Figure 1.
Gp-RBP-1 induces a Gpa2-mediated HR in Nicotiana benthamiana leaves.
(A) HA-tagged Rx and Rx2, or untagged Gpa2 driven by the Rx promoter were transiently expressed via agro-expression in wild-type N. benthamiana leaves together with 35S promoter-driven PVX CP or a G. pallida RBP-1 protein cloned from the population Chavornay (Chav-1) fused to a C-terminal EGFP fusion and epitope tag (EGFP:HA). EGFP:HA and a G. rostochiensis RBP-1: EGFP:HA fusion were included as controls. HRs were observed within 2 to 3 days of ago-expression. (B) Tagged and untagged versions of Gp-RBP-1 were also tested that included the 23 amino acid secretion signal peptide (SP) from the predicted full-length Gp-RBP-1 protein [Gp-(SP)RBP-1 and Gp-(SP)RBP-1:EGFP:HA]. HRs were observed within 2 to 3 days of ago-expression.
Figure 2.
Distribution of the Ka/Ks ratio along the RBP-1 amino acid sequence.
Analyses were conducted using the codeml module of PAML on the full data set of G. pallida and G. mexicana sequences. Amino acid variants found to be subjected to positive selection with posterior probability >95% (Table S1A) are indicated in red above each site. Amino acid variants found to be subjected to positive selection in PAML and at least one other method (Table S1B) are indicated in italic. Sequence portions corresponding to the SPRYSEC extended loops in the B30.2 protein structure are highlighted in pink. The entire B30.2 domain is indicated by a bar above the graph, with the region containing the duplicated PRY domains indicated by double bars.
Figure 3.
Analysis of Gp-RBP-1 variants from virulent and avirulent populations.
Alignment of deduced Gp-RBP-1 proteins encoded by cDNA sequences cloned from G. pallida populations D383 (avirulent; pathotype Pa-2), Rookmaker (virulent; Pa-3) and Chavornay (virulent; Pa-2/3). Variant residues are indicated with shading, with the critical proline/serine polymorphism indicated in red. PRY domain repeats are indicated by a red bar over the alignment, with the dashed segment of the bar corresponding to an extension of the repeat in two of the variants. The SPRY homology domain is overscored by the black bar.
Figure 4.
Recognition of Gp-RBP-1 by Gpa2 corresponds to avirulence, but not virulence in G. pallida populations.
(A) Gp-RBP-1 variants (shown in Figure 3) cloned into pBIN61 as HA-tagged proteins under control by the CMV 35S promoter were transiently expressed via agro-infiltration on GPAII::Gpa2 transgenic tobacco. The responses in the infiltrated patches were scored visually with a complete lack of response scored as (-). Positive HR responses were scored as follows: complete collapse and rapid desiccation of the infiltration patch within 2 days (+++), complete collapse of the infiltration patch by 3 days post-infiltration (++), or slow and incomplete collapse with residual live cells (+). HR phenotypes representative of the scale used herein are shown (B), as photographed seven days after infiltration. The presence of either a proline (P) or serine (S) residue at the position corresponding to Rook-1 residue 187 is indicated. (C) Immunoblot with horse radish peroxidase-conjugated anti-HA antibody demonstrating relative protein levels of transiently expressed RBP-1 proteins.
Figure 5.
A single residue in the Gp-RBP-1 SPRY domain is a key determinant of Gpa2 recognition.
(A) Proline 187 of Rook-1 and Chav-7 was substituted for serine, and serine 187 of Rook-4 and Gmex-1 was substituted for proline. The resulting RBP-1:HA proteins were transiently expressed in Gpa2 tobacco leaves. Note that Rook-4 S187P induced an HR of a strength equivalent to those elicited by Rook-1 and Chav-7 (+++ as per Figure 4B), whereas Gmex-1 S187P induced a much weaker response (+ as per Figure 4B). RBP-1:HA variants were also expressed in wild-type tobacco and protein extracts were subjected to anti-HA immunoblotting (IB) to determine protein expression levels (lower panel). (B) Deletions of, and fusions between, G. pallida Chav-7 and G. mexicana Gmex-1 RBP-1:HA are represented schematically. Individual proteins were expressed in wild-type tobacco and protein extracts were subjected to anti-HA immunoblotting to determine protein expression levels (lower panel). Individual proteins were scored for their ability to induce an HR on Gpa2-transgenic tobacco as per the scale in Figure 4B.
Figure 6.
Gpa2-mediated responses to PVX-RBP-1:HA requires RanGAP2.
PVX vectors were generated to express two avirulent versions (D383-2 and D383-4) of Gp-RBP-1:HA (PVX-D2 and PVX-D4) as well as two virulent (Rook-2 and Chav-4) variants (PVX-R2 and PVX-C4). (A) Virus saps containing recombinant viruses were rub-inoculated onto Gpa2-transgenic N. benthamiana that had previously been infected with the empty TRV VIGS (TV:00) vector or TRV:RGAP2. Phenotypes from a representative experiment are shown for PVX-D2 and PVX-R2, photographed two weeks after PVX inoculation. Virus spread to systemic tissues was observed either by the development of systemic lesions and necrosis (PVX-D2 and PVX-D4) or PVX symptoms typical of infected wild-type plants (PVX-R2 and PVX-C4). Necrosis on local and systemic leaves is indicated by arrows. (B) Protein extracts taken from inoculated and systemic leaves of Gpa2-transgenic N. benthamiana plants, infected as in (A), were subjected to anti-HA immunoblotting (IB) to detect Gp-RBP-1:HA accumulation.
Figure 7.
Tethering of RanGAP2 and Gp-RBP-1 enhances Gpa2-mediated HR.
(A) The open reading frames of RanGAP2, Gp-RBP-1 clone D383-2 and GUS were fused at their C termini to either the C-terminal or N-terminal fragments of YFP:FLAG (cYF and nYF, respectively). D383-2:cYF and D383-2:nYF were co-expressed, by agro-infiltration, in Gpa2-transgenic tobacco together with both complementing fusion proteins (yellow) and non-complementing YFP fusion proteins (white) as indicated (top panel). RanGAP2 with only a C-terminal FLAG tag (RanGAP2:F) was included as an additional non-complementing control. (B) Fusion proteins were also expressed in wild-type tobacco and protein extracts were subjected to anti-FLAG immunoblotting (IB) to confirm that activation in the combinations with complementing YFP fragments did not correlate with the highest RanGAP2 levels.