Dissection Of The Pathogenic Mechanism Of Cellulase In Xanthomonas Campestris Pv. Campestris

Xanthomonas campestris pv.campestris(Xcc)belongs to gram-negative bacteria.Xcc is a model strain of Xanthomonas for studying the molecular mechanism of interaction between microorganisms and plants.Xcc is an important pathogen that infects cruciferous plants.Extracellular cellulases are important for full virulence of Xcc and play a major role in the early stage of infection.However,its pathogenesis mechanism is still unclear and the relationship between cellulase activity and pathogenicity is rarely reported in Xcc.Therefore,this study intends to make a preliminary exploration of the pathogenesis of cellulase.The main results of this study are as follows.1.To investigate the effects of other cellulases in Xcc 8004 on host pathogenicity and non-host HR.Through bioinformatics analysis,we found that except 9 endo-cellulase genes(XC0026,XC0027,XC0028,XC0639,XC1727,XC0783,XC0784,XC2483,and XC0625),Xcc 8004has three putative cellulose degradation enzyme genes,i.e.,XC0626(Cellulose 1,4-β-cellobiosidase),XC1005(Cellulose 1,4-β-cellobiosidase),and XC3054(Endo-1,3-β-glucanase).In this study,the deletion mutants of these three cellulase genes were constructed.The hypersentive response(HR)was tested on non-host pepper ECW-10R(Capsicum annuum cv.ECW-10R).The results showed that there was no significant difference between the deletion mutants and the wild-type strain Xcc8004.Then pathogenicity was tested on Chinese radish(Raphanus sativus var.radiculus)cv.Manshenhong vie leaf cutting.The results indicated that the single-gene mutants did not affect pathogenicity,while the multi-genes-deletion mutants reduced slightly the virulence.2.In order to investigate the effect of structural domain of endoglucanase genes on pathogenicity and cellulase activity,the in trans-complementary strains of each domain of XC0026,XC0027,XC0028,and XC0639 on the basis of the D9(the multi-genes-deletion mutant of all 9 endo-cellulase genes)were constructed respevtively.Throught virulence assay and cellulase enzyme activity detection,we found that in pathogenicity,there were a lot of defects on functional domain of XC0026,signal domain of XC0028,and functional domain of XC0639,and there was great contribution on XC0028 functional domain,while the contribution to pathogenicity of XC0026 signal domain remains unclear.In terms of cellulase activity,the functional domains of XC0026,XC0027,and XC0028 were found to be defective.We studied the effect of the bioinformatically predicted cellulase binding domain(CBD)of XC0639 on enzyme activity by constructing XC0639 CBD deletion mutant named D0639(CBD),a complementary strain of D0639(CBD)named C0639(CBD),and all 9 annotated cellulase genes deletion mutant(D9)carrying an XC0639 harboring or lacking its CBD in trans.Interestingly,there was no significant difference among the cellulase activities produced by D0639(CBD),C0639(CBD),D9/C0639,D9/C0639(CBD~-),and the wild-type strain 8004,suggesting that the predicted CBD might be not involved in the cellulase enzyme activity of XC0639.3.For further understanding the relationship between cellulase activity and pathogenicity,we extracted the cell wall components of Chinese radish cv.Manshenhong vie leaf cutting.Through cellulase activity testing(cell wall as substrate),the result showed that there was no significant difference among the cellulase activities produced by D0639,D9,D9/C0027,D9/C0639,and the wild-type strain Xcc 8004,suggesting that the major cellulase gene XC0639 might not be the major gene for degrading cell wall,and there should be other cell wall degrade enzymes in Xcc 8004.4.In order to search the interaction between the cellulase XC0027and the host plants targets,we preliminarily screened the plant(Chines cabbage cv.Jingfeng No.1)target proteins interacting with XC0027through the co-immunoprecipitation(Co-IP)experiment.As a result,36candidate interaction proteins were initially obtained through mass spectrometry analysis.