Functional Analysis Of PhoB?phoR And Aave₂620 In Acidovorax Citrulli

Bacterial fruit blotch?BFB?of cucurbits.The pathogen is Acidovorax citrulli,The morphological feature is a single pole flagellum,no fluorescence and aerobic.Gram shows negative bacteria.BFB is a bacterial quarantine disease worldwide.It is a key quarantine object for China's entry and exit.It mainly spreads seeds through bacteria to cause severe damage to watermelons,cucurbits and other cucurbits around the world,it brings huge losses to the industrial economy.The c-di-GMP signaling pathway and two-component regulatory system in bacteria are two complex and distinct physiological processes.The effects of bacterial toxicity,motility,and biofilm formation on pathogenicity are crucial.This study investigated the effects of genes Aave₂620 and phoB and phoR on pathogenicity-related phenotypes from the perspectives of cdi-GMP signal regulation and two-component signal regulation.Through the bioinformatics analysis of Aave₂620,phoB and phoR genes,including the domain function,through the threeparent hybridization and homologous recombination double exchange,Aave₂620,phoB and phoR gene single gene deletion mutant strains and complement strains,and The phoB and phoR double-deletion mutant strains were tested for wild-type strain Aac5 as a positive control and water as a negative control.In addition,the genes Aave₂620,phoB and phoR were measured using real-time quantitative q RT-PCR technology to verify whether there is a regulatory relationship between the three,and to explore the absence of Aave₂620,phoB and phoR on virulence factors,type 3 secretory system regulatory factors,and other Impact of disease-related genes.The findings are as follows:?1?Through NCBI database and SMART software amino acid sequence alignment,a total of 43 proteins encoding GGDEF and EAL domains were found in the AAC00-1 genome of watermelon phage.The analysis results showed that gene Aave₂620 has GGDEF involved in the synthesis of c-di-GMP The domain and the EAL domain that degrades c-di-GMP,as well as the PAS domain used as a signal sensor,are functionally annotated as phosphodiesterase.Gene Aave₂631 encodes the protein PhoB and has a REC domain that receives signals and is related to the flagellar motor gene ch Y;gene Aave₂632 encodes the protein PhoR and has a signalling HK domain and a HATPase domain.The relationship between the genes Aave₂631 and Aave₂632 is predicted It is two-component..?2?Real-time fluorescence quantitative results showed that the deletion of the gene Aave₂620 significantly increased the expression of hrp E and hrp G,the major genes related to the three types of secretion system of acid bacillus Aac5,and the expression of flagellaregulated genes fli A and che A showed significant up-regulation and virulence.The factors trb C and vir B are also significantly upregulated.?Aave₂620 has a significantly lower pathogenic capacity than the wild-type Aac5,significantly reduced in vitro and in vivo growth capacity,significantly reduced morbidity and biofilm formation ability,and significantly reduced exercise capacity compared with the wild-type.The deletion of the gene Aave₂620 can cause normal tobacco allergy reaction.The deletion of the gene phoB still has tobacco allergic reactions,the mutant strain has reduced pathogenicity,and has a significant impact on biofilm formation.The hrp X and hrp G gene expression in ?phoB is significantly down-regulated,and the hrp E gene significantly up-regulates the flagella-related gene fli A.The expression level was significantly up-regulated,and the che A gene was significantly down-regulated.The expression of virulence factors trb C and vir B in ?phoR was significantly down-regulated,and the gene phoR had a positive regulation effect on hrp G and che Y.The single deletion of the gene phoR did not significantly affect the host's pathogenicity,athletic ability,in vitro and in vivo growth ability,and other disease-related phenotypes,but the double deletion of the genes phoB and phoR exacerbated the host's effect Pathogenicity,double-deletion mutant ?phoB / phoR can cause tobacco allergy,and exercise ability is almost not affected,but the ability to form biofilms is significantly enhanced,and the major related genes of the type 3 secretion system,hrp E,hrp G and hrp X plays a significant positive regulatory role.?3?The genes Aave₂620,phoB and phoR are all related to the type 3 secretion system.The gene Aave₂620 belongs to the c-di-GMP signal pathway regulatory factor.The genes phoB and phoR belong to the two-component signaling system and the three-type secretion system belong to completely different three signal pathways.However,the three can communicate with each other and work together..Many previous studies have shown that c-diGMP does not function independently in the cell,but often coordinates with other common important regulatory systems,such as type III secretion systems,quorum sensing systems,and two-component systems.Together,they play a role in regulating a variety of life activities and pathogenicity in cells.This feature was verified in watermelon acid phage by this experiment.Real-time fluorescence quantitative results show that the deletion of genes Aave₂620,phoB and phoR can cause significant changes in the expression levels of major genes related to the three types of secretory systems such as hrp G and hrp X,but the regulation direction is different,so that each regulation system has its own Regulatory characteristics and angles will also be closely combined with other signal pathways,intertwined into a complete signal transmission network to complete various physiological activities of pathogenic bacteria.?4?There are significant regulatory relationships among genes Aave₂620,phoB and phoR.After real-time quantitative q RT-PCR,the expression data showed that the gene Aave₂620 was significantly positively regulated by the phoR gene,the Aave₂620 gene was positively regulating the phoB gene,and the gene phoB was significantly up-regulated in ?phoR.