Plant phased small interfering RNAs (phasiRNAs) contribute to robust male fertility; however, specific functions remain undefined. In maize (Zea mays), male sterile23 (ms23), necessary for both 24-nt phasiRNA precursor (24-PHAS) loci and Dicer-like5 (Dcl5) expression, and dcl5-1 mutants unable to slice PHAS transcripts lack nearly all 24-nt phasiRNAs.

Based on sequence capture bisulfite-sequencing, we find that CHH DNA methylation of most 24-PHAS loci is increased in meiotic anthers of control plants but not in the ms23 and dcl5 mutants.

Because dcl5-1 anthers express PHAS precursors, we conclude that the 24-nt phasiRNAs, rather than just activation of PHAS transcription, are required for targeting increased CHH methylation at these loci.

Although PHAS precursors are processed into multiple 24-nt phasiRNA products, there is substantial differential product accumulation. Abundant 24-nt phasiRNA positions corresponded to high CHH methylation within individual loci, reinforcing the conclusion that 24-nt phasiRNAs contribute to increased CHH methylation in cis.