Functional Characterization of Two RNA Methyltransferase Genes METTL3 and METTL14 Uncovers the Roles of m 6 A in Mediating Adaptation of Plutella xylostella to Host Plants.

N 6 -methyladenosine (m 6 A) is one of the major epigenetic modifications in eukaryotes. Although increasing functions of m 6 A have been identified in insects, its role in Plutella xylostella L. for host plant adaptation remains unclear. In the current study, we show that the m 6 A content of P. xylostella was relatively low in different developmental stages and tissues, with no significant differences. Two RNA methyltransferase genes, PxMETTL3 (methyltransferase-like 3) and PxMETTL14 (methyltransferase-like 14), were identified and characterized. PxMETTL3 could be transcribed into two transcripts, and PxMETTL14 had only one transcript; both of these genes were highly expressed in egg and adult stages and reproductive tissues. The CRISPR/Cas9-mediated knockout of PxMETTL3 (Δ PxMETTL3-2 ) or PxMETTL14 (Δ PxMETTL14-14 ) confirmed their function in m 6 A installation into RNA. Furthermore, upon transfer from an artificial diet to the host plant, the mutant strains were affected in terms of larval and pupal weight or adult emergence rate, while the wildtype (WT) strain did not exhibit any difference. In addition, the fecundity and egg hatching rate of the WT strain decreased significantly, whereas only the Δ PxMETTL14-14 mutant strain displayed significantly decreased fecundity. There seemed to be a tradeoff between the stress adaptation and reproduction in P. xylostella mediated by m 6 A modification. During host transfer, the expression of PxMETTL14 was consistent with the change in m 6 A content, which implied that PxMETTL14 could respond to host plant defense effectively, and may regulate m 6 A content. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed transcripts with changes in m 6 A levels revealed that the potential functions of m 6 A-related genes may be involved in steroid biosynthesis for larval performance and metabolic pathways for adult reproduction. Overall, our work reveals an epigenetic regulation mechanism for the rapid adaptation of P. xylostella to variations in the host environment.