Arabidopsis TNL-WRKY domain receptor RRS1 contributes to temperature-conditioned RPS4 auto-immunity

Abstract

In plant effector-triggered immunity (ETI), intracellular nucleotide binding-leucine rich repeat (NLR) receptors are activated by specific pathogen effectors. The Arabidopsis TIR (Toll-Interleukin-1 receptor domain)-NLR (denoted TNL) gene pair, RPS4 and RRS1, confers resistance to Pseudomonas syringae pv tomato (Pst) strain DC3000 expressing the Type III-secreted effector, AvrRps4. Nuclear accumulation of AvrRps4, RPS4, and the TNL resistance regulator EDS1 is necessary for ETI. RRS1 possesses a C-terminal "WRKY" transcription factor DNA binding domain suggesting that important RPS4/RRS1 recognition and/or resistance signaling events occur at the nuclear chromatin. In Arabidopsis accession Ws-0, the RPS4(Ws) /RRS1(Ws) allelic pair governs resistance to Pst/AvrRps4 accompanied by host programed cell death (pcd). In accession Col-0, RPS4(Col) /RRS1(Col) effectively limits Pst/AvrRps4 growth without pcd. Constitutive expression of HA-StrepII tagged RPS4(Col) (in a 35S:RPS4-HS line) confers temperature-conditioned EDS1-dependent auto-immunity. Here we show that a high (28°C, non-permissive) to moderate (19°C, permissive) temperature shift of 35S:RPS4-HS plants can be used to follow defense-related transcriptional dynamics without a pathogen effector trigger. By comparing responses of 35S:RPS4-HS with 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 mutants, we establish that RPS4(Col) auto-immunity depends entirely on EDS1 and partially on RRS1(Col) . Examination of gene expression microarray data over 24 h after temperature shift reveals a mainly quantitative RRS1(Col) contribution to up- or down-regulation of a small subset of RPS4(Col) -reprogramed, EDS1-dependent genes. We find significant over-representation of WRKY transcription factor binding W-box cis-elements within the promoters of these genes. Our data show that RRS1(Col) contributes to temperature-conditioned RPS4(Col) auto-immunity and are consistent with activated RPS4(Col) engaging RRS1(Col) for resistance signaling.

Keywords: EDS1 signaling; biotic stress; programed cell death; resistance gene pair; temperature shift; transcriptional reprograming.

FIGURE 1

RPS4^Ws and RRS1^Ws act cooperatively in AvrRps4-triggered bacterial resistance and pcd. (A) Four-week-old plants were spray-inoculated with virulent Pst DC3000 or Pst expressing AvrRps4-HA, AvrRps4-HA-NLS, or AvrRps4-HA-NES variants. Bacterial titers at 3 dpi are shown. All bacterial strains had similar entry rates measured at 3 hpi (data not shown). Replicate values were combined from three independent experiments with similar results and SEs calculated using a linear model. ***Significant difference (p < 0.001). (B) Ion leakage measurements were recorded at the indicated time points in leaf disks of 4-week-old Ws-0, eds1-1, rps4-21, rrs1-1, and rps4-21 rrs1-1 plants after infiltration with Pfo-expressing AvrRps4-HA. Error bars represent standard errors of four samples per genotype. The experiment was performed three times with similar results.

FIGURE 2

Mutation of RRS1^Col partially suppresses 35S:RPS4-HS stunting. (A) Growth at 22°C of representative 3.5-week-old Col-0, eds1-2, and rrs1-11 and the same backgrounds containing the 35S:RPS4-HS transgene. Scale bar, 1.5 cm. (B) Quantification of rosette diameters at 3.5 weeks of lines shown in (A). (C) Immunoblot analysis of total leaf protein extracts separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) from the 3.5-week-old 35S:RPS4-HS transgenic leaf tissues in Col-0, eds1-2, and rrs1-11 backgrounds, probed with α-HA antibody. Ponceau S staining shows equal transfer of protein samples to the membrane. Two independent experiments gave similar results.

FIGURE 3

RRS1^Col contributes to enhanced basal and AvrRps4-triggered resistance of 35S:RPS4-HS at 22°C. 3.5-week-old plants of the indicated lines grown at 22°C were spray-inoculated with virulent Pst DC3000 (A) or avirulent Pst/AvrRps4 (B) bacteria in the same experiment. Bacterial titers were measured at 3 hpi (d0) indicating bacterial entry rates and at 3 dpi (d3). Standard errors were calculated from three biological samples per genotype. Letters (a,b,c,d) indicate significant differences (p < 0.05) calculated by a Student’s t-test. Experiments were performed independently three times with similar results.

FIGURE 4

A 28 to 19°C temperature shift induces RPS4-HS auto-immunity. (A) Growth of 3.5-week-old 35S:RPS4-HS plants at 28°C (upper panel) and 6 days after moving to 19°C (lower panel). Scale bars, 2 cm. (B) Semi-quantitative RT-PCR of known Pst/AvrRps4-responsive, EDS1-dependent genes over 0–6 h after temperature shift of Col-0, 35S:RPS4-HS eds1-2, and 35S:RPS4-HS Col-0 plants, as indicated. (C) Ion leakage measurements made over a 10-day period after shift from high to low temperature (dps) in leaf disks of the different 3.5-week-old 35S:RPS4-HS lines and Col-0 wild-type, as indicated. Error bars represent standard errors of four samples per genotype. Three independent experiments gave similar results. (D) Immunoblot analysis of total leaf protein extracts from 3.5-week-old 35S:RPS4-HS lines grown at 28°C and shifted to 19°C for 8 h, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with α-HA antibody. Ponceau S staining shows equal transfer of protein samples to the membrane.

FIGURE 5

RRS1^Col contributes to EDS1-dependent gene expression changes in RPS4^Col auto-immunity. Gene expression microarray analysis was performed on leaf RNAs of 3.5-week-old 35S:RPS4-HS transgenic plants in the Col-0, rrs1-11, or eds1-2 backgrounds at 0, 2, 8, and 24 h after temperature shift. (A) Induced or repressed genes (q-values < 0.01 and >2-fold changes) in 35S:RPS4-HS Col-0 upon temperature shift at any time point and the log₂ ratios compared to 0 h are plotted. Linear regression lines indicate log₂ ratios in 35S:RPS4-HS Col-0 (red) and the 35S:RPS4-HS rrs1-11 or 35S:RPS4-HS eds1-2 mutant lines (black). (B) Log₂ gene expression ratios at 8 h after temperature shift compared to 0 h in 35S:RPS4-HS Col-0 plants for previously identified Pst/AvrRps4-triggered EDS1-dependent genes, shown by Heatmap clustering analysis. (C) Heatmap clustering of 250 genes whose expression changes are RRS1-dependent in 35S:RPS4-HS after temperature shift at any time point (q-values < 0.01 and >2-fold change). Expression patterns for the 250 genes in 35S:RPS4-HS Col-0, 35S:RPS4-HS rrs1-11, and 35S:RPS4-HS eds1-2 lines are shown at 2, 8, and 24 h. Highlighted clusters 1–4 are described in the text. (D) GO term analysis of the 250 RRS1-dependent genes.

FIGURE 6

W-boxes are highly enriched in promoters of RRS1-dependent genes. POBO analysis of the motif distribution in 1000 bp promoters of RRS1-dependent genes. One thousand pseudo-clusters of the 250 RRS1-dependent genes, genes regulated by the temperature shift (Temp-shifted) and randomly selected genes from 35S:RPS4-HS (Genome-wide) are shown. Jagged lines indicate motif frequencies from which a fitted curve was derived. The W-box (TTGACY) is significantly over-represented in promoters of the RRS1-dependent genes compared to temperature-responsive genes or genes from the genome background with p-values < 0.0001.