R-Loop-Regulated Chloroplast Genome Stability In Arabidopsis Thaliana

R-loop biology has become a hot research topic in recent years.It is a special chromatin mark in the genome,which is composed of RNA:DNA hybrid and a single strand DNA.R-loop structure on the genome is involved in a variety of biological processes and affects the stability of the genome.Therefore,the control of R-loop level is crucial to genome stability maintenance.Although a large number of studies have shown that R-loop exists widely in various species,there are few studies in plants,and still blank in chloroplasts.To extend our understanding of R-loops in plant organelles,we started to analyze the biological functions of the organelle-localized RNase H1 enzymes,which can remove R-loops.We successfully identified a chloroplast located RNase H1 enzyme At RNH1 C,which can effectively remove the R-loops from the genome.Through the analysis of At RNH1 C protein interactome,we provide evidence that At RNH1 C collaborates with At Gyrases to restrict the R-loop levels formed by transcription-replication conflicts in r DNA regions,and thus to maintain the stability of chloroplast genome.This work is the first time to identify and reveal how plant organelles maintain genome stability by controlling the level of R-loops.In order to establish the regulatory network of R-loop mediated regulation of chloroplast genome stability in Arabidopsis thaliana,we took atrnh1 c mutant as the research model and adopted two screening strategies,including overexpression and EMS,to find the maintenance factors involved in chloroplast genome stability.Through screening,we successfully identified a RNA:DNA hybrid helicase RHON1,it can partially reduce the overaccumulated R-loop levels caused by atrnh1 c gene mutation,when we overexpress RHON1 in atrnh1 c mutant.Further studies have found that RHON1 can interact with plastid-encoded RNA polymerase(PEP).RHON1 can alleviate the conflicts between transcription and replication by regulating the transcriptional activity of PEP.Our results indicate that RHON1 is another R-loop removal pathway in parallel with At RNH1 C.In addition,we also performed EMS random mutagenesis in atrnh1 c mutant plants to screen the suppressor of atrnh1 c yellowish phenotype,thus to searching for new regulators or new pathways involved in R-loop removal in chloroplasts.At present,dozens of suppressors and candidate genes have been obtained,through whole genome sequencing and bioinformatics analysis.In the first two parts of this paper we first identified two R-loop mediated chloroplast genome stability maintenance pathways,and the third part will provide a valuable resource for our subsequent screening.These parts of the work will fill in the gaps in the study of chloroplast stability by regulating R-loop levels,and also open a new window to explore how plant chloroplast genome maintains its integrity.