Iden-tification And Study On Genes Regulated By HpaR1 In Xanthomonas Campestris Pv.Campestris

Xanthomonas campestris pv.campestris,Xcc is one of the model strains for studying plant pathogen.It is the cause agent of black rot disease of most crucifer,such as radish,cabbage,broccoli.Therefore,it is one of the agriculture important pathogen.GntR family protein belongs to the transcriptional regulator of HTH super family.GntR family presents widely in many different bacteria and affect various biological processes within them.In Xcc 8004,XC₂736 is annotated as a transcriptional regulator of YtrA subfamily of GntR family.Previous study revealed that mutation of XC₂736（hpaR1）can lead to less motility and reduction in extracellular endogluconase activity in the bacteria,also result in reduce of virulence in Chinese radish and of HR induction in nonhost plants.XC₂736 also appears to regulate the expression of hrp cluster.To further understand the specific role of the transcription factor in the pathogenesis,previous data from two-dimensional electrophoresis experiment discovered a group of genes which differentially expressed in HpaR1 mutant（Unpublished）.In this study GUS fusion reporter system and RT-PCR stratyge were adopted to identify target genes controlled by HpaR1.Then their biological functions has been analyzed.（1）Identify target genes:14 genes with larger expression difference as shown in two-dimensional electrophoresis data have been chosen.Their regulatory relation with HpaR1 has been examed by GUS fusion reporter system.The promoter region containing the initiation codon from the target genes were linked to pLAFR6GUS plasmid which possesses a gusA gene without its promoter.Then the constructed plasmids were introduced to both wide type strain and hpaR1 mutant.Their transcriptional level was tested in NYGB and MMX medium.The results showed XC₁358,XC₀639,XC₂921 are positively regulated by HpaRl,but XC 3680 is negatively regulated and the results were confirmed by semi-quantitative RT-PCR.XC₀638 and XC₀639 are contiguous in Xcc 8004 genome and transcribed at the same direction.Some data（unpublished）implied that HpaRl positively controls the expression of XC 0638 and it has been confirmed by the result from RT-PCR in this work.（2）Biological functions analysis of target genes:The mutants and complementary strains of XC₀638,XC₁358,XC₀639 have been constructed in this work.Phenotypical assay showed that mutation of XC₀638 and XC₁358 results in reduced motility and 40%decreased virulence;mutation of XC₁358 leads to cell aggregation but no other effect.The mutation of XC₀639 showed reduced extracellular endogluconase activity but with unaffected virulence and other phenotype.Previous study of hpaR1 mutant showed affect on motility and the activity of extracellular cellulose.We speculated it may be achieved through HpaR1 positively regulated motility related genes XC₀638 and XC₁358 and cellulase gene XC₀639.Take together this work identified 3 genes positively regulated by HpaR1,of which XC₁358 and XC₀638 showed affect to bacterial virulence and motility and XC 0639 is the gene coded of the main endogluconase.