Figure 1.
Amino acid sequence alignment of VirB2 family proteins.
Multiple amino acid sequence alignment of VirB2 homologues with ClustalW2 [50]. The organism/plasmid name for each homolog is indicated on the left of the aligned sequence. UniProt accession numbers: Agrobacterium tumefaciens pTiC58, P17792; Agrobacterium rhizogenes pRi1724, Q9F5A1; Agrobacterium tumefaciens pTiA6, P05351; Agrobacterium vitis pTiS4, B9K417; Rhizobium etli p42a, Q2K2L1; Sinorhizobium medicae pSMED02; A6UMA7, Mesorhizobium ciceri chromosome (chr.), E8TGI0; Agrobacterium radiobacter K84 chromosome (chr.), B9JE70; Agrobacterium tumefaciens pAtC58, Q7D3S1; Rhizobium etli p42d, Q8KIM6; Sinorhizobium meliloti pSymA, Q92YZ4. The amino acid residues identical in all proteins are in black and those conserved in most but not all of the proteins are in gray. The arrow indicates the processing site of VirB2 encoded by pTiC58. Each region/domain of VirB2 is indicated: SP, signal peptide; TM-1, trans-membrane domain 1; CP, cytoplasmic domain; TM-2, trans-membrane domain 2; N-PP, N-terminal periplasmic domain; C-PP, C-terminal periplasmic domain.
Figure 2.
Western blot analysis of the intracellular and extracellular S2 fractions, and tumor assays on tomato stem of VirB2 variants.
A. tumefaciens cells grown on acetosyringone (AS)-induced AB-MES (pH 5.5) agar at 19°C for 3 days [7] were collected to isolate intracellular proteins and extracellular S2 fractions. C58, A. tumefaciens wild type strain C58; V, empty vector pRL662; ΔvirB2, virB2 deletion mutant; ΔvirB2(pVirB2), expression of wild type virBp-B1-B2-B3 in ΔvirB2. Western blot analysis with antisera against VirB2 B24 peptide or B23 peptide (for variants in C-PP) or RNA polymerase RpoA, as an internal control. Unprocessed VirB2 precursor is indicated as VirB2p and processed mature VirB2 as VirB2m. Shows representative tumor assay results on tomato stems. Similar results were obtained from at least three independent experiments (3–5 plants for each mutant in each independent experiment). Each region/domain of VirB2 is indicated as described in Figure 1.
Figure 3.
Phenotype summary of VirB2 variants.
VirB2 amino acid substitutions are indicated with the levels of extracellular VirB2 (ExB2) production and occurrence or size of tumor formation on tomato stems (Vir) with wild type (+++), modest reduction (++), highly attenuation (+), or loss (−). VirB2 protein sequences with indicated conserved amino acid residues, regions/domains, and the processing site (indicated by an arrow) are presented as described in Figure 1. The 34 VirB2 variants are classified into three groups: ExB2−/Vir−, ExB2−/Vir+, and ExB2+/Vir+ shown in red, green and black, respectively.
Figure 4.
T-pilus observation by transmission electron microscopy (TEM).
A. tumefaciens cells grown on T-pilus induction condition were collected and stained with 2% uranyl acetate to visualize T-pilus by TEM. Shows representative TEM image of A. tumefaciens strains producing wild-type VirB2 (A) or VirB2 variants D55A (B), I85A (C), L94A (D), M107A (E), A110G (F), G119A (G) and G119C (H). The rigid, long T-pilus is indicated by an arrow (A, G). Scale bar: 200 nm. All samples were examined for T-pilus formation by examining hundreds of bacterial cells from at least two independent experiments.
Figure 5.
T-pilus−/Vir+ uncoupling mutants show highly attenuated transient transformation efficiency in Arabidopsis seedlings.
A. tumefaciens strains expressing the wild type or variants of VirB2 harboring the T-DNA vector pBISN1 were used to infect 4-day-old Arabidopsis seedlings. GUS activity as a reporter of transient transformation efficiency was determined by GUS staining (A and B) or quantitative activity assay (C) at 3 dpi. (A) GUS staining of T-pilus−/Vir+ uncoupling mutants and (B) T-pilus+/Vir+ mutants. (C) Quantitative GUS activity of all mutants. Data are mean±SD of 4 biological repeats from 2 independent experiments (10 seedlings in each biological repeat). The data were analyzed by ANOVA for statistical classification, which revealed two groups (groups a and b) of strains differing in transient GUS activity.
Figure 6.
Transient transformation assay in Arabidopsis seedlings with or without wounded cotyledons.
A. tumefaciens strains expressing the wild type or variants of VirB2, L94A (T-pilus−/Vir+), A110G (T-pilus−/Vir+) and G121A (T-pilus+/Vir+), harboring the T-DNA vector pBISN1 were used to infect 7-day-old Arabidopsis seedlings. GUS activity as a reporter of transient transformation efficiency was determined by GUS staining or quantitative activity assay at 3 dpi. (A) Infection of Arabidopsis seedlings with cotyledons wounded by a needle before infection. A. tumefaciens cells grown in 523 overnight culture without AS induction were used to infect the wounded Arabidopsis seedlings in the absence of AS. (B) Infection of Arabidopsis seedlings without intentional wounding, in which A. tumefaciens cells were pre-induced by AS for infection and co-cultured in the presence of AS. Data for quantitative GUS activity are mean±SD of four biological repeats from two independent experiments (10 seedlings in each biological repeat).