Functional Analysis Of Two Xrv Genes Of Xanthomonas Oryzae Pv.Oryzae Strain PXO99~A

Bacterial blight of rice is the vascular disease caused by gram-negative plant pathogenic bacteria Xanthomonas oryzae pv.Oryzae,which seriously affects the sustained growth of rice plant.Gram-negative bacteria contain a class of conserved nucleoid-structuring proteins(H-NS)which are similar to eukaryotic histones.As a kind of important regulators,H-NS proteins can bind DNA and acts as a global transcriptional repressor.H-NS proteins regulate the expression of many genes including virulence-related genes and environment-responsive genes.Three genes encoding H-NS proteins,xrvA,xrvB and xrvC(Xanthomonas regulator of virulence)were found in the genome database of PXO99A with the conserved C-terminal domain of H-NS proteins,which are all conserved in two other sequenced Xoo strains.In this study,xrv gene knock-out mutants were generated from PXO99A based on double crossover homologous recombination.The cosmid pHMxnvA,pHMxrvC and pHMxrvAxrvC containing the xrv coding sequence were electroporated into the respective mutants to construct the complementation strains.The functions of xrv genes were explored by measuring the growth rate in the NB and XOM2 media,the ability of eliciting hypersensitive response in tobacco leaves and the virulence in rice of the xrv-deleted mutants compared with the wildtype.This study has conducted the preliminary inquiry on the mechanisim that xrv genes regulate the virulence.The sucrose counter-selection vector pExrv was constructed to knock out xrv genes by overlap-PCR.Then pExrv was transformed into PXO99A to select the transformants.Xrv gene knock-out mutants were confirmed by PCR.Besides,the complementation strains of xrv genes were generated by introducing pHMbxrv into the respective xrv gene knock-out mutant.Here,we found that no matter under the nutrient-rich or nutrient-poor condition,the growth rates of xrv gene knock-out mutants were greatly slower than PXO99A.The ability of xrv mutants inducing hypersensitive response in tobacco leaves were stronger compared with PXO99A.When inoculated in rice leaves,the virulence of xrv mutants was significantly decreased on IRBB214.And there was no obvious difference in virulence of xrv mutants compared with the wildtype on other 31 rice varieties.qRT-PCR detection results revealed that the expression of hrPG,hrpX,hpa1 and most non-TAL genes were significantly decreased in xrv mutants compared with that in the wild-type.Among the three xrv mutants,the expression levels of hrpG in PXO99?xrvA,PXO99?xrvC and PXO99?xrA?rvC were only 57.6%,40.8%and 24.0%as that in PXO99A.Besides,the expression levels of hrpX in PXO99?xrvA,PXO99?xrvC and PXO99?xrvA?xrvC were only 58.2%,51.8%and 42.4%as that in PXO99A.The expression levels of hpal in PXO99?rvvA PXO99?xrvC and PXO99?xrvA?xrvC were only 60.1%,60.9%and 58.3%as that in PXO99A.The expression of rice susceptible gene Os8N3 was detected by qRT-PCR after inoculated with xrv mutants,we found that the expression levels of Os8N3 in rice leaves inoculated with PXO99?xrvA,PXO99?xrvC and PXO99?xrv?xrvC were only 29.5%,23.1%and 10.3%respectively as that in leaves inoculated with PXO99A.The detection of cAMP revealed that the loss of xrv genes did not influence the secretion of PthXol protein into plant cells.The results suggest that the losses of xrv genes reduce the growth rate of PXO99A and enhance the ability of inducing hypersensitive response in tobacco leaves.Xrv genes can positively regulate the expression of hrp genes and most non-TAL genes.Furthermore,xrv genes play a vital role in regulating virulence on rice by influcing the expression of susceptible gene Os8N3.