Functional Of The Pathognicity-related Genes Of Xanthomonas Citri Subsp. Citri And Acidovorax Citrulli

Citrus bacterial canker and bacterial fruit blotch of melon are tow destructive diseases in the field and are caused by Xanthomonas citri subsp.Citri?Xcc?and Acidovorax citrulli?Ac?,respectively.In this report,we isolated pathogenicity-related genes from these two pathogens and characterized their roles in pathogenesis in plants as demonstrated in the following.1.Identification of new Xcc TALE targets in citrusTALE?transcription activator-like efector,TALE?proteins of X.citri subsp.citri are secreted via a type III secretion system?T3SS?into plant cell nuclei to bind an EBE?TALE-binding element?of a target gene promoter which promote disease development.We found that there are,at least,5 new tal genes in Xcc.To identify these TALE targets in citrus?Citrus sinensis?L.?Osbeck?,these TALE genes were individually transferred into a tal-free strain Xcc049E.Then,the transconjugants of Xcc049E with single TALE were infiltrated into citrus leaves using needleless syringes.Pathogenicity test showed that these five tal genes did not contribute obvious virulence to the pathogen as that by PthA.However,these TALE proteins promoted bacterial growth in planta.Citrus tissues infiltrated with bacteria for 24 h were collected to use for transcriptome analysis by deep genomic sequencing.The profiles of these tissues challenged by five TALE proteins displayed up-regualted genes in citrus compared to the tal-free strain Xcc049E.Candidate genes were screened from the up-regulated genes of citrus when the predicted DNA sequences,possibly bound by the RVDs?repeat-variable diresidues?of these TALE proteins,matched regions of gene promoters.Electrophoresis mobility-shift assay?EMSA?was explored to test whether the promoter regions of the candidates were bound by the TALE proteins or not.The results indicated that the TALE protein 086-78 bound to the promoter region of Cs2g22130 and 003-4.4 to Cs3g16800 and Cs5g34120,respectively,implying that Cs2g22130 is the target of 086-78,Cs3g16800and Cs5g34120 are the targets of 003-4.4.These findings provide a fundamental basis to understand molecular mechanisms of Xcc-citrus interaction.2.virulence roles of glyceraldehyde-3-phophoate de-hydrogenase of Xcc in citrusThe presence of PthA?a major virulent protein?in Xcc bacterial cells prompted us to postulate that there is a potential PthA target in the pathogen.We found that there is a possible EBE site in the promoter region of glyceraldehyde-3-phophoate de-hydrogenase gene?gpd1?in a strain Xcc021that almost matched a predicted DNA binding sequence of PthA.To identify whether the gpd1 is the PthA target or not,the gpd1 gene was knocked out in strains Xcc049E?tal-free,hypovirulent?and Xcc021?hypervirulent?.Pathogenicity assays demonstrated that the mutation in gpd1 reduced Xcc021's virulence and impaired bacterial growth in planta.Biological feature tests showed that the mutation in gpd1 reduced bacterial aggregation and swimming ability but increased bacterial biofilm formation comparing to the wild-type.The above results indicated that gpd1 contributes bacterial virulence in citrus.3.Characterization of avrRxo1_Acc gene of A.citrulli in pathogenicity in plantsBioinfoirmatics indicated that there is a homolous gene avrRxo1_Acc in the genome of A.citrulli to avrRxo1 of X.oryzae pv.oryzicola.Previous reports show that AvrRxo1 of X.oryzae pv.oryzicola matches an R gene Rxo1 to activate plant resistance in maize.To know whether or not AvrRxo1_Acc can trigger hypersentive response in maize containing Rxo1,avrRxo1_Acc was cloned from A.citrulli and then transferred into a mutant in which avrRxo1gene of X.oryzae pv.oryzicola was deleted.The transconjugant of the mutant with was infiltrated into maize leaves containing Rxo1.The results showed that avrRxo1_Acc of A.citrulli could not restore HR induction in maize to the avrRxo1 mutant of X.oryzae pv.oryzicola.Trancient expression of avrRxo1_Acc in maize,mediated by Agrobacterium,did not show HR as that by avrRxo1_Xoc.These results demonstrated that the relation of avrRxo1_Ac-Rxo1does not follow“gene-for-gene”manner,implying that the transfer of Rxo1into melon may not generate resistance to bacterial fruit blotch disease.