Transcriptional And Functional Studies Of Agrobacterium VirD5 Effector Protein

Agrobacterium tumefaciens is a ubiquitous soil-borne pathogen that is responsible for crown gall.During the transformation process,Agrobacterium delivers a single-strand DNA segment(known as T-DNA)from its Ti plasmid into plant cells.Then,the T-DNA is integrated into the chromosomal DNA of the host plant cells,causing genetic transformation of the host plant cells.Thus,Agrobacterium has usually been used as a tool for genetically engineering plants.The molecular mechanisms of Agrobacterium infection process have been studied for years.But there are still many unresolved issues,especially the molecular mechanism of T-DNA integration process.During the infection process,several virulence proteins were transferred to a host cell,including VirD2、Vir E2、Vir E3、VirD5 and Vir F.These virulence proteins play important roles in the infection process.However,little is known about the function of VirD5.It was reported that VirD5 may maintain the stability of Vir F.In our laboratory,we found that VirD5 may function as a potential transcription factor,can bind to a specific DNA element(D5RE),and may maintain the stability of VIP1.In this work,we intended to explore the function of VirD5.To this end,we constructed the mutant Agrobacterium strains of A281 and EHA105 strains lacking vir D5.The vir D5 mutant strains and the wild-type Agrobacterium were used to infect Arabidopsis thaliana,rice callus and tomato.In our results,the efficiency of stable transformation by vir D5 mutant strains significantly reduced when compared with the control group.However,the efficiency of transient transformation by vir D5 mutant strains has no significant difference compared with the control group.These results indicated that mutation of vir D5 affected Agrobacterium-mediated stable transformation but not transient transformation.Furthermore,to explore the transcription factor function of VirD5,we infected leaves of Arabidopsis using vir D5 mutant strains or wild-type strains.After 48 h,we checked the expression of candidate genes,which contain putative D5 RE elements in their promotors and may be regulated by VirD5.We found that wild-type Agrobacterium could upregulated the expression of the At3g49480 gene,while the vir D5 mutant strain couldnot.The results indicated that VirD5 may regulate the expression of the At3g49480 gene.Indeed,the results of yeast one-hybrid experiment showed that VirD5 directly binds the promoter of the At3g49480 gene.Therefore,our results indicated that VirD5 may function as a transcription factor,and may regulate the expression of At3g49480.As At3g49480 encodes an F-box protein,which may mediate degradation of other proteins,we hypothesize that VirD5 and the At3g49480 gene-encoded protein may be involved in removing the coat proteins of T-complex.In summary,we found that mutation of vir D5 affects Agrobacterium-mediated stable transformation but not transient transformation.VirD5 function as a transcription factor which can bind to the promoter and regulate the expression of the At3g49480 gene.