Interaction of HSP90 with RAR1 and SGT1. (A) Domain structures of HvHSP90 constructs. Numbers refer to amino acids encoded. N, N-terminal ATPase domain; M, middle substrate binding domain; C, C-terminal for dimerization and cochaperone binding. (B) In vivo interaction analysis of RAR1 and HSP90 by the yeast two-hybrid system. (Left) Domain structures of plant RAR1 constructs. Interactions were performed by using the LexA system with the lacZ reporter gene, expressing RAR1 proteins as binding domain fusions and HvHSP90 proteins as activator domain fusions. (C) In vitro binding assay using HsHsp90 and S-tag RAR1 fusion derivatives. The precipitated proteins were immunoblotted with the indicated antibodies. Molecular mass markers are indicated (in kDa). (D) Sequence alignment of the TPR domain from SGT1 and PP5 proteins: human SGT1 (Hs, residues 10–123, GenBank accession no. AAD30062), barley SGT1 (Hv, 5–118, AAL33610), Arabidopsis SGT1a and SGT1b (At, 1–114, At4g23570 and At4g11260, respectively), human PP5 (Hs, 27–140, AAD22669), Schizosaccharomyces pombe PP5 (Sp, 4–117, T40391), Saccharomyces cerevisiae PPT1 (Sc, 11–124, S52571). Green indicates 100% conserved residues, and blue indicates >50% conserved residues. (E) In vivo interaction analysis of SGT1 and HSP90 by the yeast two hybrid system. Domain structures of HvSGT1 and AtSGT1b made in pLexA vector are shown on the left. Interactions were detected as shown in B. (F) Coimmunoprecipitation of HvSGT1 and HvHSP90 in barley. Protein extract from HvRAR1 or mutant hvrar1–2 barley plants were immunoprecipiated with SGT1 or preimmune (PI) antibodies. Samples of eluted fractions were analyzed by immunoblotting with antibodies to SGT1 and HSP90.

AtHSP90.1 mRNA expression is induced by Pst DC3000 strains. RT-PCR analysis was performed on mRNA isolated from leaves inoculated with Pst DC3000 strains (1 × 10⁷ cfu/ml) containing vector only, or clones expressing avrRpm1 or avrRpt2 or leaves subjected to mock inoculation. RT-PCR from an actin gene was used as a control to verify evenness of RNA template amounts.

Geldanamycin inhibits RPS2-dependent HR and resistance. (A) The HR test by trypan blue staining. The right half of the leaves from 6-week-old Arabidopsis Col-0 plants (containing both RPM1 and RPS2) were infiltrated with Pst DC3000 strains (1 × 10⁷ cfu/ml) containing vector only, or clones expressing avrRpm1 or avrRpt2. GDA (10μM) or mock solutions lacking GDA were infiltrated together with the bacterial pathogens. Leaves were stained with trypan blue 8 or 20 hpi. Fractions indicate numbers of leaves exhibiting HR and total number of leaves tested. (B) Bacterial growth analysis of Pst DC3000 strains (1 × 10⁵ cfu/ml) containing vector only, or clones expressing avrRpm1 or avrRpt2, inoculated into Arabidopsis Col-0, together with GDA (pink line) or solution lacking GDA (blue line). These experiments were performed three times with similar results.

AtHSP90.1 is required for RPS2-dependent resistance. (A) Relative position of T-DNA insertions within the AtHSP90.1 gene. Exons are indicated by black boxes. Primers used for RT-PCR were indicated. (B) Bacterial growth analysis of Pst DC3000 (vector) (1 × 10⁵ cfu/ml), which was hand-infiltrated with the needleless syringe into the leaves of 6- to 7-week-old Arabidopsis Col-0, or mutants athsp90.1-1 and athsp90.1-2. Leaves were harvested immediately after infiltration (white column) or at 3 dpi (black column). (C) Same as in B, except with Pst DC3000 (avrRPM1). (D) Same as in B, except with Pst DC3000 (avrRpt2). *, Significantly different from wild-type control at P < 0.05. (E) Disease phenotype of athsp90.1-1 and athsp90.1-2. The right halves of leaves were hand-infiltrated with Pst DC3000 (vector) or Pst DC3000 (avrRpt2) (1 × 10⁵ cfu/ml), and photographs were taken 6 dpi.