CHH DNA methylation increases at 24-PHAS loci depend on 24-nt phasiRNAs in maize meiotic anthers

Plant phasiRNAs contribute to robust male fertility, however, specific functions remain undefined. male sterile23 (ms23), necessary for both 24-nt phasiRNA precursor (24-PHAS) loci and Dicer-like5 (Dcl5) expression, or dcl5-1 mutants unable to slice PHAS transcripts, lack nearly all 24-nt phasiRNAs. Based on capture sequence bisulfite sequencing, we find that CHH DNA methylation of most 24-PHAS loci is identical in control and mutant anthers prior to PHAS transcriptional activation. However, increased CHH methylation that is present in fertile anthers expressing 24-PHAS is absent in these mutants. Since dcl5-1 anthers express PHAS precursors, we conclude that the 24-nt phasiRNAs, but not the activation of PHAS transcription is required for targeting CHH methylation to these loci. Although PHAS precursors are processed into multiple 24-nt phasiRNA products, there is dramatic differential product accumulation. Higher CHH methylation is correlated with 24-nt phasiRNA abundance within individual loci, reinforcing the conclusion that 24-nt phasiRNAs contribute to increased CHH methylation at complementary sites.