Functional Study Of Effector Ace1 & Analysis Of Pathogenicity Differences Under Light And Dark Conditions In Acidovorax Citrulli

Acidovorax citrulli is a Gram-negative bacterium.It mainly threatens watermelons and melons,causing serious economic losses.Its pathogenesis is not yet well understood.Type III effector proteins(T3Es)secreted by T3 SS in plant pathogenic bacteria can inhibit plant immune response.It plays a key role in pathogenesis.In addition,previous reports have proven that T3 SS is a determinant in the pathogenesis of A.citrulli.However,limited research on T3 Es in A.citrulli has been published.Based on this,this study developed the identification and functional analysis of T3 Es of A.citrulli.(1)The functional study of hrpG and hrpX predicted as the key regulators of T3 SS of A.citrulliThe results have shown that hrpG and hrpX regulates the formation of biofilm,which enhances the capability of HR of non-host Nicotiana tabacum,and ROS burst of host Nicotiana benthamiana induced by A.citrulli.Moreover,the hrpG and hrpX contribute significantly to the pathogenesis of A.citrulli.More importantly,the hrpG and hrpX involve in the regulation of T3 E expression.(2)Preliminary screening of candidate T3 Es in A.citrulliUpon confirming the participation of hrpG and hrpX in the regulation of T3 Es,we then conducted transcriptome sequencing on the wild-type strains Aac5,hrpG and hrpX mutant strain.This is to detect potential T3 Es in differential genes.Combining the bioinformatics predictions with that of transcriptome sequencing,20 candidate genes were selected for analysis.A preliminary screening of 20 genes was performed,and 4 more likely candidate genes were obtained as the next research object.(3)Identification and functional analysis of Ace1 predicted as a type 3 effector of A.citrulliTaking ace1 as the research object,Western blot,qPCR and promoter activity assay were used to verify ace1 as a new type 3 effector.The results are as follows: Ace1 promotes the infecting process of A.citrulli in its natural host watermelon;Ace1 inhibits ROS burst and callose deposition in the host N.benthamiana;In the host N.benthamiana,the Ace1 protein could be located throughout the cells;in Pseudomonas syringae DC3000 strain,Ace1 inhibits PCD of N.benthamiana induced by AvrPto effector proteins in Pseudomonas syringae pv.tomato DC3000 strain.With IP and mass spectrometry,we obtained the potential mutually-targeting protein in N.benthamiana with Ace1.Utilizing bimolecular fluorescence complementation of luc and qPCR technologies,we postulate an interaction between the two,but further verification is needed.(4)Analysis of pathogenicity difference of A.citrulli under light and dark conditionsUnder light and dark conditions,the expression of virulence factors of A.citrulli and the host resistance response induced by A.citrulli were significantly different.Light conditions inhibited the pathogenicity of A.citrulli.Under light conditions,lov gene inhibited the swimming ability of A.citrulli,but the effect of lov deletion on the overall pathogenicity was not significant.iTRAQ analysis showed that there were 77 differentially expressed proteins in A.citrulli under light and dark conditions involving multiple pathogenetic metabolic pathways,and screening a potential type 3 effector that was induced by dark induced expression.In summary,this study determined that hrpG and hrpX in A.citrulli are involved in regulating the expression of T3Es;Preliminary screening obtained four potential T3 Es in A.citrulli,in which Ace1 was identified as a type III effector.And Ace1 was found to be able to inhibition of plant immune responses;Differences in the pathogenicity of A.citrulli were found under light and dark conditions.These laid an important foundation for the subsequent analysis of the pathogenic mechanism of A.citrulli.