RNA N6-methyladenosine (m~6A) Modification Involves In Replication Of Rice Black Streaked Dwarf Virus In Laodelphax Striatellus

Rice black streaked dwarf virus(RBSDV,genus Fijivirus)is transmitted by the small brown planthopper(SBPH,Laodelphax striatellus)in a persistent propagative manner and outbreak intermittently.After the rice plant is infected by RBSDV,it will be dwarf and can’t head sprouting,which threaten rice yield severely.At present,the most effective method for managing RBSDV-induced diseases is controlling vector insects due to the lack of disease-resistant varieties.m~6A methylation(N6-methyladenosine)modification is a type of RNA methylation with rich biological functions that exists widely in nature and synergically regulated by methyltransferases,demethylases and binding proteins.It has been reported that m~6A modification can also influence the virus life cycle in the host.For example,the cell metabolism and immune system of host are regulated by m~6A modification,and thus affect the virus replication.However,the effect of m~6A modification on the vector and virus transmission has not been reported.We found that the level of m~6A decreased after RBSDV infection using quantitative determination and immunofluorescence labeling.MERIP-seq technique was used to analyze the m~6A modification at the transcriptome level between nonviruliferous and viruliferous SBPHs.It was found that the total number of m~6A sites in the viruliferous group was less than that in the nonviruliferous group,and there were 686 different m~6A sites between the two groups.These results suggest that m~6A modification may be related to the life activity of RBSDV in vector SBPH.Then we cloned the full-length CDS of Ls METTL3 and Ls METTL14 in SBPH,which are two kind of methyltransferase,and verified their conservation in evolution by bioinformatics analysis such as constructing phylogenetic trees and multiple sequence alignments.RNA interference(RNAi)was performed by microinjection of ds RNA to silence the expression of Ls METTL3 and Ls METTL14 of SBPH(injection of ds GFP as the control).The silencing effect was significant(decreased by 61-79%)and could last for more than 9 d confirmed by RT-q PCR.After injection,the m~6A level declined by 21.12%compared with the control,which verified its methyltransferase function.Finally,RNAi was used to reduce the m~6A level of SBPH in nonviruliferous and viruliferous groups that were fed on RBSDV-infected rice plants for a 3-d acquisition access period(AAP),and then RT-q PCR,Western blot,immunofluorescence labeling and other methods were used to detect the titers of RBSDV.The results showed that the RBSDV titers in SBPH injected with ds Ls METTL3+14 was significantly higher than that in SBPH injected with ds GFP.In conclusion,m~6A level of SBPH decreased after infected with RBSDV,and the accumulation of RBSDV significantly increased after the m~6A level was inhibited by silencing Ls METTL3 and Ls METTL14.Therefore,m~6A modification may have a negative effect on the replication of RBSDV in SBPH.However,how the regulation is carried out in detail needs more research.