Agrobacterium rhizogenes GALLS Protein Contains Domains for ATP Binding, Nuclear Localization, and Type IV Secretion

Abstract

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of singlestranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.

The Agrobacterium rhizogenes GALLS Gene Encodes Two Secreted Proteins Required for Genetic Transformation of Plants

“…Plasmids containing mutant or wild-type GALLS genes were transformed into A. tumefaciens mx358 (virE2:: Tn3-lacZ) (36). A. tumefaciens strains were tested for virulence on carrot root disks, Kalanchoe daigremontiana leaves, and Arabidopsis thaliana root explants as described previously (16,28). The basal surfaces of 10 to 18 carrot root slices were inoculated with overnight cultures of each bacterial strain (10 l/slice); five replicates were performed.…”

“…A plasmid containing the GALLS gene with M808I and M815V mutations was constructed as follows. Plasmid pLH389 (16) contains the coding sequence and promoter of the wildtype GALLS gene in a derivative of pBluescript SK(Ϫ) (Stratagene) with a loxP site inserted into its KpnI site. The NcoI fragment of the GALLS gene, which contains PstI site 3 ( Fig.…”

“…To compare levels of GALLS proteins in bacterial cells, A. tumefaciens mx358 cells harboring mutant or wild-type GALLS genes were cultured as described previously (16). Harvested cells were lysed in a French pressure cell, and soluble proteins were prepared as described previously (16).…”

“…To compare levels of GALLS proteins in bacterial cells, A. tumefaciens mx358 cells harboring mutant or wild-type GALLS genes were cultured as described previously (16). Harvested cells were lysed in a French pressure cell, and soluble proteins were prepared as described previously (16). Samples containing 18 g of protein were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) through 4 to 20% gradient gels (Bio-Rad), and proteins were transferred to a polyvinylidene difluoride membrane using a Bio-Rad miniProtean II electroblotting cell.…”

“…1) (15), which is important for its ability to substitute for VirE2 (16). This indicates that GALLS-FL performs a critical function inside the nucleus or at the nuclear membrane.…”