Broader functions of TIR domains in Arabidopsis immunity

Abstract

TIR domains are NAD-degrading enzymes that function during immune signaling in prokaryotes, plants, and animals. In plants, most TIR domains are incorporated into intracellular immune receptors termed TNLs. In Arabidopsis, TIR-derived small molecules bind and activate EDS1 heterodimers, which in turn activate RNLs, a class of cation channel-forming immune receptors. RNL activation drives cytoplasmic Ca²⁺ influx, transcriptional reprogramming, pathogen resistance, and host cell death. We screened for mutants that suppress an RNL activation mimic allele and identified a TNL, SADR1. Despite being required for the function of an autoactivated RNL, SADR1 is not required for defense signaling triggered by other tested TNLs. SADR1 is required for defense signaling initiated by some transmembrane pattern recognition receptors and contributes to the unbridled spread of cell death in lesion simulating disease 1. Together with RNLs, SADR1 regulates defense gene expression at infection site borders, likely in a non-cell autonomous manner. RNL mutants that cannot sustain this pattern of gene expression are unable to prevent disease spread beyond localized infection sites, suggesting that this pattern corresponds to a pathogen containment mechanism. SADR1 potentiates RNL-driven immune signaling not only through the activation of EDS1 but also partially independently of EDS1. We studied EDS1-independent TIR function using nicotinamide, an NADase inhibitor. Nicotinamide decreased defense induction from transmembrane pattern recognition receptors and decreased calcium influx, pathogen growth restriction, and host cell death following intracellular immune receptor activation. We demonstrate that TIR domains can potentiate calcium influx and defense and are thus broadly required for Arabidopsis immunity.

Keywords: Arabidopsis; NLR; TIR domains; immunity.

Fig. 1.

SADR1 is required for the constitutive immunity phenotypes of the activation mimic RNL ADR1-L2 D484V. (A) Mutations sadr1-26.6 and 30.4 fully suppress the stunted growth phenotype of adr1-l2-4 pADR1:ADR1-L2 D484V (hereafter ADR1-L2 DV). Introduction of a loss-of-function mutation in SADR1 by CRISPR-Cas9 (sadr1-c1) suppresses ADR1-L2 DV. (B) Schematic representation of SADR1 shows conserved protein domains and the location of mutations identified in the screen or introduced with CRISPR-Cas9. Mutants 12.6, 17.4, and 30.3 are partial suppressors identified in the screen. The fonts indicate the genetic background; blue is ADR1-L2 DVand black and red fonts indicate a wild-type background. (C) Suppressed ADR1-L2 DV sadr1-c1 plants express wild-type levels of ADR1-L2 mRNA. Data are from four independent experiments (N = 4). Letters indicate statistical significance (two-tailed t test, P < 0.05). (D) sadr1-c1 suppresses most of the PR1 expression induced by ADR1-L2 DV. ADR1-L2 DV-induced overaccumulation (E) is also suppressed by sadr1-c1 (F). All experiments were performed at least three times. (G) SADR1 protein structure modeled onto the TNL RPP1 structure (7CRC). PR: Ponceau red staining.

Fig. 2.

SADR1 is required for NLP20/RLP23 signaling, contributes to lsd1 runaway cell death, and regulates PR1 expression around the infection site. (A) SADR1 is required downstream of NLP20/RLP23. Plants were challenged with Pst DC3000 EV 24 h after water or NLP20 1 µM treatment (N = 4) (23). (B) SADR1 is partially required for lsd1 runaway cell death. Fresh:dry weight ratio measurements indicating the proportion of dead tissues two weeks after induction of runaway cell death with 300 µM BTH. Data are from six independent experiments (N > 70). (C) Representative pictures of plants in (B). (D) SADR1 and RNLs are required for PR1 expression at the margin of infection sites. Representative pictures of pPR1:YFP^NLS-expressing leaves of the indicated genotype infected with Pst DC3000 AvrRps4 mCherry (OD = 0.2) at 24hpi. Notably, adr1 adr1-l1 adr1-l2 and helperless mutants cannot induce strong PR1 expression on the infection border (white arrows). See SI AppendixFig. S5. (E) PR1 expression on the margin of the infection site 24 h after infection with Pst DC3000 AvrRps4 (N = 4). Data presented in (E) are from five independent experiments. Letters indicate statistical significance [(A and B) ANOVA with the post hoc Tukey (E) two-tailed t test, P < 0.05].

Fig. 3.

ADR1s limit Pst DC3000 propagation and prevent systemic disease from localized infections. (A) Schematic representation of the experimental procedure used in (B) to measure the extent of pathogen propagation in planta. (B) Bacterial growth at 10 dpi in noninfiltrated tissues (N = 4, ANOVA with the post hoc Tukey, P < 0.01). (C) Representative pictures of leaves infiltrated with Pst DC3000 EV on one half (white asterisks) at 10 dpi. Leaves of the adr1 adr1-l1 adr1-l2 triple mutant and RNL-free helperless plants exhibit expanding lesions into noninfiltrated tissues. (D) Representative pictures of plants infiltrated with Pst DC3000 on four half-leaves at 28 dpi. The adr1 adr1-l1 adr1-l2 triple mutant and helperless plants show systemic disease symptoms.

Fig. 4.

SADR1 potentiates residual ADR1-L2 D484V activity independently of EDS1. (A) Representative pictures of 6-wk-old plants with the genotypes indicated above. ADR1-L2 D484V-driven growth inhibition (N = 48) (B), defense against Pst DC3000 (N = 12) (C), and resistance to Hpa isolate Noco2 (N = 9) (D) are fully suppressed by sadr1-c1 and partially by eds1-12. Data are from three independent experiments. Letters indicate statistical significance (ANOVA with the post hoc Tukey, P < 0.05).

Fig. 5.

Inhibition of TIR enzymatic activity with NAM regulates NLP20 response, as well as defense and cell death resulting from NLR activation. (A) 50 mM NAM treatment inhibits PR1 expression following NLP20 treatment and defense against virulent Pst DC3000 EV (N = 3) (B), avirulent Pst DC3000 AvrRps4 (C), AvrRpm1 (D), and HopZ1a (E) (N = 12). NAM treatment also delays cell death induction after inoculation of Pst DC3000 (OD₆₀₀ = 0.2) expressing AvrRps4 (F) or AvrRpm1 (G) but not HopZ1a (H). Numbers indicate the number of HR+ leaves. * Loss of turgor was observed in some leaves that did not exhibit autofluorescence characteristic of HR cell death. Data from (A) to (E) are from three independent experiments and from one representative experiment in (F) to (H). Letters indicate statistical significance (ANOVA with the post hoc Tukey or two-tailed t test [in (A)], P < 0.05).

Fig. 6.

Impact of NAM 50 mM on cytosolic calcium elevation during NLR-mediated ETI. (A) Impact of NAM on [Ca²⁺]_cyt during ETI by Pst DC3000 EV, Pst DC3000 AvrRps4 (triggering TNL RPS4), Pst DC3000 AvrRpm1 (triggering CNL RPM1), and Pst DC3000 HopZ1a (triggering CNL ZAR1) inoculated at OD₆₀₀ = 0.2. Bars represent SEM (N = 8). Data from the four panels are from a single experiment and have been split into four for clarity. (B) Data from four experiments showing [Ca²⁺]_cyt at 10 h after inoculation. Letters indicate statistical significance (two-tailed t test, P < 0.05).