tomato(Solanum lycopersicum)is an important economic vegetable crop and viruses drastically harm tomato production.Elucidating antiviral RNAi in tomato has important theoretical and practical significance.Our laboratory used the cucumber mosaic virus(CMV)with defective VSR to establish a genetic screening system to study plant antiviral RNAi.Tomato is one of CMV’s natural hosts.The system may also provide a good platform for studying antiviral RNAi in tomato.Preliminary studies have found that wild-type CMV can replicate efficiently in tomato Microtom,and causes serious diseases.However,CMV with defective VSR cannot efficiently replicate in Microtom,and the plants did not show disease symptoms.These results indicated that not only antiviral RNAi also plays an important role in tomato antiviral immunity,but also suggests that CMV-Δ2b provides a powerful weapon for studying tomato antiviral immunity.We use CRISPR to knock out RDR1 and AGO1 in tomato AC,and DCL2 and DCL4 in tomato Microtom to further explore the function of antiviral RNAi in tomato.This study will lay a foundation of antiviral RNAi mechanism of tomato and provide a new strategy for agricultural production practice. |
