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 single-stranded 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.

FIG. 1.

Domains in the GALLS protein. Boxes indicate the locations of helicase motifs I, II, and III, TraA-like domains 1 to 5, the NLS, GALLS repeats 1 to 3, and the type IV secretion signal. Wild-type and mutant amino acid sequences are shown for Walker A and B ATP-binding sites in the GALLS protein encoded by pRi1724; amino acids that match the Walker box consensus sequences are in bold type, and mutations are underlined. Helicase motif III in the GALLS protein from pRi1724 is aligned with TraA from A. tumefaciens pTiC58 (accession no. NC_003065) and RecD (a subunit of the RecBCD helicase/nuclease) from Mycoplasma pulmonis (accession no. CAC13955); amino acids that match the helicase motif III consensus sequence are in bold type, and dashes indicate amino acids deleted from the GALLSΔhelicase III protein. The nuclear localization signal in the GALLS protein encoded by pRi1724 is aligned with the NLS in VirD2 from pTiA6 (accession no. AF242881); basic amino acids are in bold type.

FIG. 2.

Virulence tests on carrot disks inoculated with an A. tumefaciens virE2 mutant harboring wild-type or mutant GALLS genes. Carrot disks were left uninoculated (D), or carrots were inoculated with derivatives of an A. tumefaciens virE2 mutant (MX358) containing plasmids that encode the following: (A) wild-type GALLS (pLH338), (B) wild-type GALLS (pJNM389), (C) GALLSΔNLS (encoded by gallsΔ705-723; pJNM390), (E) GALLS::TEV-NLS (pJNM392), (F) vector-only control (pVK100), (G) GALLS Walker A mutant (K172E; pLH372), (H) GALLS Walker B mutant (D239N; pLH373), or (I) GALLSΔhelicase motif III (gallsΔ273-282; pLH371).

FIG. 3.

Immunoblot detection of mutant and wild-type GALLS proteins and Cre::GALLS fusion proteins. Numbers beside each panel indicate molecular weight standards (in thousands). Panel A shows an immunoblot probed with polyclonal rabbit antibodies raised against the purified GALLS protein. Soluble proteins were extracted from A. tumefaciens MX358 expressing the following: no GALLS protein (vector-only control; lane 1), wild-type GALLS (lane 2), GALLSΔ273-282 (helicase motif III deletion; lane 3), GALLS-K172E (Walker A mutation; lane 4), GALLS-D239N (Walker B mutation, lane 5), wild-type GALLS (lane 6), GALLSΔ705-723 (NLS deletion, lane 7), or GALLS plus TEV NLS (lane 8). Protein samples in lanes 2 to 5 were extracted from strains that contain the GALLS gene in a multicopy IncP plasmid (pVK100), whereas samples in lanes 6 to 8 were extracted from strains that contain the GALLS gene in a single-copy replicon based on an Ri plasmid ori (pDM12), which may explain the lower levels of the GALLS protein in these samples. Panel B shows an immunoblot probed with Cre-specific antibodies. Soluble proteins were extracted from A. tumefaciens LBA1100 expressing the following: Cre::GALLS-27R>Dx2 (lane 1), Cre::GALLS-27ΔR (lane 2), Cre::GALLS-27Δ6 (lane 3), Cre::GALLS-27Δ2 (lane 4), Cre::GALLS-27R>E/E>R (lane 5), Cre::GALLS-11 (lane 6), Cre::GALLS-27 (lane 7), or Cre (lane 8). Cre::GALLS-27 protein levels in A. tumefaciens LBA2587 were comparable to those in LBA1100 (data not shown).

FIG. 4.

The carboxy terminus of GALLS contains a type IV secretion signal. GFP fluorescence is an indicator of Cre-Vir protein transfer from A. tumefaciens into root cells. Roots from A. thaliana line CB1 were cocultivated with A. tumefaciens LBA1100 expressing Cre (B) or Cre fused to the C-terminal 27 (A) or 11 (E) amino acids of GALLS. Root explants were cocultivated with an A. tumefaciens virE2 mutant (LBA2573) containing Cre fused to the last 27 (C) or 11 (D) amino acids of GALLS. Panel F shows roots infected with an A. tumefaciens virE1 mutant (LBA2571) containing Cre fused to the last 11 amino acids of GALLS.