Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory

Abstract

Endogenous peptides regulate plant immunity and growth. Systemin, a peptide specific to the Solanaceae, is known for its functions in plant responses to insect herbivory and pathogen infections. Here, we describe the identification of the tomato (Solanum lycopersicum) PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the TOMATO PROTEIN KINASE1b (TPK1b) interacting protein and demonstrate its biological functions in systemin signaling and tomato immune responses. Tomato PORK1 RNA interference (RNAi) plants with significantly reduced PORK1 expression showed increased susceptibility to tobacco hornworm (Manduca sexta), reduced seedling growth sensitivity to the systemin peptide, and compromised systemin-mediated resistance to Botrytis cinerea. Systemin-induced expression of Proteinase Inhibitor II (PI-II), a classical marker for systemin signaling, was abrogated in PORK1 RNAi plants. Similarly, in response to systemin and wounding, the expression of jasmonate pathway genes was attenuated in PORK1 RNAi plants. TPK1b, a key regulator of tomato defense against B. cinerea and M. sexta, was phosphorylated by PORK1. Interestingly, wounding- and systemin-induced phosphorylation of TPK1b was attenuated when PORK1 expression was suppressed. Our data suggest that resistance to B. cinerea and M. sexta is dependent on PORK1-mediated responses to systemin and subsequent phosphorylation of TPK1b. Altogether, PORK1 regulates tomato systemin, wounding, and immune responses.

Figure 1.

PORK1 Interaction and Phosphorylation with TPK1b. (A) TPK1b interacts with PORK1 kinase domain (KD). TPK1b-MYC and the kinase domain of PORK1 (PORK1-KD-HA) were coexpressed in N. benthamiana through Agrobacterium tumefaciens-mediated transient expression. PORK1-KD-HA was immunoprecipitated (IP) with anti-HA antibody coupled agarose beads along with TPK1b-MYC. The bound proteins were detected by immunoblot with the indicated antibodies. (B) Direct interaction between PORK1 and TPK1b in in vitro binding assay. Equal amounts of recombinant proteins were mixed as shown in the input. After washing, GST-TPK1b was bound with MBP-PORK1-KD and MBP-PORK1-KD^K30A, but not with the MBP-PORK1-LRR, which is used as a negative control. (C) PORK1 is a functional kinase with autophosphorylation and transphosphorylation activities. (D) PORK1 phosphorylates TPK1b in in vitro kinase assay. In (C) and (D), MBP-PORK1-KD, MBP-PORK1-KD^K30A, or GST-TPK1b^K106A were expressed and purified from E. coli using amylose resin columns. MBP-PORK1-KD or MBP-PORK1-KD^K30A was incubated with Myelin Basic Protein (C) or TPK1b^K106A (D) in a kinase buffer containing [γ-³²P]ATP. Phosphorylation was detected by autoradiography. Coomassie blue staining shows equal loading of protein samples. EV, empty vector. The experiments were repeated at least two times with similar results.

Figure 2.

PORK1 Mediates (Pro)systemin-Induced Resistance to B. cinerea. (A) RT-qPCR analysis of PORK1 transcript levels in wild-type and PORK1 RNAi plants. (B) and (C) B. cinerea disease lesion size (B) and disease symptoms in mock (water) or systemin-pretreated plants (C). Data represent mean ± se (n ≥ 90). Detached leaves were evenly sprayed with water (mock) or 2 µM systemin, in both cases containing 0.01% of Silwet L-77. Different letters indicate statistically significant differences. Multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post-hoc test (P < 0.05; Supplemental File 1). (D) VIGS of tomato PORK1 in the background of 35S-Prosystemin tomato plants. (E) PORK1-silenced 35S-Prosystemin plants show enhanced disease symptoms. Pictures of disease symptoms are form 3 d after inoculation with B. cinerea. Disease assays were conducted on plants with reduced PORK1 expression shown under (D). (F) Mean lesion diameter at 3 d after inoculation. Data represent means ± se (n ≥ 70). Asterisks indicate statistically significant differences (Student’s t test, P < 0.001; Supplemental File 1). (G) PI-II expression in GFP- or PORK1-silenced 35S:Prosystemin plants. GFP (mock)- or PORK1-silenced plants were spray inoculated with B. cinerea spore suspension and RNA extracted from leaf tissues at different time points. In (A), (D), and (G), gene expression was analyzed by RT-qPCR and relative expression levels were calculated by the comparative cycle threshold method using tomato Actin as a reference. Mean values represent ± sd (n = 9) from three independent biological replicates and three technical repeats. Different letters indicate statistically significant differences. Multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post-hoc test (P < 0.05; Supplemental File 1). hpi, hours postinoculation. The presented data are representative of at least three independent experiments.

Figure 3.

PORK1 RNAi Plants Show Reduced Resistance to Tobacco Hornworm Larvae. (A) Wild-type and PORK1 RNAi plants at the beginning and end of tobacco hornworm (M. sexta) feeding trial. (B) Size of larvae recovered 10 d after feeding trial. (C) Average weights of larvae at the beginning and at 10 d after feeding trial. Data represent mean ± se (n = 10). Multiple comparisons were calculated by one-way ANOVA test (P < 0.05; Supplemental File 1). Different letters indicate statistically significant differences. DAI, days after inoculation. (D) and (E) B. cinerea disease lesion size (D) and disease symptoms in wounded (W) and unwounded (UW) plants (E). Data represent means ± se (n ≥ 120). Asterisks indicate statistically significant differences (Student’s t test, P < 0.001). The experiments were repeated at least three times with similar results.

Figure 4.

PORK1 Contributes to Systemin-Mediated Seedling Growth Responses and Gene Expression. (A) and (B) Tomato PORK1 RNAi lines display hypocotyl growth insensitivity (A) and loss of growth responses (B) to systemin. Seeds were geminated and grown on MS medium with or without 10 nM systemin. The hypocotyl lengths were measured at 12 d. Values represent mean ± se (n > 10) from each genotype. Multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post-hoc test (P < 0.05). Different letters indicate statistically significant differences. (C) to (E) The expression of PORK1 (C) and PI-II ([D] and [E]) genes in response to systemin. In (C), detached leaves of wild-type tomato plants were immersed in 50 nM systemin or water (mock). Values represent ± sd (n = 9) from three independent biological replicates and three technical replicates. Asterisks indicate significant difference (two-way ANOVA, P < 0.001). In (D) and (E), values represent mean ± sd (n = 9) from three independent biological replicates and three technical replicates. Quantification of each gene expression level was normalized with tomato Actin gene. The multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post-hoc test (P < 0.05; Supplemental File 1). Different letters indicate statistically significant differences. hpt, hours post-treatment. At least two independent experiments were conducted showing similar results.

Figure 5.

PORK1 Is Required for Systemin-Induced Gene Expression. The expression of AOS2 (A), OPR3 (B), MYC2 (C), PR1a (D), LoxD (E), CaM6 (F) ACS6 (G), ERF1b (H), and LRR22 (I) genes in response to systemin in leaf tissues. Values represent mean ± sd (n = 9) from three independent biological replicates and three technical replicates. Expression is presented relative to expression level of each gene after normalization to the Actin gene. The multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post-hoc test (P < 0.05; Supplemental File 1). Different letters indicate statistically significant differences. hpt, hours post-treatment. Data are representative of at least three independent experiments.

Figure 6.

PORK1 Is Required for Wound-Induced Gene Expression. (A) RT-qPCR showing the induction of PORK1 expression after mechanical wounding. (B) to (H) The expression of PI-II (B), OPR3 (C), AOS2 (D), LoxD (E), LRR22 (F), ACS6 (G), and ERF1b (H) in responses to mechanical wounding was reduced in PORK1 RNAi plants. Values represent mean ± sd (n = 9) from three independent biological replicates and three technical repeats. Quantification of each gene expression level was normalized to the level of Actin expression. Different letters indicate significant differences. The multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post-hoc test (P < 0.05; Supplemental File 1). hpt, hours post-treatment. Data are representative of at least three independent experiments.

Figure 7.

Regulation of PORK1 and TPK1b Phosphorylation. (A) Protein accumulation and phosphorylation of PORK1 (left panel) and TPK1b (right panel) are enhanced in response to wounding and systemin. PORK1-KD-HA and TPK1b-HA were infiltrated into N. benthamiana leaves. At 36 h, water (ddH₂O) or 1 µM systemin was infiltrated, and tissue samples collected at different time points. Immunoblots were used to detect protein levels and mobility shifts of proteins. Ponceau S staining shows equal loading of total protein. M, mock; S, systemin. At least two independent experiments were repeated showing the similar results. (B) RT-qPCR analysis of PORK1 transcript levels in GFP- or PORK1-silenced TPK1b-HA overexpressing plants. The expression of PORK1 was normalized to that of Actin. Values represent mean ± sd (n = 9) from three independent biological replicates and three technical replicates. Different letters indicate statistically significant differences. Multiple comparisons were calculated by one-way ANOVA followed by Bonferroni post-hoc test (P < 0.05; Supplemental File 1). Data are representative of two independent experiments. (C) PORK1 is required for systemin-induced TPK1b phosphorylation. GFP- and PORK1-silenced TPK1b-overexpressing plants selected from (B) were infiltrated with water or systemin to induce TPK1b phosphorylation. TPK1b expression and phosphorylation were detected by immunoblotting. Mobility shifts of the proteins are owing to changes in phosphorylation. The experiment was repeated three times with similar results.