GENETIC COMPLEMENTATION ANALYSIS OF THE AGROBACTERIUM-TUMEFACIENS VIRB OPERON - VIRB2 THROUGH VIRB11 ARE ESSENTIAL VIRULENCE GENES

The Agrobacterium tumefaciens virB gene products are proposed to assem ble into a transport system capable of exporting complexes of DNA and protein across the bacterial envelope en route to plant cells. Nonpola r null mutations were constructed in each of the 11 virB genes of the A. tumefaciens pTiA6NC plasmid. In tumorigenicity assays, Delta virB1 mutants exhibited severely attenuated virulence and Delta virB2 throug h Delta virB11 mutants exhibited avirulence. NdeI restriction sites in troduced at the predicted translational start sites of the virB genes were used to subclone each of the virB genes downstream of the lncZ or virB promoter on broad-host-range plasmids. virB gene expression plas mids were used to define promoter and general sequence requirements fo r genetic complementation of the deletion mutations. Whereas virB1 and virB2 complemented Delta virB1 and Delta virB2, respectively, only wh en expressed in trans from the virB promoter, virB3 through virB11 com plemented the corresponding deletion mutations when expressed in trans from either the lacZ or virB promoter. Several virB genes required ad ditional upstream or downstream sequences for complementation: (i) vir BZ complemented the Delta virB2 mutation only when the complementing p lasmid coexpressed virB1 and virB2, (ii) virB6 and virB9 complemented the Delta virB6 and Delta virB9 mutations only when the complementing plasmids carried at most 55 and 230 bp of sequences residing 5' of the se genes, respectively, and (iii) virB7 and virB8 complemented the Del ta virB7 and Delta virB8 mutations only when the complementing: plasmi d coexpressed virB7 and virB8. These studies established that virB1 is an accessory virulence determinant and virBZ through virB11 are absol utely essential for the A. tumefaciens infection process.