LRR Receptor-like Protein in Rapeseed Confers Resistance to Sclerotinia sclerotiorum Infection via a Conserved Ss NEP2 Peptide

Abstract

Brassica napus is one of the most extensively cultivated oilseed crops in China, but its yield is significantly impacted by stem rot caused by Sclerotinia sclerotiorum. Receptor-like proteins (RLPs) and receptor-like kinases (RLKs) play essential roles in plant-pathogen interactions; however, their regulatory mechanisms remain largely unknown in B. napus. In this study, we investigated the function of the leucine-rich repeat receptor-like protein BnaRLP-G13-1 in Brassica napus immunity. Previous observations indicated that B. napus plants expressing BnaRLP-G13-1 exhibited enhanced resistance to Sclerotinia sclerotiorum. We hypothesized that BnaRLP-G13-1 mediates pathogen recognition and immune signaling. To test this, we employed mitogen-activated protein kinase (MAPK) activity assays, transgenic overexpression analyses, and pathogen infection assays. Our results demonstrated that BnaRLP-G13-1 recognizes the conserved necrosis- and ethylene-inducing peptide Ssnlp24_SsNEP2 derived from S. sclerotiorum, triggering MAPK cascades and subsequent immune responses. Furthermore, protein interaction studies revealed that BnaRLP-G13-1 physically interacts with the receptor-like kinase BnaSOBIR1, which is essential for full antifungal defense activation. These results elucidate the molecular basis of BnaRLP-G13-1-mediated immunity, providing insights into improving disease resistance in oilseed crops.

Keywords: BnaSOBIR1; Brassica napus; LRR-RLP; Sclerotinia sclerotiorum; SsNEP2; plant immunity.

Figure 1

BnaC04g56380D overexpression enhances plant immunity to S. sclerotiorum. (A) Relative expression levels of BnaC04g56380D in XY15 after S. sclerotiorum infection for 48 or 96 h. (B) Subcellular localization of BnaC04g56380D protein. BnaC04g56380D-eGFP transiently expressed in N. benthamiana leaves with 35S-eGFP construct as negative control. Merge: Merged via GFP and bright-field microscopy. (C,D) S. sclerotiorum (wild-type) inoculation on detached leaves of A. thaliana Col-0 and 35S-BnaC04g56380D/Col-0. Data recorded at 48 hpi. Bar = 10 mm. ImageJ v1.51 used to analyze lesion areas. Three leaves from Col-0 or 35S-BnaC04g56380D/Col-0 plants selected for each infection experiment. (E,F) S. sclerotiorum (wild-type) inoculation on detached leaves of B. napus XY15 and 35S-BnaC04g56380D/XY15. One leaf from XY15 or 35S-BnaC04g56380D/XY15 selected for each infection experiment, and two mycelial plugs were inoculated on each leaf. Data recorded at 48 hpi. Bar = 50 mm. ImageJ used to analyze lesion areas. Experiments conducted three times, with similar results. Error bars represent SD (** p < 0.01).

Figure 2

BnaC04g56380D functionally complements loss of AtRLP23. (A,B) S. sclerotiorum (wild type) inoculation on detached leaves of A. thaliana Col-0, rlp23-1, rlp23-3. Data recorded at 48 hpi. Bar = 10 mm. ImageJ used to analyze lesion areas. (C) nlp24-induced MAPK activation in A. thaliana. Seedlings treated with 1 μM of nlp24. Western blot analysis with anti-pERK antibody used for evaluation of MAPK activation. Ponceau staining served as internal reference. Experiments conducted three times, with similar results. (D,E) Lesion areas of Col-0, 35S-BnaC04g56380D/rlp23-1 and 35S-Empty vector/rlp23-1 on leaves after S. sclerotiorum infection. Bar = 10 mm. ImageJ used to analyze lesion areas. Three leaves of Col-0, rlp23-1, rlp23-3, or 35S-BnaC04g56380D/rlp23-1 plants selected for each infection experiment. Experiments conducted three times with similar results. Error bars represent SD. Statistical significance between Col-0 and mutant analyzed using Student’s t-test (* p < 0.05, ** p < 0.01).

Figure 3

BnaRLP-G13-1 recognizes conserved sequences of NEP2 from S. sclerotiorum. (A) Relative expression levels of AtRLP23, AtSOBIR1, and AtBAK1 in Col-0 after treatment with 1 μM Ssnlp24_SsNEP2 for 4 or 24 h. Experiments conducted three times, with similar results. Error bars represent SD (** p < 0.01). (B) Analysis of interactions between AtRLP23/BnaRLP-G13-1 and SsNEP2/Ssnlp24_SsNEP2 by BiFC in N. benthamiana. AtRLP23 and BnaRLP-G13-1 fused to YNE, and Ssnlp24_SsNEP2 or SsNEP2 fused to YCE. Merge: Merged via YFP and bright-field microscopy.

Figure 4

Ssnlp24_SsNEP2induces plant resistance to S. sclerotiorum, which requires AtRLP23. (A) The MAPK activation induced by Ssnlp24_SsNEP2 in A. thaliana Col-0, rlp23-1, and rlp23-3. The seedlings were treated with 1 μM nlp24 or Ssnlp24_SsNEP2. Western blot analysis with an anti-pERK antibody was used for the evaluation of MAPK activation. Ponceau staining served as the internal reference. The experiments were conducted three times. (B) Four-week-old soil-grown A. thaliana Col-0, rlp23-1, and rlp23-3 plants were pretreated with H₂O and 1 μM Ssnlp24_SsNEP2 and inoculated with S. sclerotiorum (wild-type) 24 h later. The data were recorded at 36 hpi. The scale bar = 10 mm. ImageJ was used to analyze the lesion areas. (C) The lesion areas of A. thaliana Col-0, rlp23-1, and rlp23-3 on the leaves after pretreatment with H₂O and 1 μM Ssnlp24_SsNEP2 and S. sclerotiorum infection. ImageJ was used to analyze the lesion areas. Three leaves of the Col-0, rlp23-1, rlp23-3 plants were selected for each infection experiment. The error bars represent SD (** p < 0.01). (D) Leaf disks from the true leaves of five-week-old Arabidopsis Col-0, rlp23-1, and rlp23-3 mutant plants were treated with 1 µM of nlp24, 1 µM of Ssnlp24_SsNEP2, or H₂O. The data are reported in relative light units (RLUs) and the error bars represent SD. The experiment was repeated three times, with similar results.

Figure 5

The identification of SOBIR1-encoding genes in B. napus. (A) The phylogenetic tree of SOBIR1 was constructed based on amino acid sequences from A. thaliana and B. napus. The data were downloaded from Ensembl plants (http://plants.ensembl.org/index.html, accessed on 1 January 2022). (B) The morphological phenotypes of the A. thaliana Col-0, bir1-1, sobir1bir1-1, and 35s-BnaSOBIR1/sobir1bir1-1 seedlings. All the plants were grown in soil at 22 °C in parallel and photographed when they were 4 weeks old. The scale bar = 10 mm. (C) The DAB staining of true leaves of A. thaliana Col-0, bir1-1, sobir1bir1-1, and 35s-BnaSOBIR1/sobir1bir1-1. All the plants were grown on 1/2 MS medium plates at 22 °C in an incubator and stained when they were 2 weeks old. The scale bar = 500 μm. (D,E) The relative expression levels of AtPR1 and AtPR2 in A. thaliana Col-0, bir1-1, sobir1bir1-1, and 35s-BnaSOBIR1/sobir1bir1-1. The experiments were conducted three times, with similar results. The error bars represent SD (ns: non-significant, * p < 0.05, ** p < 0.01).

Figure 6

BnaSOBIR1 enhances plant immunity to S. sclerotiorum. (A) Relative expression levels of BnaSOBIR1 Group A and BnaSOBIR1 Group B in XY15 after S. sclerotiorum infection for 48 or 96 h. (B,C) Inoculated S. sclerotiorum (wild-type) on detached leaves of A. thaliana Col-0 and 35S-BnaSOBIR1/Col-0. Data recorded at 48 hpi. Bar = 10 mm. ImageJ used to analyze lesioned areas. Three leaves of Col-0 or 35S-BnaSOBIR1/Col-0 plants selected for each infection experiment. (D,E) Inoculated S. sclerotiorum (wild-type) on detached B. napus XY15 and 35S-BnaSOBIR1/XY15 leaves. Data recorded at 48 hpi. One leaf from XY15 or 35S-BnaSOBIR1/XY15 plant selected for each infection experiment, and two mycelial plugs inoculated on each leaf. Bar = 50 mm. ImageJ used to analyze lesion areas. Experiments conducted three times, with similar results. Error bars represent SD (** p < 0.01).

Figure 7

The analysis of the interactions between BnaRLP-G13-1 and BnaSOBIR1. (A) BnaRLP-G13-1 was fused to YNE, and BnaSOBIR1-1 and BnaSOBIR1-2 were fused to YCE. YNE + BnaSOBIR1-1-YCE and YNE + BnaSOBIR1-2-YCE were the negative controls. Merge: Merged via YFP and bright-field microscopy. (B) BnaRLP-G13-1 were fused to pBT3-STE, and BnaSOBIR1-1 and BnaSOBIR1-2 were fused to pPR3N. pNubG-Fe65 + pTSU2-APP served as the positive control, and pPR3N + pTSU2-APP served as the negative control. The data were recorded after 5 days.

Figure 8

A model of BnaRLP-G13-1 enhancing plant resistance to S. sclerotiorum. S. sclerotiorum promotes infection by secreting a toxic protein (SsNEP2). To resist the infection, plants cleave SsNEP2 to generate a small peptide (Ssnlp24_SsNEP2) through an as yet unknown mechanism. After recognizing Ssnlp24_SsNEP2, BnaRLP-G13-1 forms a complex with SOBIR1 and BAK1 to activate the MAPK cascade reaction and promote immunity-related gene expression, enhancing plant resistance to S. sclerotiorum.