Xanthomonas Campestris Promotes Diffusible Signal Factor(DSF) Biosynthesis And Pathogenicity By Utilizing Sugars Of Host Plant

Plant pathogen Xanthomonas campestris pv.campestris?Xcc?,which is the causal agent of black rot of crucifers.Xcc is a vascular pathogen that usually enters the xylem system through hydathodes and wound.It produces diffusible signal factor?DSF?quorum sensing?QS?signals to regulate diverse biological functions such as biofilm formation,motility,virulence and antibiotic resistance.SWEET?Sugars Will Eventually be Exported Transporters?mediated sugar transport is not only essential for plants carbohydrate distribution but also for pathogen resistance.Xanthomonas can live in the intercellular space?apoplasm?of plants,where they acquire carbohydrates for energy and carbon.Previous study showed that Xcc utilizes metabolites of host plant to enhance biosynthesis of DSF-family signals.However,it is unclear how Xcc benefits from the metabolic products of host plant.Our results in this study further suggested the complex interaction between Xcc and the host plant,the main work includes the following four parts:1.Analysis of the transcriptome profiles suggested that the host plant juice affected the expression levels of many genes in Xc1.Compared the transcriptome profiles of the DSFdeficient rpfF mutant and the wild-type Xc1 strain with addition of Chinese cabbage juice and found substantial overlap in the genes that were affected in expression.Quantitative RTPCR analysis of the selected genes confirmed the RNA-seq results.From this we concluded that Chinese cabbage juice plays an important effect on the DSF QS system in Xcc.2.Exogenous addition of the cabbage juice to the rpfC deletion mutant,slightly increased the bacterial growth rate,but remarkably increased the production of both DSF and BDSF after inoculation.UPLC-MS analysis showed that the two major sugars in the juice are sucrose and glucose.We found that exogenous addition of 15 mM glucose significantly enhanced the production of DSF and BDSF at 4 h after inoculation,respectively.And addition of 15 m M sucrose showed no detectable effect on the DSF-family signal synthesis in Xcc at the early stage.But at 24 h post-inoculation,addition of sucrose increased the production of DSF and BDSF,respectively.3.Exogenous addition of 10% glucose and sucrose increased the CFU?colony forming units?of Xc1 from 2.87692×10⁶ mL^-1 to 5.9125×10⁶ mL^-1 and 7.25667×10⁶ mL^-1 at 3 d postinoculation.Quantitative RT-PCR analysis showed that Xc1 infection caused an increase of the expression levels of some transporter genes in A.thaliana.Mutation of AtSWEET4 or AtSWEET8 decreased the virulence and reproduction of pathogens.It was shown that the decreased pathogenicity and reproduction of Xc1 in AtSWEET mutants was increased with exogenous addition of 10% sugars at 3 d post-inoculation.So the decreased pathogenicity of Xcc in AtSWEET mutants may be caused by the limitation of sugars.4.Quantitative RT-PCR analysis revealed that infection by Xc1 caused an induction of some sucrose hydrolase gene expression levels in A.thaliana.Expression of three sucrose hydrolase genes in Xc1 was also induced with exogenous addition of cabbage juice.In vitro enzyme activity analysis showed that glucose and fructose were released after sucrose being catalyzed by CINV1 or XC₀805.In the absence of sucrose,there was no obvious difference of DSF production among ?rpf C,?rpf C?CINV1?and ?rpfC?XC₀805?strains.On the contrary,with addition of sucrose,overexpression of CINV1 and XC₀805 in ?rpfC increased DSF production by 30.95% and 34.27%,at 4h after inoculation,respectively,suggesting overexpression of sucrose hydrolases significantly increased the production of glucose for DSF biosynthesis in the presence of sucrose.In conclusion,the work reported here has allowed us to identify for the first time that Xcc targets the sugar transport and sucrose hydrolases to utilize sugars of host plant as nutrients and substrates of DSF signal biosynthesis to promote the pathogenicity.