Identification And Biological Function Of The Effector Ace0201 And Ace1242 In Acidovorax Citrulli

The bacterial fruit blotch(BFB)caused by Acidovorax citrulli is a devastating seed-borne disease,mainly affecting watermelon,melon and other cucurbit crops.The epidemic of BFB can cause huge economic losses,which seriously restricts the healthy development of the watermelon and melon industries.Because the pathogenic mechanism of A.citrulli is unclear,there are still no effective means to prevent and control BFB.Therefore,analyzing the pathogenic mechanism of A.citrulli is the key to solve the problem of preventing and controlling BFB.It is well known that the type 3 effectors(T3Es)secreted by the Type III secretion system(T3SS)play important roles in the pathogenesis of phytopathogenic bacteria.So screening,identification and functional studies of the effectors can help to understand the pathogenesis of pathogens.However,there are few reports about effectors in A.citrulli.Therefore,in this study,Aac5 was used as a research object,bioinformatics,tobacco transient expression system,Western blot,q RT-PCR and other techniques were used to screen and identify the specific effectors in A.citrulli and the putative effector which homologous to Avr Rpt2.Then we carried out a preliminary analysis of their biological functions,the results were as follows:1.Identification of the effector Ace0201 and preliminary analysis of its biological functionsThrough bioinformatics analysis,we found a putative protein Ace0201 in A.citrulli which homologous to the effector Avr Rpt2.The result of q RT-PCR showed that the expression of gene ace0201 was regulated by T3 SS core genes,hrp G and hrp X.The result of western blotting further confirmed that Ace0201 had a secretory function and was regulated by T3 SS.These all indicates that Ace0201 is a type III effector.Further studies showed that Ace0210 located in the cell membrane and nucleus of tobacco.Besides,Ace0201 had the functional characteristics of inhibiting BAX-induced PCD reaction and inhibiting ROS burst.However,the pathogenic ability of A.citrulli to natural host watermelon was enhanced afterdeleting gene ace0201.2.Screening of specific effectors in Acidovorax citrulli31 candidate specific effectors were obtained through bioinformatics prediction.Further screening of candidate specific effectors using a tobacco transient expression system,the candidate effector Ace1242 was successfully screened to inhibit BAX-induced PCD response.BLAST analysis showed Ace1242 did not share homology with other pathogenic bacteria effectors.These all indicates that Ace1242 has the functional characteristic of inhibiting plant immune response and may be a specific effector of A.citrulli.3.Identification of the candidate effector Ace1242 and preliminary analysis of its biological functionsThe candidate effector Ace1242 contains 95 amino acids.It was found that the T3 SS core gene hrp G and hrp X regulated the expression of the gene ace1242.So Ace1242 is regulated by T3 SS.The pathogenicity test results showed that the pathogenicity of the bacteria to the natural host watermelon decreased significantly after the gene ace1242 was deleted,indicating that Ace1242 played an important role in the pathogenesis.Through observing the subcellular localization,we found Ace1242 located in the cell membrane and nucleus of tobacco.The determination result of reactive oxygen species(ROS)showed that Ace1242 significantly inhibited the outbreak of ROS,indicating that Ace1242 inhibited the PTI response of tobacco.In conclusion,we successfully identified a effector Ace0201 which homologous to Avr Rpt2 and screened a potential specific effector Ace1242 in A.citrulli,in this study.Both of them play important roles in the pathogenesis of A.citrulli.