The Assembly Mechanism And RdDM-independent Function Of RNA Polymerase V In Arabidopsis

RNA polymerase V(Pol V),a plant-specific RNA polymerase,plays an important role in the RNA-directed DNA methylation(Rd DM)pathway.In Rd DM,Pol V transcribes long non-coding RNAs,which act as scaffolds and recruit Rd DM components to catalyze DNA methylation at Rd DM target loci.In this study,we investigate the mechanism of Pol V assembly and Rd DM-independent roles of Pol V.We performed a forward genetic screen and identified mutant alleles of MINIYO(IYO)and QUATRE-QUART 2(QQT2)that cause defects in Rd DM in Arabidopsis thaliana.NRPE1,the largest subunit of Pol V,failed to associate with other Pol V subunits in the iyo-4 and qqt2-2 mutants,suggesting the involvement of IYO and QQT2 in Pol V assembly.IYO associates with the NRPE3 subas sembly and QQT2 associates with the NRPE2 subassembly.IYO and QQT2 interact to bridge the NRPE3 and NRPE2 subassemblies prior to the assembly of Pol V holoenzyme.We performed RNA-sequencing(RNA-seq)of Arabidopsis wild-type Col-0,and Pol IV and Pol V largest subunit null mutants,nrpd1 and nrpe1 and Chromatin-Immunoprecipitation-sequencing(Ch IP-seq)of Col-0 and nrpe1.We found that Pol V transcripts,produced upstream of ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 5(ERF5)and ERF6,respectively,positively regulate ERF5 and ERF6 expression independently of Rd DM.ERF5 and ERF6 positively regulate jasmonate-mediated plant defense against necrotic fungi in a redundant manner.The induction of jasmonate-responsive gene PDF1.2a by methyl jasmonate(Me JA)was compromised in nrpe1,but not in nrpd1.Furthermore,the nrpe1 mutant,but not the nrpd1 mutant,showed increased susceptibility to the beet armyworm Spodoptera exigua and necrotic fungus Botrytis cinerea.Our results suggest that Pol V can enhance plant resistance against insect and fungal pathogens independently of Rd DM.In summary,this study provides insights into how Pol V is assembled and reveals Rd DM-independent regulation of gene expression by Pol V.This study also offers new targets for the improvement of pathogen resistance in crops.