HyperTRIBE mapping of the RNA m6A demethylase ALKBH9 binding sites in bamboo reveals its role in plant defense

Abstract RNA demethylation plays an important role in diverse biological processes. Intriguingly, RNA demethylation has not been reported in bamboo, which is known for its rapid growth. PheALKBH9, an m6A demethylase in bamboo, was stably transformed into rice and increased its susceptibility to rice blast disease. Heterologous expression of PheALKBH9 reduced the overall m6A modification levels in rice. Using HyperTRIBE (Targets of RNA-binding proteins Identified By Editing), we identified evolutionarily conserved PheALKBH9 target RNAs in both rice and Moso bamboo. Overexpression of PheALKBH9 led to higher protein expression and shorter poly(A) tails. Notably, PheALKBH9 directly bound to CCR4-associated factor1 (CAF1G) and poly(A)-binding genes (PABPC1 and PABPC2), potentially modulating poly(A) tail lengths. In addition, PheALKBH9 also bound to and removed m6A modifications from Perox4, JAZ7, and METS2, key players in plant immunity, suggesting that PheALKBH9 plays a role in plant disease resistance. In summary, our study unveils a previously unknown role of PheALKBH9-mediated m6A demethylation in response to blast disease and provides insights into its mechanisms in monocotyledonous plants.