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Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors - PubMed

  • ️Mon Jan 01 2018

Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors

Yan Ma et al. Proc Natl Acad Sci U S A. 2018.

Abstract

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R-RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector's authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY-domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY-domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a "reversibly closed" conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors.

Keywords: effector target; effector-triggered immunity; integrated decoy; paired NLR immune receptors; plant-disease resistance.

Copyright © 2018 the Author(s). Published by PNAS.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

RRS1 WRKY domain (DOM5) is required for autoinhibition and domain 4 (DOM4) for activation of the RRS1–RPS4 complex. (A) Successive deletions of RRS1-R compromise effector responsiveness or autoinhibition. Each tobacco leaf section was coinfiltrated to transiently express RPS4 and a truncated RRS1-R with either mCherry (mCh), AvrRps4:mCh or PopP2:mCh. ∆C83, deletion of C-terminal 83 aa of RRS1-R; ∆D56, deletion of the WRKY domain (DOM5) and domain 6 of RRS1-R (DOM6-R). (B) Replacement of DOM5 and/or DOM6 of RRS1 with that of RRS1B causes RPS4-dependent autoactivity. Each leaf section was infiltrated to express a chimeric RRS1 with or without RPS4. The chimeras are represented with domains from RRS1 as A’s, domains from RRS1B as B’s, bacterial LexA shown as L, and the WRKY domain and C-terminal amino acids of WRKY41 shown as W41. (C) Loss of effector responsiveness in the RRS1-R chimeras where NB-ARC, LRR, or domain 4 (DOM4) is replaced by an equivalent domain of RRS1B, when coexpressed with RPS4. Each section represents the presence (yellow) or absence (green) of HR in tobacco leaves at 4 d postinfiltration (dpi). HRs were assessed at 4 dpi. Phenotypes are representative of at least three consistent replicates.

Fig. 2.
Fig. 2.

Distinct genetic requirements in RPS4 and RRS1 for PopP2 and AvrRps4 responsiveness, and for autoactivity. (A) RPS4 C-terminal domain (CTD) is required for RPS4 + RRS1-R∆D56–triggered HR. Each leaf section was coinfiltrated to express RRS1-R∆D56 with a CTD variant of RPS4. Deletion of CTD (∆CTD), CTD swap with RPS4B [RPS4(AAAB)], or several mutations in CTD (C887Y, S914F, G952E, G997E) impair the HR activity of RPS4 + RRS1-R∆D56. (B) DOM4 swap with RRS1B and/or CTD swap with RPS4B influence effector responsiveness of RRS1-R/RPS4. Each leaf section was coinfiltrated to express wild-type or chimeric RRS1-R and RPS4 with mCh, AvrRps4, or PopP2. (C–E) Mutations S983F, E1070K in RRS1-R DOM4, and C887Y in RPS4 CTD primarily impair PopP2-, but not AvrRps4-triggered HR. Each leaf section was coinfiltrated to express wild-type or mutant RRS1-R, RPS4 with mCh, AvrRps4, or PopP2. (G) DOM6 of RRS1-R is required to compensate for the noncognate RRS1 DOM4(A) and RPS4B CTD(B) combination. Each leaf section was coinfiltrated to express either RPS4 or RPS4(AAAB) with an autoactive RRS1 variant. Only the RRS1 variants possessing a DOM6-R trigger HR with RPS4(AAAB). For A–E and G, HRs were assessed at 4 dpi. Photographs are representative of three consistent replicates. (F and H) Percentage representations of cell death scores in C–H at 4 dpi. Stacked bars are color-coded to show the proportions (in percentage) of each cell death scale (0–5) out of the total infiltrated panels scored. Panel (0) in cell death score is reused from the second row of G. Total panels scored are 7–19 (F) and 9–11 (H).

Fig. 3.
Fig. 3.

Interactions between DOM4 and D56 of RRS1 are influenced by effectors, mutations and domain swaps that activate the complex. (A) Co-IP assays to assess RRS1-R DOM4(A):GFP association with HF-tagged D56, DOM5, and DOM6-R of RRS1-R (A) and RRS1B (B) after transient coexpression in Nb leaves. DOM4(A) coimmunoprecipitates more strongly with A compared with B domains. (B) Co-IP assays to assess RRS1-R DOM4:GFP association with different alleles or mutants of D56:HF. D56-R and D56-S are of RRS1-R and RRS1-S, respectively. K2Q and K2R are acetyl-mimic and acetyl-null mutations of K1221 in RRS1-R WRKY domain. slh1 is a leucine insertion in RRS1-R WRKY domain. (C and D) Co-IP assays reveal effector interference with DOM4:GFP–D56-R:HF or DOM4:GFP–D56-S:HF association. Effectors are tagged with mCherry (mCh). Controls include AvrRps4 mutants E187A, E187A/E175A (EEAA) and KRVYAAAA (KRVY), a PopP2 mutant C321A, and mCherry. AvrRps4 inhibits DOM4–D56-R association, and PopP2 weakly interferes with both DOM4–D56-R and DOM4–D56-S association. Immunoblots show protein accumulations in total extracts (input) and after IP with anti-FLAG(IP-FLAG) or anti-GFP(IP-GFP) beads. Asterisks mark bands that indicate (lack of) associations. These were repeated three times with similar results.

Fig. 4.
Fig. 4.

Interactions between RPS4 CTD and RRS1 D456 are influenced by effectors and mutations that activate the complex. (A) Co-IP assays to assess RPS4 CTD:HF association with GFP-tagged DOM4, D56-R, D456-R of RRS1-R after coexpression in Nb leaves. CTD coimmunoprecipitates strongly with D456-R and more weakly with DOM4, but not with D56-R. (B) Co-IP assays to show RPS4 CTD:GFP association with mutants of D456-R:HF (K2Q, K2R, slh1). CTD coimmunoprecipitates more weakly with these mutants than with D456-R. (C) Co-IP assays reveal effector interference of RPS4 CTD:GFP and RRS1-R D456-R:HF associations. Effectors are tagged with mCh. Controls include AvrRps4 mutant EEAA, PopP2 mutant C321A, and mCherry. AvrRps4 but not PopP2 interferes with CTD–D456-R associations. Immunoblots show protein accumulation in input and after IP-FLAG or IP-GFP. Asterisks mark bands that indicate association. These were repeated three times with similar results.

Fig. 5.
Fig. 5.

BiFC analyses reveal effector-dependent conformational differences in RRS1 D456. (A) RRS1-R(cCFP-nVenus) + RPS4 respond to AvrRps4 (weaker than RRS1-R:HF + RPS4), but not PopP2, in tobacco transient assays. Diagram of RRS1-R(cCFP-nVenus) illustrates a cCFP between LRR and DOM4, and an nVenus at the C terminus. HRs were assessed at 3 dpi. Photographs are representative of three consistent replicates. (B–D) Nuclear BiFC signal of RRS1-R(cCFP-nVenus) is reduced in the presence of AvrRps4:mCh, but not AvrRps4 mutants, after coexpression in Nb leaves at 2 dpi. Representative images are shown (B). Box plots show quantifications of YFP signals (C) and mCh signals (D). (E–G) Nuclear BiFC signals of cCFP:D456-R:nVenus or cCFP:D456-S:nVenus remain unaltered in the presence of PopP2:mCh or C321A:mCh, compared with mCherry control. Representative images are shown (E). Box plots show quantifications of YFP signals (F) and mCh signals (G). Signal intensity of YFP (B and F) or mCh (C and G) was quantified as average gray value of each nucleus, and then each normalized to the mean intensity (YFP or mCh) of the mCh control sample within each biological replicate. Data points, color-coded for different biological replicates, represent Log10 of the normalized values. Linear mixed-effects model (lme) and tests for general linear hypotheses (glht) with Tukey comparisons were used for statistical analysis. Means with the same letter are not significantly different (P < 0.001).

Fig. 6.
Fig. 6.

FRET analyses reveal differences in RRS1 D456 conformation preactivation and postactivation. (A) Cartoon illustrates how FRET reflects possible conformational differences of eCFP:D456-R:YFP. (B) Diagrams illustrate plasmid design for FRET assays. LB and RB indicate T-DNA left and right borders, respectively. For simplicity, details of the promoters and terminators are omitted from the cartoon and are in

SI Appendix

. (C) FRET efficiency of eCFP:D456:YFP is significantly reduced in the presence of AvrRps4 and AvrRps4(KRVY), but not AvrRps4(EEAA), compared with mCherry(mCh) control. FRET analyses were performed after transient expression of described constructs (B) in Nb leaves at 2 dpi. Data points, pooling several biological replicates, each represents a single-cell FRET efficiency (in percentage) quantified by FRET-AB. Linear mixed-effects model (lme) and tests for general linear hypotheses (glht) with Tukey comparisons were used for statistical analysis. Means with the same letter are not significantly different (P < 0.001).

Fig. 7.
Fig. 7.

Engineered RRS1-R with a reversibly closed D456-R shows reversible loss of defense activation. (A) Schematic overview of RRS1-R engineering. Tagging the N and C termini of D456-R within a full-length RRS1-R with high-affinity proteins Im9 and E9 imposes a closed D456-R conformation. A TEV cleavage site at the N terminus of E9 is designed to allow cleavage and thus relieves this closed conformation upon coexpression of TEV protease. Interface mutants Im9YYAA and E9F86A that abolish Im9–E9 interactions were used to engineer a control open RRS1-R. (B) Engineered RRS1-R with a reversibly closed D456-R shows reversible loss of defense activation by AvrRps4. Each tobacco leaf section was coinfiltrated to transiently express RPS4 and an engineered RRS1-R with mCherry (mCh) or AvrRps4. RRS1-R(Im9) carries an Im9 between LRR and DOM4. RRS1-R(E9) contains an E9 fused to the C terminus. RRS1-R(Im9-E9) and RRS1-R(Im9YYAA-E9F86A) are simultaneously tagged with Im9 and E9 or their mutants, respectively. (C and D) Engineered RRS1-Rslh1 or RRS1-RK2Q with a reversibly closed D456-R shows reversible loss of autoactivity. Each leaf section was coinfiltrated to express RPS4 with an engineered RRS1-Rslh1 or RRS1-RK2Q. HRs were assessed at 4 dpi. Photographs are representative of three consistent replicates.

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