The RIN4-like/NOI proteins NOI10 and NOI11 modulate the response to biotic stresses mediated by RIN4 in Arabidopsis

Abstract

NOI10 and NOI11 are two RIN4-like/NOI proteins that participate in the immune response of the Arabidopsis plant and affect the RIN4-regulated mechanisms involving the R-proteins RPM1 and RPS2. The immune response in plants depends on the regulation of signaling pathways triggered by pathogens and herbivores. RIN4, a protein of the RIN4-like/NOI family, is considered to be a central immune signal in the interactions of plants and pathogens. In Arabidopsis thaliana, four of the 15 members of the RIN4-like/NOI family (NOI3, NOI5, NOI10, and NOI11) were induced in response to the plant herbivore Tetranychus urticae. While overexpressing NOI10 and NOI11 plants did not affect mite performance, opposite callose accumulation patterns were observed when compared to RIN4 overexpressing plants. In vitro and in vivo analyses demonstrated the interaction of NOI10 and NOI11 with the RIN4 interactors RPM1, RPS2, and RIPK, suggesting a role in the context of the RIN4-regulated immune response. Transient expression experiments in Nicotiana benthamiana evidenced that NOI10 and NOI11 differed from RIN4 in their functionality. Furthermore, overexpressing NOI10 and NOI11 plants had significant differences in susceptibility with WT and overexpressing RIN4 plants when challenged with Pseudomonas syringae bacteria expressing the AvrRpt2 or the AvrRpm1 effectors. These results demonstrate the participation of NOI10 and NOI11 in the RIN4-mediated pathway. Whereas RIN4 is considered a guardee protein, NOI10 and NOI11 could act as decoys to modulate the concerted activity of effectors and R-proteins.

Keywords: Arabidopsis; Biotic stress; Plant defense; Pseudomonas syringae; RIN4-like/NOI proteins; Tetranychus urticae.

Fig. 1

Gene expression after T. urticae infestation and subcellular location of RIN4-like/NOIs. (A) Correlation between RNAseq data (lines) and RT-qPCR validation (bars) in Arabidopsis for NOI3, NOI5, NOI10, NOI11, and RIN4 after 30 min, 1 h, 3 h, and 24 h of mite infestation. Data are means of three biological replicates. (B) Log₁₀ normalized counts of RIN4-like/NOIs according to RNAseq data (Santamaria et al. 2021). (C) Confocal images of N. benthamiana leaves transiently transformed with 35S::GFP or 35S::RIN4-like/NOIs-GFP constructs. A scale bar common for all images is shown

Fig. 2

Plant damage and callose quantification after mite feeding on Arabidopsis RIN4-like/NOI genotypes. (A) The foliar damaged area was quantified after 4 d of mite infestation. Data are mean ± SE of ten replicates. (B) Callose deposition in leaves after 24 h of mite infestation. Representative images (left) and callose quantification (right). Data are mean ± SE of ten replicates. Different letters indicate significant differences (P < 0.05, One-way ANOVA followed by Duncan multiple comparisons test)

Fig. 3

Gene coexpression and protein interaction analyses of RIN4-like/NOIs. (A) Venn diagram showing the number of specific and shared genes present in the lists of the 100 most coexpressed genes with NOI10, NOI11, and RIN4 in the ATTED II database. The percentage of these genes induced at 30 min of T. urticae infestation is included. (B–D) Networks of the most enriched biological processes using the 100 most coexpressed genes with NOI10 (B), NOI11 (C), and RIN4 (D), created with the ClueGO tool in Cytoscape. (E) Network of the physical interactors of RIN4 obtained in the STRING database and visualized in Cytoscape. (F) Differential gene expression (log2FC) of RIN4 interactors after T. urticae treatment according to RNAseq data (Santamaria et al. 2021). (G) MYTH results of protein–protein interaction using RIN4-like/NOIs as prey and RIN4 interactors as baits

Fig. 4

HR associated with RPM1-RIN4-like/NOIs interactions. (A) Cell death phenotype of RPM1 and indicated RIN4-like/NOI combinations transiently expressed from the 35S promoter in N. benthamiana. (B) Co-immunoprecipitation assays of N. benthamiana extracts from leaves transiently expressing GFP-RIN4-like/NOIs and RPM1-HA under the control of the 35S promoter. The presence of proteins in the crude extracts and the immunoprecipitated fractions was determined by western blot analysis using anti-GFP and anti-HA antibodies. The experiments were independently repeated three times with similar results

Fig. 5

HR associated with RPM1-RIPK-RIN4-like/NOIs interactions. (A) Cell death phenotype of RPM1-RIPK and indicated RIN4/NOIs transiently expressed from the 35S promoter in N. benthamiana. (B) Co-immunoprecipitation assays of N. benthamiana extracts from leaves transiently expressing GFP-RIN4-like/NOIs and RPM1-HA under the control of the 35S promoter. The presence of proteins in the crude extracts and the immunoprecipitated fractions was determined by western blot analysis using anti-GFP and anti-HA antibodies. The experiments were independently repeated three times with similar results

Fig. 6

HR associated with RPS2-RIN4-like/NOIs interactions. (A) Cell death phenotype of RPS2 and indicated RIN4-like/NOIs transiently expressed from the 35S promoter in N. benthamiana. (B) Co-immunoprecipitation assays of N. benthamiana extracts from leaves transiently expressing GFP-NOI11 and RPS2-HA under the control of the 35S promoter. The presence of proteins in the crude extracts and the immunoprecipitated fractions was determined by western blot analysis using anti-GFP and anti-HA antibodies. The experiments were independently repeated three times with similar results

Fig. 7

NOI10 and NOI11 affect resistance to bacteria. (A) Line graph showing the expression levels of NOI10, NOI11, and RIN4 genes after Pst DC3000 (AvrRpt2) inoculation (left axis, circle markers, dark lines) and the number of the 100 most coexpressed genes for each gene that were induced by Pst DC3000 (AvrRpt2) (right axis, square markers, light lines). Data were obtained from the PlaD database. (B) Bacterial quantification after 24 h of leaf infiltration with different Pst DC3000 genotypes in WT and RIN4-like/NOI overexpressing lines. Data are representative of two independent experiments with similar results. Data are means ± SE of six biological replicates. Different letters indicate significant differences (P < 0.05, One-way ANOVA followed by Duncan multiple comparisons test). (C) Schematic model showing the participation of NOI10 and NOI11 in the RIN4-mediated defense responses. PTI pattern-triggered immunity, ETI effector-triggered immunity. Pointed head arrows indicate induction, blunt head arrow repression, and dashed lines no effect