Identification And Analysis Of The Relationship Between ?-aminobutyric Acid And Xanthomonas Campestris Pv. Campestris

Xanthomonas campestris pv.campestris(Xcc)is a very important pathogenic bacterium.It can infect all the cruciferous plant(Brassicaceae)in the world and cause black rot disease in the plants,causing a lot of loss in agricultural production.?-Aminobutyric acid(GABA)is an very important ubiquitous non-protein amino acid in both prokaryotic and eukaryotic,and plays an important role in the organisms.In response to abiotic and biotic stresses,GABA can be synthetized or degradated rapidly in plant and bacteria.In the organisms,GABA is usually metabolized via a short pathway,which is called GABA shunt.GABA shunt also has an important influence of organisms.In this work,we attempt to investrigate the influence of GABA and GABA shunt in Xcc 8004,and identify the genes annotated as relevant enzymes of GABA shunt in Xcc 8004.This work will contribute to dissect the pathogenic mechanism of the phytopathogenic bacteria,which should offer an insight for prevention the loss in economic.In this work,we found that GABA reduced the secretion of XopXccN,a type ? secreted(T3S)effector encoded by XC0241,through GUS activty test in 8004/pLgus0241.Furthermore,3 mM GABA could inhibit the growth of Xcc 8004 in NYG medium.However,from plant assay results,exogenous GABA couldn't inhibit the virulence of Xcc 8004 in Chinese radish,not matter by spraying and irrigating GABA.Xcc 8004 was unable to utilize GABA as a sole carbon and nitrogen source.Consistently,GABA was barely detected in Xcc 8004.From KEGG's Pathway(http://www.genome.jp/kegg-bin/show_pathway?xcb00250),only XC1780 was annotated as succinic semialdehyde dehydrogenase,the other two enzymes of GABA shunt are still unknow.By using prokaryotic expression vector,the recombination proteins XC1780 we expressed and purified.Via determination of the enzymatic activity of the recombination proteins or the total proteins of Xcc 8004,we confirmed that XC1780 harbors the activity of succinic semialdehyde dehydrogenase and it localizes in the cytosol.But we couldn't detect the enzymatic activity of glutamate decarboxylase and GABA transaminase,hinting that glutamate decarboxylase and GABA transaminase might be absent in the Xcc 8004,or the activites of these enzymes are very low.To confirm the speculation,the GABA transaminase of Pseudomonas syringae pv.tomato DC3000 was introduced into Xcc 8004 by triparental conjugation.The yielding strain could utilize GABA as a sole nitrogen source.The plant assay results demonstrated that XC1780 was required for virulence and in planta growth of Xcc 8004.However,the mutation of XC1780 didn't affect the production of extracellular enzyme and EPS.XC1780 couldn't be identified as a T3S effector gene.The rssults of GUS activty examination showed that the expression of XC1780 is induced in XCM medium and in planta but reppressed in NYG medium,and not regulated by hrpG and hrpX.Vice versa,the expression hrpG and hrpX is not controlled by XC1780 either.