Salicylic acid receptors activate jasmonic acid signalling through a non-canonical pathway to promote effector-triggered immunity

Abstract

It is an apparent conundrum how plants evolved effector-triggered immunity (ETI), involving programmed cell death (PCD), as a major defence mechanism against biotrophic pathogens, because ETI-associated PCD could leave them vulnerable to necrotrophic pathogens that thrive on dead host cells. Interestingly, during ETI, the normally antagonistic defence hormones, salicylic acid (SA) and jasmonic acid (JA) associated with defence against biotrophs and necrotrophs respectively, both accumulate to high levels. In this study, we made the surprising finding that JA is a positive regulator of RPS2-mediated ETI. Early induction of JA-responsive genes and de novo JA synthesis following SA accumulation is activated through the SA receptors NPR3 and NPR4, instead of the JA receptor COI1. We provide evidence that NPR3 and NPR4 may mediate this effect by promoting degradation of the JA transcriptional repressor JAZs. This unique interplay between SA and JA offers a possible explanation of how plants can mount defence against a biotrophic pathogen without becoming vulnerable to necrotrophic pathogens.

Figure 1. JA synthesis and signalling are activated through the RPS2 immune receptor and required for ETI and associated PCD.

(a) Expression levels of JA-responsive genes in wild-type (WT) and the rps2 mutant at 4 h post inoculation (h.p.i.) with Psm ES4326/avrRpt2 at OD_600nm=0.2. The final data were normalized to the expression with 10 mM MgSO₄ (Control) treatment. qRT-PCR was performed on LOX3 (LIPOXYGENASE 3), AOS (ALLENE OXIDE SYNTHASE), OPR3 (OPDA REDUCTASE 3), JAZ1 (JASMONATE ZIM-DOMAIN PROTEIN 1), JAZ10, MYC2 (ARABIDOPSIS MYELOCYTOMATOSIS ONCOGENE HOMOLOG 2) with UBQ5 (UBIQUITIN 5) as a reference. Data from three biological replicates were combined using linear mixed-effects model. Significant difference was detected using Student's t-test. Data are presented as Mean±s.d. (b) Three-week-old plants were first infiltrated with Psm ES4326/avrRpt2 at OD_600nm=0.01, and 3.5 h later water (Mock) or 100 μM MeJA (MeJA) was sprayed. Starting at 0.5 h post inoculation (h.p.i.), leaf discs were collected for conductivity assay. Data are shown as mean±s.d. (n=3 biological replicates). (c) Representative leaves of WT, aos, jaz1Δjas, and rps2 plants 15 h.p.i. by Psm ES4326/avrRpt2 at OD_600nm=0.01 (upper panel). Conductivity measurements were performed 0.5 h.p.i. with Psm ES4326/avrRpt2 (lower panel). Data are shown as mean±s.d. (n=3 biological replicates). (d) Representative leaves and conductivity measurements of WT, coi1 and rps2 plants. The pathogen inoculation and conductivity measurements were as described in c. Data are shown as mean±s.d. (n=3 biological replicates). (e) Plants were infiltrated with Psm ES4326/avrRpt2 at OD_600nm=0.002 and pathogen growth was measured at day 0 and day 3. c.f.u., colony forming unit. Significant difference was detected by two-way ANOVA. Data are shown as mean±s.d. (n=8 biological replicates). (f) The growth of Psm ES4326/avrRpt2 in WT, coi1, and rps2. The same method was used as described in e. All experiments were repeated three times with similar results. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS, no significant difference. qRT-PCR, quantitative reverse transcription PCR.

Figure 2. The levels of SA and JA in WT and rps2 during ETI.

Three-week-old WT and rps2 plants were infiltrated with Psm ES4326/avrRpt2 at OD_600nm=0.01. Samples were collected at 0, 4, 8, 12 h.p.i. The levels of (a) free SA; (b) total SA; (c) JA-Ile; and (d) JA were measured. Significant difference was detected using Student's t-test. Data are shown as mean±s.d. (n=5–6 biological replicates). All experiments were repeated twice with similar results. *P<0.05; **P<0.01; ****P<0.0001; NS, no significant difference.

Figure 3. ETI-mediated early induction of JA-responsive genes is dependent on SA and NPR3 and NPR4 but independent of NPR1 and the JA receptor COI1.

Leaves from corresponding plants were harvested 4 h.p.i. with Psm ES4236/avrRpt2 (avrRpt2) at OD_600nm=0.2 or 10 mM MgSO₄ (Control). qRT-PCR was performed on LOX3, JAZ1, JAZ10, with UBQ5 as a reference. Gene expression (a) in WT, sid2 and rps2; (b) in WT, npr1 and rps2; (c) in WT, npr3 npr4 (n3n4), npr1 npr3 npr4 (n1n3n4) and rps2; (d) in WT, coi1 and rps2 was measured. Data from three biological replicates were combined using linear mixed-effects model. Significant difference was detected using Student's t-test. Data are shown as mean±s.d. *P<0.05; **P<0.01; ***P<0.001; NS, no significant difference. qRT-PCR, quantitative reverse transcription PCR.

Figure 4. SA facilitates NPR3 and NPR4 interactions with JAZ proteins.

Yeast two-hybrid assay (Y2H) was performed to study (a) interactions between NPR3 and JAZs and (b) interactions between NPR4 and JAZs. For SA treatment, 100 μM SA was added in the yeast media. (c) The split-luciferase assays were performed on JAZ1 with NPR3 or NPR4. The left half of the N. benthamiana leaf was co-infiltrated with cLuc and NPR3-nLuc or cLuc and NPR4-nLuc as control. The right half of the leaf was co-infiltrated with cLuc-JAZ1 and NPR3-nLuc or cLuc-JAZ1 and NPR4-nLuc. After two days of incubation, luciferin was sprayed onto the inoculated leaves and chemiluminescence images were taken by a CCD camera 0 and 3 h after 1 mM SA treatment. (d) The co-immunoprecipitation (co-IP) assay on JAZ1 with NPR3 or NPR4 in Arabidopsis. Samples were collected at 4 h.p.i. with Psm ES4326/avrRpt2, at OD_600nm=0.2, plus 40 μM MG115. The co-IP assay was carried out using the GFP-Trap_A beads for 2 h at 4 °C. The HA-JAZ1 protein was detected by western blot using HA antibody. The NPR3-GFP, NPR4-GFP or GFP protein levels were measured by western blots using the GFP antibody. All experiments were repeated three times with similar results.

Figure 5. NPR3 and NPR4 promote ETI-induced reduction of JAZ1 level and de novo JA synthesis.

(a) Representative leaves and conductivity measurements of WT, the 35S:HA-JAZ1 overexpressing (JAZ1 OE) transgenic line, and the rps2 mutant. The same methods were used as in Fig. 1c. Data are shown as mean±s.d. (n=3 biological replicates). (b) HA-JAZ1 protein levels were determined in WT, npr3 npr4 (n3n4) and the rps2 mutant 4 h.p.i. with 10 mM MgSO₄ (Control), Psm ES4326/avrRpt2 (avrRpt2) or Psm ES4326/avrRpt2 together with 40 μM MG115 (avrRpt2 MG115). (c) HA-JAZ1 protein levels were measured in WT, sid2 and the rps2 mutant 4 h.p.i. with 10 mM MgSO₄ (Control), or Psm ES4326/avrRpt2 (avrRpt2). (d) HA-JAZ1 protein levels were determined in WT, npr3 npr4 (n3n4) and the rps2 mutant 4 h after being sprayed with water (Mock) or 1 mM SA. For (b,c,d) the western blots were performed using the HA antibody. β-Tubulin served as a loading control. KD, kilodalton. (e) The JA-Ile levels in WT, npr3 npr4 and rps2 at 0 and 12 h.p.i. with Psm ES4326/avrRpt2 at OD_600nm=0.01. Significant difference was detected using Student's t-test. Data are shown as mean±s.d. (n=5–6 biological replicates). (f) Plants were infiltrated with Psm ES4326/avrRpt2 at OD_600nm=0.01 and 3.5 h later water (Mock) or 100 μM MeJA (MeJA) was sprayed. Pathogen growth was measured at 1 day post inoculation. Significant difference was detected by two-way ANOVA. Data are shown as mean±s.d. (n=8 biological replicates). All experiments were repeated three times with similar results. *P<0.05; ****P<0.0001; NS, no significant difference.

Figure 6. Working model of the interplay between SA and JA during ETI.

Activation of an NB-LRR immune receptor in plants by a pathogen effector leads to induction of multiple signalling pathways. A major event of the induction is the accumulation of SA at the infection site. At the high level of SA, NPR3 can interact with its substrate NPR1 to remove its repression on ETI and on crosstalk inhibition of the JA signalling pathway. Both NPR3 and NPR4 can also interact with JAZ proteins in an SA-enhanced manner leading to the degradation of JAZs. This results in activation of de novo JA synthesis of JA and amplification of the JA signalling through the canonical pathway. Activation of both SA- and JA-signalling pathways during ETI enables plants to use this PCD-associated defence strategy against biotrophic pathogens without making them vulnerable to necrotrophic pathogens. The blue triangle shape in the graph represents an unknown signal that may affect the degradation of JAZ1 through NPR3 and NPR4.