The ARID1-AGO9 complex orchestrates easiRNA movement during sperm maturation in Arabidopsis [RIP-seq]

We report that profiles of small RNAs bound by ARID1 and AGO9 in inflorescences by RIP-small RNA sequencing technology. Sperm maturation triggers the reactivation of heterochromatic transposable elements (TEs) in the companion vegetative cell, which results in easiRNA (epigenetically reactivated siRNA) production to silence TEs in sperm via movement in pollen. How easiRNA movement is initiated during sperm maturation and when TEs are reactivated are open questions. Here, we report that TE reactivation initiates after the first asymmetric pollen mitosis and the shuttle-localized transcription factor ARID1 triggers easiRNA movement by associating with AGO9 from the vegetative cell to sperm. Dysfunction of both ARID1 and AGO9 causes over-reactivation of a subset of heterochromatic TEs in pollen. Mechanistically, ARID1 interacts with AGO9, in which ARID1 facilitates the recruitment of AGO9 to TE chromatin and instead AGO9 regulates the transient localization of ARID1 in the generative nucleus. Moreover, ARID1-bound easiRNAs are highly overlapped with those of AGO9, and the association of ARID1 with easiRNA is AGO9-dependent. In consequence, mutations of both ARID1 and AGO9 attenuate easiRNA movement from the vegetative cell to germline cells, thus finally leading to de-repression of TE in sperm. Collectively, these results fill the gap how siRNAs move in gametes and uncover a delicate collaboration between a gametic-specific transcription factor and AGOs to maintain genome integrity during fertilization. Overall design: Immunoprecipitation assay was performed using proARID1::ARID1-GFP Col-0 and proARID1::ARID1-GFP ago9-2 plants, in which GFP-Trap beads and anti-AGO9 antibody were used for immunoprecipitated RNA associated with ARID1 and AGO9, respectively. Total RNAs were purified from ARID1- and AGO9 asscociated precipitates, and purified RNAs were analyzed by small RNA sequencing, using Illumina Hiseq 2000. Two biological replicates were done.