Research On The Mechanism Of HrpD5 Positively Regulates The Expression Of HrpF In Xanthomonas Oryzae Pv.oryzicola

Xanthomonas oryzae pv.oryzicola(Xoc)is a gram-negative plant pathogenic bacterium in rice.Xoc can infect the host rice and cause bacterial leaf streak(BLS),which mainly relys on its type III secretion system(T3SS)encoded by hrp gene cluster.The hrp gene cluster of Xoc is about 27 kb and consists of 10 hrp genes,9 hrc(hrp-conserved)genes and 8 hpa(hrp-associated)genes mainly regulated by two key hrp regulators HrpG and HrpX,which is also known as hrp-hrc-hpa gene cluster.Previous studies found that the hrpF gene was down-regulated in hrpB1,hpa2,hrcT,hpaP,hrpB5,hrcU,hrcQ,hrpD6,hrcS,hrpD5,hrpX and hrpG mutants by studying the expression of hrpF gene in 29 hrp gene mutants.In order to reveal the mechanism of the decrease of hrpF transcription in these 12 mutants.The mechanism of the decrease of hrpF expression in 12 mutants compared to RS105 was studied from the level of transcription and translation using hrp::gusA fusion expression system and complementary vector pML123.The results of hrpF GUS activity assay showed that the GUS activity of hrpF in R△hpa2,R△hpaP,R△hrcQ,R△hrcS,R△hrpD5,R△hrpD6,R△hrpG and R△hrpX was significantly lower than that of RS105,which was significantly increased or no significant change in other mutants.The results of hrpX and hrpG GUS activity assay showed that the GUS activity of hrpX and hrpG in R△hpa2,R△hpaP,R△hrcS,R△hrpD5,R△hrpD6,R△hrpG and R△hrpX was significantly lower than that of RS105.The results of real-time PCR revealed that the mRNA levels of hrpF,hrpX and hrpG was significantly reduced in R△hpa2,R△hpaP,R△hrcS,R△hrpD5,R△hrpD6,R△hrpG and R△hrpX compared with RS105.The results of injection of non-host tobacco and seedling susceptible rice IR24 showed that R△hrpD5 lost the pathogenicity in susceptible host rice and the ability to trigger HR in non-host tobacco.There was no water soaking after injecting seedling susceptible rice IR24 with R△hrpD5,but the complementary strain CR△hrpD5 can produce water soaking in host rice and trigger HR in non-host tobacco.The results of real-time PCR revealed that the mRNA levels of hrpF,hrpX and hrpG in the complementary strain CR△hrpD5 was restored to the wild type strain RS105.These results indicate that the hrpD5 gene can positively regulate the expression of hrpF gene by affecting the HrpG-HrpX pathway.These provide a new clue for the analysis of Xanthomonas hrp regulatory network.