A receptor for dual ligands governs plant immunity and hormone response and is targeted by a nematode effector

Abstract

In this study, we show that the potato (Solanum tuberosum) pattern recognition receptor (PRR) NEMATODE-INDUCED LEUCINE-RICH REPEAT (LRR)-RLK1 (StNILR1) functions as a dual receptor, recognizing both nematode-associated molecular pattern ascaroside #18 (Ascr18) and plant hormone brassinosteroid (BR) to activate two different physiological outputs: pattern-triggered immunity (PTI) and BR response. Ascr18/BR-StNILR1 signaling requires the coreceptor potato BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (StBAK1) and perception of either ligand strengthens StNILR1 interaction with StBAK1 in plant cells. Significantly, the parasitically successful potato cyst nematode (Globodera pallida) utilizes the effector RHA1B, which is a functional ubiquitin ligase, to target StNILR1 for ubiquitination-mediated proteasome-dependent degradation, thereby countering Ascr18/BR-StNILR1-mediated PTI in potato and facilitating nematode parasitism. These findings broaden our understanding of PRR specificity and reveal a nematode parasitic mechanism that targets a PTI signaling pathway.

Keywords: Ascr18; BR; RHA1B; StNILR1; dual receptor.

Fig. 1.

StNILR1 recognizes Ascr18, mediating PTI responses. (A) ITC assay indicates the StNILR1 ectodomain (Ecto-StNILR1) binds to Ascr18. 100 μM Ascr18 was injected into Ecto-StNILR1 solution in the ITC cell. The area of each single injection peak corresponds to the total heat released from that injection. The integrated heat is plotted against the molar ratio of Ascr18 titrated into the cell containing Ecto-StNILR1. The binding constant (Kd) and the calculated stoichiometry (N) (± fitting error) are indicated. (B) The C-terminal five LRRs of Ecto-StNILR1 are dispensable for StNILR1 binding to Ascr18 in the ITC assay. (C–F) StNILR1 plays an important role in basal-level resistance to G. pallida, as manifested by that, in comparison to the WT potato plants, StNILR1-OX lines displayed resistance to G. pallida (C) and (D), whereas StNILR1-KD lines were more susceptible (E) and (F). Four-week-old vegetatively propagated WT or transgenic potato plants were inoculated with G. pallida. Nematodes (male and female) were counted 6 wk after the inoculation using acid fuchsin staining. Data are presented as mean ± SD (n = 10). (G) StNILR1 mediates Ascr18-triggered PTI signaling. Leaves of 2-wk-old vegetatively propagated WT or StNILR1-KD transgenic potato seedlings were soaked in 0.2 μM Ascr18 or the Mock solution containing 0.01% Tween 80 for 5 s and the roots were analyzed by RT-PCR at 72 h after treatment. Ascr18-induced expression of StPR1 and StPDF1.2 was compromised in the StNILR1 knockdown transgenic potato roots. StACT41 served as an internal reference for normalization. Data are presented as mean ± SD (n = 3). (H) and (I) StNILR1 is required for Ascr18-triggered immunity against potato cyst nematode G. pallida. Four-week-old vegetatively propagated WT or transgenic potato plants were inoculated with G. pallida cysts and leaf-sprayed with 0.2 μM Ascr18 or Mock solution every other day for 6 wk. Nematodes (male and female) were counted 6 wk after inoculation using acid fuchsin assay. Data are presented as mean ± SD (n = 10). Experiments were repeated two times with similar results. Data were analyzed by two-sided Student’s t test (D, F, and I), one-way ANOVA (G), or two-way ANOVA (C, E, and H) followed by Tukey’s test. Statistical analysis was performed within the same genotype (D, F, and I). n = biologically independent samples.

Fig. 2.

StNILR1 recognizes BR, mediating BR responses. (A) BL responsiveness is compromised in StNILR1 knockdown lines. Seven-day-old vegetatively propagated WT or StNILR1-KD potato seedlings were grown on ½ MS medium containing 100 nM BL or the Mock DMSO solution for 10 d under light condition (Left). Root length was measured (Right). Data are presented as mean ± SD (n = 16). (Scare bar, 1 cm.) (B) StNILR1 mediates BL-induced repression of BR biosynthesis genes. Roots of 7-d-old vegetatively propagated WT or StNILR1-KD potato seedlings were socked in 0.1 μM BL or the Mock solution for 5 s. 48 h after treatment, total RNA was extracted from root tissues for qRT-PCR analysis of StCPD, StDWF4, StNIA1, and StROT3 expression. StACT41 served as an internal reference for normalization. Data are presented as mean ± SD (n = 3). (C) StNILR1 ectodomain directly binds to BL in ITC assay. 50 μM BL was injected into Ecto-StNILR1 solution in the ITC cell. The area of each single injection peak corresponds to the total heat released from that injection. The integrated heat is plotted against the molar ratio of BL titrated into the cell containing Ecto-StNILR1. The binding constant (Kd) and the calculated stoichiometry (N) (± fitting error) are indicated. (D) AtNILR1 ectodomain dose not bind to BL in ITC assay. (E) Arabidopsis nilr1 mutant shows root growth inhibition upon BL treatment. Six-day-old Col-0, bri1-301, or nilr1 Arabidopsis seedlings were grown on the ½ MS medium supplemented with 100 nM BL or equal volume of mock solution under light condition. After 7 d, root lengths were measured (Top) and statically analyzed (Bottom). Data are presented as mean ± SD (n = 24). (Scare bar, 1 cm.) (F) Arabidopsis bri1-301 mutant expressing StNILR1 under the AtBRI1 native promoter partially rescues the phenotypic defects of the bri1-301 mutant. Five-week-old plants were photographed as shown in the two Top panels. Root lengths of 6-d-old seedlings were measured and statistically analyzed as shown in the two Bottom panels. Data are presented as mean ± SD (n = 20). (Scare bar, 1 cm.) (G) Expression pattern of StNILR1, StBRI1, and StBRL3 in different potato tissues. RNA was extracted from leaves, stems, and roots of 2-wk-old vegetatively propagated potato seedlings. Relative transcript levels of StNILR1, StBRI1, and StBRL3 were analyzed using qRT-PCR. StACT41 served as an internal reference for normalization. Data are presented as mean ± SD (n = 3). (H) The StNILR1 gene is specifically repressed in StNILR1 knockdown (StNILR1-KD) potato lines. StNILR1, StBRI1, StBRL1, and StBRL3 mRNA levels were analyzed in WT and StNILR1-KD potato roots by qRT-PCR assay. Root tissues of 7-d-old potato seedlings were collected for RNA extraction and qRT-PCR analyses. StACT41 served as an internal reference for normalization. Data are presented as mean ± SD (n = 3). Experiments were repeated three (A, B, E, G, and H) or two (C and D) times with similar results. Data were analyzed by two-sided Student’s t test (A and E), or one-way ANOVA (B, G, F, and H) followed by Tukey’s test. n = biologically independent samples.

Fig. 3.

Recognition of either ligand by StNILR1 activates both PTI and BR responses. (A) Ascr18 does not inhibit root growth of Arabidopsis seedlings. Six-day-old wild type Arabidopsis seedlings were grown on 1/2 MS medium supplemented with various concentrations of Ascr18 under light conditions. Seven days after treatment, root lengths were measured (Left) and statistically analyzed (Right). Data are presented as mean ± SD (n = 20). (Scare bar, 1 cm.) (B) StNILR1 is required for Ascr18-triggered root growth inhibition in potato. Seven-day-old vegetatively propagated WT or StNILR1-KD potato seedlings were grown on ½ MS medium containing 1 μM Ascr18 or Mock solution for 10 d under light condition (Top). Root length was measured (Bottom). Data are presented as mean ± SD (n = 16). (Scare bar, 1 cm.) (C) Arabidopsis nilr1 mutant expressing StNILR1 under the AtNILR1 native promoter exhibits sensitivity to Ascr18. Six-day-old WT, pAtNILR1::StNILR1/nilr1, or nilr1 Arabidopsis seedlings were grown on ½ MS medium containing 1 μM Ascr18 or the Mock DMSO solution under light condition for 7 d (Top). Root length was measured (Bottom). Data are presented as mean ± SD (n = 20). (Scare bar, 1 cm.) (D) StNILR1 mediates Ascr18/BL-induced repression of BR biosynthesis genes. Roots of 7-d-old vegetatively propagated potato seedlings were socked in 1 μM Ascr18 or the Mock solution for 5 s. 48 h after treatment, total RNA was extracted from root tissues for qRT-PCR analysis of StCPD, StDWF4, StNIA1, and StROT3 expression. StACT41 served as an internal reference for normalization. Data are presented as mean ± SD (n = 3). (E) StNILR1 mediates BR-induced PTI responses. Leaves of 2-wk-old vegetatively propagated WT or StNILR1-KD potato seedlings were socked in 0.2 μM BL or equal volume of mock solution containing 0.01% Tween 80 for 5 s. Roots were collected at 72 h after treatment for RNA extraction and qRT-PCR analyses. StACT41 was used as an internal reference for normalization. The expression levels of StPR1 and StPDF1.2 in the roots of BL-treated potato seedlings were normalized to those in the roots of Mock-treated potato seedlings. Data are presented as mean ± SD (n = 3). (F) and (G) StNILR1 is required for BL-triggered immunity against potato cyst nematode G. pallida. Four-week-old vegetatively propagated potato plants were inoculated with G. pallida cysts and leaf-sprayed with 0.2 μM BL, or Mock solution every other day for 6 wk. Nematodes (female and male) numbers were counted 6 wk after inoculation using acid fuchsin assay. Data are presented as mean ± SD (n = 10). Experiments were repeated three (A–E) or two (F and G) times with similar results. Data were analyzed by two-sided Student’s t test (G), or one-way ANOVA (A, D, and E), or two-way ANOVA (B, C, and F) followed by Tukey’s test. Statistical analysis was performed within the same genotype (G). n = biologically independent samples.

Fig. 4.

Both Ascr18-StNILR1 and BR-StNILR1 signaling functions through StBAK1. (A) StNILR1 interacts with StBAK1 in vivo. Epitope-tagged StNILR1 (Myc-StNILR1) was coexpressed with StBAK1 (StBAK1-HA) or StBAK1-like (StBAK1L-HA) in N. benthamiana leaves. Protein extracts were immunoprecipitated with anti-HA affinity matrix, followed by western blotting using anti-Myc antibody to verify the interaction between StNILR1 and StBAK1 in plant cells. It is notable that a weak Myc-StNILR1 band was detected in the immunoprecipitated protein complex by the anti-Myc antibody, suggesting a weak interaction between StNILR1 and StBAK1 in plant cells. (B) StBAK1 directly interacts with StNILR1 in vitro. The epitope-tagged CD of StBAK1 (MBP-StBAK1-CD) recombinant protein was incubated with StNILR1-CD (GST-StNILR1-CD) or an unrelated protein SlWOX14 (Gene ID: Solyc02g082670; GST-SlWOX14) and captured with anti-GST affinity beads. Western blotting using anti-MBP antibody confirmed that MBP-StBAK1-CD directly interacts with GST-StNILR1-CD, but not the unrelated GST-SlWOX14, in vitro. (C) The in planta interaction between StBAK1 and StNILR1 is strengthened in the presence of BL and Ascr18. Myc-StNILR1 was coexpressed with StBAK1-HA in N. benthamiana leaves. 2 μM Ascr18, 2 μM BL, or Mock solution was infiltrated into preinfiltrated leaf tissues 8 h before tissue collection. Protein extracts were immunoprecipitated with anti-HA affinity matrix, followed by western blotting using anti-Myc antibody to verify the enhanced association of StNILR1 with StBAK1 by Ascr18 or BL. (D) Ascr18- or BL-triggered immune signaling is compromised in the StBAK1 knockdown transgenic potato plants. Leaves of 4-wk-old vegetatively propagated WT or StBAK1-KD transgenic potato plants were soaked in 0.2 μM BL, 0.2 μM Ascr18, or Mock solution containing 0.01% Tween 80 for 5 s and analyzed by RT-PCT at 72 h after treatment. StACT41 was used as an internal reference for normalization. The expression levels of StPR1 and StPDF1.2 in Ascr18- or BL-treated roots were normalized to those in Mock-treated roots. Data are presented as mean ± SD (n = 3). (E) and (F) StBAK1 is required for BL/Ascr18-triggered resistance against the potato cyst nematode G. pallida. Experiments were conducted as in Fig. 1 H and I. Data are presented as mean ± SD (n = 10). (G) BL or Ascr18 responsiveness is compromised in StBAK1 knockdown (StBAK1-KD) transgenic potato seedlings. Seven-day-old vegetatively propagated WT or StBAK1-KD potato seedlings were grown on ½ MS medium containing 0.1 μM BL, 1 μM Ascr18, or Mock solution for 10 d under light condition (Left). Root length was measured (Right). Data are presented as mean ± SD (n = 16). (Scare bar, 1 cm.) (H) StBAK1 is essential for the BL/Ascr18-induced BR responses. The roots of 7-d-old vegetatively propagated WT or StBAK1-KD seedlings were soaked in 0.1 μM BL or 1 μM Ascr18 for 5 s and analyzed by qRT-PCR at 48 h after treatment. StACT41 was used as an internal reference for normalization. The expression levels of StCPD, StDWF4, StNIA1, and StROT3 in roots of Ascr18- or BL-treated potato seedlings were normalized to those in Mock-treated roots. The repression of StCPD, StDWF4, StNIA1, and StROT3 caused by Ascr18 or BL treatment was abolished in StBAK1-KD transgenic potato roots. Data are presented as mean ± SD (n = 3). Experiments were repeated three (D, G, and H) or two (A, B, E, and F) times with similar results. Data were analyzed by two-sided Student’s t test (F), or two-way ANOVA (E, D, H, and G) followed by Tukey’s test. Statistical analysis was performed within the same genotype (F). n = biologically independent samples.

Fig. 5.

G. pallida effector RHA1B promotes nematode parasitism by disrupting StNILR1-mediated immunity. (A) and (B) RHA1B interacts with StNILR1 (A) and StBAK1 (B) in vivo. (C) and (D) RHA1B interacts with StNILR1 (C) and StBAK1 (D) in vitro. (E) RHA1B ubiquitinates StNILR1 in vitro. Polyubiquitination of the recombinant MBP-StNILR1-CD protein, as indicated by the smear banding pattern detected by anti-MBP antibody (Top), only occurred when MBP-StNILR1-CD-Myc was incubated with a proper protein combination of ubiquitin E1 (E1-His), E2 (E2-His), RHA1B (GST-RHA1B), and free ubiquitin (Ub-Flag). The smear banding pattern detected by anti-Flag antibody represents self-ubiquitination of MBP-RHA1B (Bottom). (F) RHA1B promotes degradation of StNILR1 in plant cells, dependent on the ubiquitin ligase activity. Coexpression of StNILR1 with RHA1B, but not the ligase-deficient HA-RHA1B^C135S mutant or the empty vector (E.V.) in N. benthamiana leaves, led to degradation of StNILR1 in plant cells. (G) Coexpression of RHA1B with StNILR1 in N. benthamiana leaves with or without the presence of proteasome-specific inhibitor MG115. Addition of increasing amounts of MG115 abolished RHA1B-triggered protein degradation. Protein degradation levels were quantified by normalization to the relevant protein without degradation. Data are presented as mean ± SD (n = 3). (H) Upregulated expression of BR biosynthetic genes in RHA1B-OX transgenic potato roots. Total RNA was extracted from roots of 14-d-old vegetatively propagated WT and RHA1B-OX potato seedlings. Transcript levels of StCPD and StDWF4 were determined by qRT-PCR. StACT41 served as an internal reference for normalization. Data are presented as mean ± SD (n = 3). (I) Overexpression of RHA1B in transgenic potato plants interferes with the BL- and Ascr18-elicited BR hormone responses in roots. The roots of 7-d-old vegetatively propagated WT or RHA1B -OX seedlings were soaked in 0.1 μM BL or 1 μM Ascr18 for 5 s and analyzed by qRT-PCR at 48 h after treatment. StACT41 was used as an internal reference for normalization. The expression levels of StCPD, StDWF4, StNIA1, and StROT3 in roots of Ascr18- or BL-treated potato seedlings were normalized to those in Mock-treated roots. The repression of StCPD, StDWF4, StNIA1, and StROT3 caused by Ascr18 or BL treatment was abolished in RHA1B -OX transgenic potato roots. Data are presented as mean ± SD (n = 3). (J) Overexpression of RHA1B in transgenic potato compromises root growth inhibition triggered by BL or Ascr18. Seven-day-old vegetatively propagated WT or RHA1B -OX potato seedlings were grown on ½ MS medium containing 0.1 μM BL, 1 μM Ascr18, or Mock solution for 10 d under light condition (Left). Root length was measured (Right). Data are presented as mean ± SD (n = 16). (Scare bar, 1 cm.) (K) Overexpression of RHA1B in transgenic potato compromises BL- or Ascr18-triggered immune signaling. Experiments were performed as in Fig. 4D. Data are presented as mean ± SD (n = 3). (L) and (M) Overexpression of RHA1B in transgenic potato compromises BL- or Ascr18-induced resistance against the cyst nematode G. pallida. Experiments were performed as in Fig. 4 I and J. Data are presented as mean ± SD (n = 10). Experiments were repeated three (A–D and F–K) or two (L and M) times with similar results. Data were analyzed by two-sided Student’s t test (M), one-way ANOVA (H, I, and K), or two-way ANOVA (J and L) followed by Tukey’s test. Statistical analysis was performed within the same genotype (M). n = biologically independent samples.