Analysis Of The IAA Synthesis And Its Action Mechanism By Xanthomonas Oryzae Pv.oryzicola

Bacterial leaf streak is an important bacterial disease on rice and one of the phytosanitary diseases in China.Its pathogenic bacterium is Xanthomonas oryzae pv.oryzicola(Xoc),a pathogenic variant of rice streak,which has become a major bacterial disease of rice in southcentral and southern China.Although bacterial leaf streak seriously affects rice yield and quality during rice production,the molecular mechanisms of Xoc pathogenesis have been less studied.Therefore,further exploration of the pathogenesis of Xoc at the molecular level is of great theoretical and practical significance for ensuring food security and for gaining a deeper understanding of the pathogen-plant interactions.Indoleacetic acetic acid(IAA),a natural growth hormone,plays an important role in plant growth and development.During the co-evolution of plants and microorganisms,many microorganisms have also acquired the ability to synthesize IAA,which can influence and modify the physiological processes of plants.Although some studies have shown that Xanthomonas can synthesize IAA,the IAA synthesis ability and IAA action mechanism of Xoc,a pathogenic variant of Xanthomonas oryzae,are not yet clear.In this study,we identified two nitrile hydrolase family(nitrilase,NIT)genes,AKO15524.1 and AKO15829.1,by searching the whole genome of Xoc wild-type strain RS105 against the currently published genes related to enzymes in the IAA synthesis pathway in plants,and further explored their gene functions and aspects of IAA synthesis in Xoc,and the main findings were as follows:(1)To investigate the gene functions of NIT genes AKO15524.1 and AKO15829.1,mutant strain RS105＿Δ23 with deletion gene AKO15524.1,mutant strain RS105＿Δ67 with deletion gene AKO15829.1,and functional backfill strains RS105＿CPΔ23,RS105＿CPΔ67 of both mutant strains were constructed.In the assay of physiological and biochemical properties of Xoc mutant strains,it was found that deletion of the NIT genes AKO15524.1 or AKO15829.1,which are associated with IAA synthesis,significantly reduced the synthesis of extracellular polysaccharide(EPS)in the mutant strains,and supplementation with exogenous IAA improved EPS synthesis in the mutant strains,suggesting that the NIT genes associated with IAA synthesis AKO15524.1 and AKO15829.1 are regulating the synthesis of the pathogenic factor EPS in Xoc.(2)Since the NIT genes AKO15524.1 and AKO15829.1 are genes related to IAA synthesis,IAA production of RS105,the two mutant strains and the backfill strain was further quantified by ELISA.It was found that the IAA production of RS105 reached 2.180 μg/m L with the addition of 1 mg/m L tryptophan,while the IAA yields of the mutant strains RS105＿Δ23 and RS105＿Δ67 decreased by 60.09% and 71.55%,respectively,and the IAA yields of their complementary strains recovered to the level of wild-type RS105,indicating that the NIT genes AKO15524.1 and AKO15829.1 are involved in IAA synthesis in Xoc and that a nitrile hydrolase-dependent IAA synthesis pathway exists in Xoc.(3)The pathogenicity of the mutant strains RS105＿Δ23 and RS105＿Δ67 was further examined by inoculation assay,and it was found that their pathogenicity was significantly reduced on the host plant rice,while the application of IAA significantly increased the pathogenicity of the mutant strains,indicating that the NIT genes AKO15524.1 and AKO15829.1,which are associated with IAA synthesis,are regulating the pathogenicity of Xoc,and IAA may act as a virulence factor in the infestation of Xoc.(4)To investigate the reason for the reduced pathogenicity of the mutant strains,the immune defense response on rice was examined,and it was found that mutant strains RS105＿Δ23 and RS105＿Δ67 could provoke more intense reactive oxygen species burst,callus deposition and defense-related gene expression,while IAA could significantly inhibit the above immune responses induced by the mutant strains on rice.This suggests that the reduced pathogenicity of the mutant strain may be related to its reduced IAA synthesis.(5)Further,RT-q PCR with ELISA was used to detect changes in IAA levels in rice after inoculation with mutant strains RS105＿Δ23 and RS105＿Δ67,and it was found that the expression of the growth hormone signaling gene Os ARF8 induced by the mutant strains and the accumulation of IAA in the leaves were reduced compared with wild-type RS105,which indicated that Xoc-dependent NIT genes AKO15524.1 and AKO15829.1 to induce the expression of Os ARF8 and the local accumulation of IAA in leaves in rice.In summary,this study found that Xoc rely on nitrile hydrolase to synthesize IAA,and the synthesized IAA can act as a virulence factor when the pathogen infects rice,and participate in suppressing the immune defense responses caused by Xoc on rice,inducing upregulation of IAA signaling levels in rice,making the host more susceptible to the disease to facilitate Xoc infestation colonization.In this study,the mechanism of IAA produced by the metabolism of Xoc-dependent nitrile hydrolase family genes AKO15524.1 and AKO15829.1was preliminarily resolved,which can provide a certain research basis for the subsequent exploration of the IAA synthesis pathway in Xoc and help to deeply understand the pathogenic mechanism of Xoc regulated by IAA synthesis genes.