Analysis Of The Influence Of The Identified Type Ⅲ Secretion Effector Genes On The Pathogenicity In Xanthomonas Campestris Pathovar Campestris Strain 8004

Xanthomonas campestris pv.campestris(Xcc)is a kind of plant pathogenic bacteria.It can causes black rot disease on all cruciferous plants worldwide.Type Ⅲ secretion effector(T3SE)is a kind of important virulence factor of many animal and plant pathogens.In Xcc 8004,17 type Ⅲ secretion effector genes have been identified.They are XC0052,XC0241,XC0541,XC0542,XC1210,XC1553,XC2004,XC2081,XC2210,XC2602,XC2994,XC2995,XC3160,XC3176,XC3177,XC3802,and XC4273,respectively.In this study,we analyzed the influence of effector genes on the pathogenicity of Xcc 8004 by constructing deletion mutants.Seventeen single gene deletion mutants and 28 multiple genes deletion mutants have been constructed in this study.The 16-genes deletion mutant(△ 16)has deleted all the 17 effector genes except XC2210 and the 17-genes deletion mutant(△ 17)has deleted them all.The results of pathogenicity test showed that XC0052,XC0241,XC0541,XC0542,XC1210,XC2994,XC2995,XC3160,XC3177,and XC4273 contribute to the pathogenicity of Xcc 8004,for their respective deletion mutants showed significant reduced virulence.The respective deletion mutants of other 7 genes showed no significant difference with Xcc 8004 on pathogenicity.They are XC1553,XC2004,XC2081,XC2210,XC2602,XC3176,and XC3802.The strains △16 and △17 reduced virulence remarkablly.Each of their vieulence classification is of grade one.Sixteen single effector gene complementary strains were constructed based on △17.The pathogenicity test showed that no single effector gene can restore the full virulence of △17.Through leave-cutting vaccination,a plant resistant to Xcc 8004 was screened out,and was named as radish 3.In planta growth test of bacteria showed that radish 3 can inhibit the growth and reproduction of bacteria to a certain extent.Virulence tests and hypersensitive reaction experiments indicated that except for the 17 effector genes,other effector genes not identified should be present in Xcc 8004,whose functionalities are avr genes which can be recognized by the resistant genes in radish 3.By Agrobacterium-based transient expression assay,we have confirmed that XC0241,XC0541,XC0542,XC2081,XC2602,XC2994,XC2995,XC3177,XC3802,XC4273,XC1210,XC1553,XC2210,and XC3160 are not corresponding avr genes that can be recognized by the mesophyll resistance genes in turnip 3.By leave-cutting vaccination of Xcc 8004,33913,and 20 Chinese Xcc strains and in radish 3,we found that turnip 3 can be infected by Changchun xc-2,Ganyou xc-1,Shunyi xc-6,008,Guangxi Guilin xc-3,Guangxi Nanning xc-1,and Guangxi Nanning xc-2.On the otherhand,radish 3 can resist the infection of Xcc 8004,Heihe xc-1,Dalian xc-3,Daxing xc-1,Tianjin xc-1,Shanxi turnip xc-1,Xian xiangyu xc-1,Xian Duqu xc-1,Henan Nanyang xc-1,005,Guangxi Guilin xc-1,Guangxi Guilin xc-2,Guangxi Nanning xc-3,Guangxi Nanning xc-4,and 33913.