Functional Analysis Of CopZ And CusA Involved In Copper Metabolism Of Acidovorax Citrulli

Bacterial fruit blotch is a seedborne disease that causes significant yield losses in melon and watermelon industry worldwide.The causal agent of BFB is Acidovorax citrulli,a gram-negative bacterium with a single polar flagellum.At present,copper-based bactericides are commonly used to control BFB,which usually lead to increased copper resistance of pathogenic bacteria.Gene copZ and cusA play critical roles in the copper metabolism of bacteria.In order to explore their functions in A.citrulli,in this study,we took A.citrulli Aac-5 as the wild-type strain,and constructed two mutant strainsΔcopZ andΔcusA,as well as their complementary strains.The copper resistance,pathogenicity,mobility and hypersensitive response on tobacco of the subsequent strains were determined.In addition to the phenotypes,we also analyzed the expression level of genes in the mutant strains by RT-q PCR,including those related to copper-metabolism,biofilm formation ability and pathogenicity.The results were as follows:1.Obtained the bioinformation of copZ and cusA in A.citrulli.Cop Z harbors an HMA structural domain,which was conserved in bacteria and participated in heavy metal transport or protein detoxification.HMA contains two conserved cysteines related to metal binding.Cus A harbors an ACR-tran domain,which is related to drug efflux.Cop Z has no transmembrane structure,while Cus A contains 12 transmembrane structures,indicating different functions they play in the copper-resistance of A.citrulli.2.Constructed mutant and complementary strains of A.citrulli Aac-5 strainBy using the triparental conjugal method,we constructed mutant strainsΔcopZ andΔcusA,and also constructed complementary strainsΔcopZ-comp andΔcusA-comp.The mutant and complementary strains were continued cultured and verified by PCR,indicating the successful construction of mutant and complementation strains.3.Determined the growth ability,pathogenicity,motility,biofilm formation ability and hypersensitive response on tobacco of wild-type,mutant and complementation strains of A.citrulli.（1）Compared to the wild-type strain Aac-5,the growth ability ofΔcopZ at a concentration of 1.25 mmol/L Cu SO₄was significantly reduced,and that ofΔcusA was significantly reduced at a concentration of 2.5 mmol/L Cu SO₄.（2）The mutant strainsΔcopZ andΔcusA,and their complementation strains showed very similar mobility to the wild-type strain Aac-5,when no copper added into the medium.However,when the Cu SO₄was added at concentration of 1.25 mmol/L and 2.5 mmol/L,the wild-type strain Aac-5,mutant and complementary strains were unable to form motility colonies on the plates,indicating the lost of motility of the A.citrulli strains.（3）Compared to the wild-type strain Aac-5,the mutant strainsΔcopZ andΔcusA were significantly decreased in biofilm formation when no copper added in to the KB medium.With the increase of Cu SO₄concentration,the biofilm formation ability of each strain gradually decreased.However,the mutant strainsΔcopZ andΔcusA were significantly increased from the wild-type strain Aac-5.（4）The elimination ofΔcopZ andΔcusA does not affect the pathogenicity of A.citrulli Aac-5.The watermelon seedlings inoculated with wild-type,mutant and complementration strains of A.citrulli showed water-soaking necrosis,and similar disease index.The watermelon cotyledon showed obvious symptoms of BFB at 48 h after inoculated with wild-type,mutant and complementration strains of A.citrulli.The wild-type,mutant and complementration strains all induced hypersensitive response when infiltrated into tobacco leaves.4.Determined the expression of genes involved in copper resistance,biofilm formation ability and pathogenicity in A.citrulli strains.The deletion of gene copZ and gene cusA had an effect on the expression of copper metabolism related genes of wild-type Aac-5.Among them,Cue R protein is a Mer R transcription regulator,and Cop A protein is a heavy metal transport P-ATPase.The experiment found that cue R was highly expressed in the mutant strainsΔcopZ andΔcusA treated with 1.25 mmol/L Cu SO₄,indicating that cue R played a role at this copper concentration;According to the changes in the expression of cop A in mutant strainΔcopZ treated with different copper,it was found that cop A and copZ have a synergistic effect to cope with copper stress.According to the changes in the expression of copZ in mutant strainΔcusA with different copper concentration,it was found that copZ is closely related to cusA.The deletion of genes copZ and cusA had an effect on the formation of wild-type Aac-5formation ability and pathogenicity-related genes.The expression changes in the mutant strainΔcusA with different copper treatments showed that hrp G is closely related to cusA.The above results indicate that copZ and cusA not only play an important role in the copper metabolism,but also affect the ability of biofilm formation of A.citrulli.The copZ and cusA do not affect the pathogenicity,motility and hypersensitive reaction on tobacco of A.citrulli.The copZ plays a more important role in low copper environment than cusA.This study deepened the insight in copper mechanism of A.citrulli,and also provided a theoretical basis for the prevention and control of bacterial fruit blotch.