HSP90 Contributes to chs3-2D-Mediated Autoimmunity

Abstract

Plants employ multi-layered immune system to fight against pathogen infections. Different receptors are able to detect the invasion activities of pathogens, transduce signals to downstream components, and activate defense responses. Among those receptors, nucleotide-binding domain leucine-rich repeat containing proteins (NLRs) are the major intracellular ones. CHILLING SENSITIVE 3 (CHS3) is an Arabidopsis NLR with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C terminus. The gain-of-function mutant, chs3-2D, exhibiting severe dwarfism and constitutively activated defense responses, was selected as a genetic background in this study for a forward genetic screen. A mutant allele of hsp90.2 was isolated as a partial suppressor of chs3-2D, suggesting that HSP90 is required for CHS3-mediated defense signaling. In addition, HSP90 is also required for the autoimmunity of the Dominant Negative (DN)-SNIPER1 and gain-of-function ADR1-L2 D484V transgenic lines, suggesting a broad role for HSP90 in NLR-mediated defense. Overall, our work indicates a larger contribution of HSP90 not only at the sensor, but also the helper NLR levels.

Keywords: CHILLING SENSITIVE 3; CHS3; CSA1; HSP90; SNIPER1; helper NLR; plant immunity; sensor NLR.

FIGURE 1

Identification and characterization of chs3-2D suppressors. (A) Morphology of 3-week-old soil-grown plants of chs3-2D suppressors at 18 °C. (B) Weights of 3-week-old soil-grown plants of chs3-2D suppressors at 18 °C. (C) Quantification of H.a. Noco2 sporulation on the leaf surface of chs3-2D suppressors. (D) Morphology of 3-week-old soil-grown plants of the mutant chs3-2D hsp90.2 complementary transgenic lines at 18 °C. (E) Weights of 3-week-old soil-grown plants of the mutant chs3-2D hsp90.2 complementary transgenic lines at 18 °C. (F) Quantification of H.a. Noco2 conidia growth on the leaf surface of the mutant chs3-2D hsp90.2 complementary transgenic lines. (G) Amino acid alignments of selected HSP90 homologs from eukaryotic organisms. The amino acids highlighted in green are the nucleotide binding site. The mutated amino acid is labeled with a red box. For panel (G), List of the organisms included: mouse-ear cress Arabidopsis thaliana HSP90.2, maize Zea mays (C3UZ63), rice Oryza sativa (Q0J4P2), rapeseed Brassica napus (A0A078GRJ1), human Homo sapiens (CAA33259.1), yeast Saccharomyces cerevisiae (AJS65103.1), nematode Caenorhabditis elegans (NP_506626.1), alga Chlamydomonas reinhardtii (XP_001695264.1), fruit fly Drosophila melanogaster (NP_001261362.1), and moss Physcomitrium patens (XP_024385281.1). Protein sequences were aligned using CLUSTAL. (H) Schematic diagram of the HSP90 domains. Hsp90 is comprised of three domains: an N-terminal ATP-binding domain (N, green) that may contain a peptide binding element; a middle domain (M, yellow) that interacts with client proteins and also contains a loop that catalyzes ATP hydrolysis; and a C-terminal dimerization domain (C, blue). A charged region exists between the N and M domains (linker region, white). Data information: For panels (C,F), Two-week-old soil-grown seedlings were sprayed with a spore suspension of H.a. Noco2 at a concentration of 100,000 spores/ml of water. The plants were then covered and incubated for seven days in a high humidity growth chamber. Spores were counted in water suspension using a hemocytometer (bars represent means of n replicates ± SD, n = 3 with 4 plants each). One-way ANOVA followed by Tukey’s post hoc test were performed for panels (A–F). Statistical significance is indicated by different letters (P < 0.05). Error bars represent mean SD (n = 5).

FIGURE 2

Chaperone protein HSP90s and SGT1b contribute to the autoimmunity caused by overaccumulation of sensor NLRs. (A) Morphology of 3-week-old soil-grown plants of DN-SNIPER1 hsp90, DN-SNIPER1 sgt1b double mutant at 23 °C. (B) Weights of 3-week-old soil-grown plants of DN-SNIPER1 hsp90, DN-SNIPER1 sgt1b double mutant at 23 °C. (C) Quantification of H.a. Noco2 conidia growth on the leaf surface of DN-SNIPER1 hsp90, DN-SNIPER1 sgt1b double mutant. Data information: For panel C, the experimental procedure was carried out as described in Figure 1. One-way ANOVA followed by Tukey’s post hoc test were performed for panels (B,C). Statistical significance is indicated by different letters (P < 0.05). Error bars represent mean SD (n = 5).

FIGURE 3

Mutations at HSP90s or SGT1b partially suppress the autoimmunity of ADR1-L2 D484V. (A) Morphology of 3-week-old soil-grown plants of ADR1-L2 D484V hsp90, ADR1-L2 D484V sgt1b double mutant at 23 °C. (B) Weights of 3-week-old soil-grown plants of ADR1-L2 D484V hsp90, ADR1-L2 D484V sgt1b double mutant at 23 °C. (C) Quantification of H.a. Noco2 conidia growth on leaf surface of ADR1-L2 D484V hsp90, ADR1-L2 D484V sgt1b double mutant. The experiment was carried out as described in Figure 1. One-way ANOVA followed by Tukey’s post hoc test were performed for panels (B,C). Statistical significance is indicated by different letters (P < 0.05). Error bars represent mean SD (n = 5).