Roles Of Demethylase Gene Family OsRos1 In Controlling Resistance To Bacterial Leaf Streak In Rice

DNA methylation is an important way of epigenetic modification in plant growth and development,regulating genome stability and functional reversibility.Studies in arabidopsis thaliana have shown that DNA demethylation plays an important role in plant disease resistance,but the regulation mechanism is still not clear,needing to be further studied.Bacterial leaf streak(BLS)is one of the most destructive diseases in rice.Studies have shown that BLS resistance in rice is quantitatively inherited,but knowledge about its genetic basis is very limited.In the present study,the regulatory role of demethylase gene OsRos1 gene in BLS was investigated.The main results are as follows:(1)It was found from public databases that there are four members in OsRosl gene family(OsRosla,OsRos1b,OsRos1c and OsRos1d),which all contain the DNA glycosylase domain and 4Fe-4Se domain and are involved in the epigenetic regulation of gene expression and are expressed in seedlings,leaves,flowers and seeds.The expression of OsRos1a and OsRoslc in rice leaves is high,while the expression of OsRos1bis lower,and OsRos1dis almost not expressed in leaves.(2)The responses of these four genes to BLS pathogen were analyzed using qRT-PCR.Results indicated that while the expression of OsRos1dwas too low to be detected,OsRos1a,OsRos1b and OsRos1c allshowed certain levels of response to the infection of BLS pathogen,and OsRosla and OsRos1c showed upregulated expression at 3h after inoculation.(3)The knockout expression vectors of OsRos1a and OsRos1c were constructed using the CRISPR/Cas9 gene editing system and then transfered into japonica variety Nipponbare,generating 29 and 25 transgenic positive plants,respectively.Eight Osros1a mutants and seven Osms1c mutants were identified by sequencing.Among them,2 and 3 were Osrosla and Osros1c homozygous mutants,respectively.(4)The Osros1a mutants exhibited normal vegetative growth but could not produce seeds,making the mutant alleles not inheritable to the next generation.The Osros1c mutants were fertile,but showed reduced plant height and increased tiller number,suggesting that the gene positively regulates plant height but negatively regulates tiller number,respectively.(5)Inoculation test showed that the BLS resistance of Osros1c mutants was significantly lower than that of wild type.The results suggest that OsRos1c positively regulates BLS resistance;the OsROSlc-mediated demethylation is related with the resistance to BLS disease in rice.(6)MSAP analysis of genomic DNA methylation showed that the genome methylation level of Osroslc mutants was similar to that of control,but had some changes in demethylation and methylation.The results imply that OsRoslc mutation only affects the methylation status of specific sites and has little effect on the methylation level of the whole genome.