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. 2014 Oct 23;10(10):e1004655.
doi: 10.1371/journal.pgen.1004655. eCollection 2014 Oct.

The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana

Kee Hoon Sohn  1 , Cécile Segonzac  1 , Ghanasyam Rallapalli  2 , Panagiotis F Sarris  2 , Joo Yong Woo  3 , Simon J Williams  4 , Toby E Newman  5 , Kyung Hee Paek  3 , Bostjan Kobe  4 , Jonathan D G Jones  2
Affiliations

The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana

Kee Hoon Sohn et al. PLoS Genet. .

Abstract

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific "avirulent" pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PopP2 triggers RPS4/RRS1/EDS1-dependent hypersensitive response and immunity when delivered from Pseudomonas.
(A) AvrRps4N-PopP2 fusion construct. (B) Pseudomonas fluorescens Pf0-1(T3S)-delivered AvrRps4N:PopP2149–488 triggers an EDS1-dependent hypersensitive response (HR) in resistant Arabidopsis accessions. Five week-old Arabidopsis leaves were infiltrated with Pf0-1(T3S) strains expressing indicated avirulence proteins. Empty vector (EV) indicates AvrRps4N encoded by pEDV5 (see Figure S1). The photograph was taken at 24 hours post-infection (hpi). The red asterisks indicate the leaves showing HR. (C) Pf0-1(T3S)-delivered AvrRps4N:PopP2149–488 triggers an RPS4/RRS1-dependent HR in Ws-2 accession. (D) Pseudomonas syringae pv. tomato (Pto) DC3000-delivered AvrRps4N:PopP2149–488 triggers RPS4/RRS1-dependent immunity in accession Ws-2. Five week-old Arabidopsis leaves were infiltrated with Pto DC3000 strains and samples were taken at 4 dpi to recover bacteria from infected leaves. The results presented are the mean and standard error of the number of bacterial colonies recovered. Means labeled with the same letter are not statistically different at the 5% confidence level based on Tukey's test.
Figure 2
Figure 2. Pseudomonas-delivered PopP2 induces RRS1- and acetyltransferase activity-dependent transcriptional changes early after bacterial infection.
(A) Hierarchical clustering of RRS1- or PopP2-dependent gene expression. Fold-change values of 719 genes (differentially expressed at least in one time point) from all time points show the predominance of gene induction at early time points. Black, red and green colours indicate no change, up-regulated and down-regulated, respectively. C321A, an inactive PopP2 variant carrying an Alanine mutation at one of the catalytic core residues, Cysteine 321 (B) Confirmation of selected PopP2-induced genes by qRT-PCR. Five week-old plants were infiltrated with Pf0-1(T3S) expressing the indicated AvrRps4, HopA1 or PopP2 variants. Samples were taken at 8 hpi for total RNA extraction. The numbers on the Y-axis indicate fold induction compared to mock treated samples.
Figure 3
Figure 3. Low temperature-dependent transcription profiling of the slh1 mutant.
(A) Hierarchical clustering of No-0 and slh1 temperature-dependent differential gene expression. Fold-change values of 5611 genes (differentially expressed at least in one time point) are shown. The numbers on top of the heat map indicate the time (h) after temperature shift. Black, red and green colours indicate no change, up-regulated and down-regulated, respectively. (B) qRT-PCR analysis of selected RRS1SLH1-regulated genes following the temperature shift (28°C to 19°C) in 4 week-old No-0 and slh1 plants. Transcript accumulation is presented relative to No-0 before temperature shift (28°C).
Figure 4
Figure 4. Percentage pairwise overlap of genes differentially expressed during the time course of PopP2 or PopP2C321A on Ws-2 and rrs1-1 and the time course of temperature shift on No-0 and slh1.
Each time course response is categorized based on underlying response (PTI, ETI, temperature shift, auto-immunity and combinations). Each cell represents percentage of genes differentially expressed from the column experiment that were also differentially expressed in the row experiment. Green boxes highlight genes regulated by heat stress and PTI, PTI+ETI responses; blue box highlights genes regulated by PTI, ETI and PTI+ETI; black boxes highlight genes regulated by auto-immunity, heat stress and by PTI, ETI and PTI+ETI. The number of gene differentially expressed in each time course is indicated on the right. PTI, PopP2C321A-regulated genes; ETI, PopP2WT- but not PopP2C321A-regulated genes; temperature shift, temperature shift-regulated genes in No-0 wild-type; auto-immunity, temperature shirt-regulated genes in slh1 mutant but not in No-0 wild-type.
Figure 5
Figure 5. Identification of sushi (suppressor of slh1 immunity) mutants.
Fully rescued sushi mutant (M3), wild type No-0 and slh1 plants were grown at 21°C under short-day condition for four weeks. (A) Plant morphology. (B) qRT-PCR analysis of selected RRS1SLH1-regulated genes. Transcript accumulation is presented relative to No-0.
Figure 6
Figure 6. RPS4 is required for RRS1SLH1-mediated activation of immunity.
(A) Schematic presentation of SUSHI mutations in RPS4. The asterisk indicates premature stop codon. (B) The RRS1SLH1-induced growth restriction phenotype of sushi mutants is RPS4-dependent. The F1 hybrids between rrs1-1 or rrs1-1 rps4-21 and sushi were grown for five weeks at 21°C before the photograph was taken. (C) Growth restriction of F1 hybrids (shown in (B)) correlates with PR1 transcript accumulation as determined by qRT-PCR. PR1 transcript accumulation is presented relative to rrs1-1 and rrs1-1 rps4-21 respectively. (D) RPS4SUSHI variants do not confer RRS1SLH1-induced hypersensitive response or recognition of AvrRps4 or PopP2 when transiently expressed in tobacco leaf cells. Photographs were taken 3 days after agroinfiltration.
Figure 7
Figure 7. Functional analysis of SUSHI mutations in the RPS4 TIR domain.
(A) SUSHI mutations within the RPS4 TIR domain structure (PDB ID 4c6r) in cartoon (top) and surface (bottom) representation (figures were generated using PyMOL (Delano Scientific)). Molecules are rotated 90° around the y-axis from left to right. Mutated residues are labelled R28 (Blue – sushi52), A38 (Teal – sushi14), E88 (Green – sushi22), L101 (Lime – sushi71), P105 (Orange – sushi89) and G120 (Red – sushi29). (B) The SUSHI mutations abolish RPS4 TIR-induced HR in tobacco agroinfiltration assay. (C) Analysis of the full-length RPS4 variants carrying SUSHI mutations in the TIR domain for recognition of AvrRps4 or PopP2 in tobacco agroinfiltration assay. The photographs were taken 3 days after agroinfiltration.
Figure 8
Figure 8. Characterization of RRS1SLH1-induced hypersensitive response.
RRS1 (R1), RPS4 (R4), AvrRps4 (A4) and PopP2 (P2) were C-terminally epitope-tagged with His-Flag, HA, GFP and GFP, respectively. The photographs were taken 3 days after agroinfiltration. (A) The nuclear localization of RPS4 is required for RRS1SLH1-induced HR. NES and NLS indicate nuclear export signal and nuclear localization signal, respectively. (B) RRS1SLH1-dependent HR requires RPS4 P-loop (K242A) and TIR-TIR domain heterodimerization (SH-AA) but not RRS1 P-loop (K185A). (C) The interference of RRS1SLH1-induced HR by wild type RRS1 requires the P-loop but not the SH-motif. (D) RRS1SLH1 does not fully interfere with AvrRps4 or PopP2 recognition by wild type RRS1.
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