Extensin arabinosylation is involved in root response to elicitors and limits oomycete colonization

Abstract

Background and aims: Extensins are hydroxyproline-rich glycoproteins thought to strengthen the plant cell wall, one of the first barriers against pathogens, through intra- and intermolecular cross-links. The glycan moiety of extensins is believed to confer the correct structural conformation to the glycoprotein, leading to self-assembly within the cell wall that helps limit microbial adherence and invasion. However, this role is not clearly established.

Methods: We used Arabidopsis thaliana mutants impaired in extensin arabinosylation to investigate the role of extensin arabinosylation in root-microbe interactions. Mutant and wild-type roots were stimulated to elicit an immune response with flagellin 22 and immunolabelled with a set of anti-extensin antibodies. Roots were also inoculated with a soilborne oomycete, Phytophthora parasitica, to assess the effect of extensin arabinosylation on root colonization.

Key results: A differential distribution of extensin epitopes was observed in wild-type plants in response to elicitation. Elicitation also triggers altered epitope expression in mutant roots compared with wild-type and non-elicited roots. Inoculation with the pathogen P. parasitica resulted in enhanced root colonization for two mutants, specifically xeg113 and rra2.

Conclusions: We provide evidence for a link between extensin arabinosylation and root defence, and propose a model to explain the importance of glycosylation in limiting invasion of root cells by pathogenic oomycetes.

Keywords: Arabinosylation; cell wall; defence; extensin; immunocytochemistry; monoclonal antibodies; root.

Fig. 1.

Distribution of extensin epitopes in elicited and non-elicited Arabidopsis thaliana root tips. Immunolabelling was performed on 9-day-old roots using the anti-extensin monoclonal antibodies LM1 (A, B), JIM11 (C, D), JIM12 (E, F) and JIM20 (G, H). Roots were either elicited with 1 μm Flg22 (B, D, F, H) or not elicited (A, C, E, G). Images are 3-D reconstructions of 1 μm section stacks and were obtained with an inverted confocal laser scanning microscope Leica SP2 (λ_excitation, 488 nm; λ_emission, 507–550 nm). For each condition, five plant root tips were observed. Scale bars = 50 μm. RT, root tip; BLC + M, order-like cells and mucilage.

Fig. 2.

Quantification of fluorescence observed for Arabidopsis thaliana root tips immunolabelled with different anti-extensin antibodies. Immunolabelling was performed on 9-day-old roots using the anti-extensin monoclonal antibodies LM1, JIM11, JIM12 and JIM20. Roots were either elicited with 1 μm Flg22 or not elicited. The fluorescence area was measured and was related to the observed root area to calculate the corresponding ratio. For each condition, five plant root tips were observed and quantified. Bars show the s.d. Populations do not follow a normal distribution (d’Agostino test at α = 5 %, n = 5). Comparison of elicited with non-elicited: Mann–Whitney test at α = 5 %. n.s., not significant; *P <0.05; **P < 0.01.

Fig. 3.

Distribution of extensin epitopes in Arabidopsis thaliana rra2 mutant root tips. Immunolabelling was performed on 9-day-old rra2 mutant roots using the anti-extensin monoclonal antibodies LM1 (A, B) JIM11 (C, D), JIM12 (E, F) and JIM20 (G, H). Roots were either elicited with 1 μm Flg22 (B, D, F, H) or not elicited (A, C, E, G). Images are 3-D reconstructions of 1 μm section stacks and were obtained with an inverted confocal laser scanning microscope Leica SP2 (λ_excitation, 488 nm; λ_emission, 507–550 nm). For each condition, five plant root tips were observed. Scale bars = 50 μm. RT, root tip; BLC + M, border-like cells and mucilage.

Fig. 4.

Quantification of fluorescence observed for Arabidopsis thaliana mutant root tips immunolabelled with different anti-extensin antibodies. Immunolabelling was performed on 9-day-old rra1, rra2, xeg113, p4h2 mutant and wild-type (WT) roots using the anti-extensin monoclonal antibodies LM1, JIM11, JIM12 and JIM20. Roots were either elicited with 1 μm Flg22 or not elicited. The fluorescence area was measured and was related to the observed root area to calculate the corresponding ratio. For each condition, five plant root tips were observed. Graphs and statistical analyses are presented for each antibody in Supplementary data Figs S6–S9.

Fig. 5.

Accumulation of zoospores over Arabidopsis thaliana mutant roots after 3 h of inoculation. Zoospores accumulated in an area between the end of the meristematic zone and the beginning of the elongation zone. (A) Accumulation of zoospores in a wild-type (WT) root observed by fluorescence and bright-field microscopy. Images were taken with an inverted confocal laser scanning microscope Leica SP2 (λ_excitation, 561 nm; λ_emission, 570–612 nm). Scale bars = 100 μm. RT, root tip; Z, zoospores; EZ, elongation zone; MZ, meristematic zone. (B) Zoospores stained with propidium iodide were counted in a 400 μm long zone between the end of the meristematic zone and the beginning of the elongation zone, combining data from five independent experiments. Eighteen values out of 243 were considered as aberrant and removed by the ROUT method (Q = 10 %). Bars show the s.d. Each population follows a normal distribution (d’Agostino test at α = 5 %, n ≥36). Variances are not supposed equal (Fisher test at α = 5%). Comparison with the WT: t-test with Welch’s correction at α = 5%. *P < 0.05; **P < 0.01.

Fig. 6.

Monosaccharide composition and linkage analysis of AIRs from 7-day-old mutant A. thaliana roots. (A) Monosaccharide composition was analysed by GC-FID using AIRs prepared from 100 roots for both mutants and the wild type (WT). Experiments were performed twice for the rra2 mutant and four times for the WT and xeg113 mutant. Bars show the s.d. Values are relative proportions; therefore, statistical analyses cannot be performed. (B) Major results from the preliminary linkage analysis performed by GC-MS using AIRs extracted from 200 roots for the xeg113 mutant and the WT and from 100 roots for the rra2 mutant. Experiments were performed twice for both mutants and the WT. Values are ratios of relative proportions using the WT as a control.

Fig. 7.

Suggested epitope structures recognized by the anti-extensin monoclonal antibodies LM1, JIM11, JIM12 and JIM20. Epitope structure recognized by the LM1, JIM11, JIM12 and JIM20 anti-extensin mAbs, based on our immunocytochemical observations performed on Arabidopsis thaliana mutants impaired in extensin arabinosylation and wild-type roots. The JIM12 epitope may comprise part or the entire structure containing the two first arabinoses and the galactose from the extensin glycan moiety. The LM1, JIM11 and JIM20 epitopes may include the third arabinose and/or following arabinose residues on the extensin glycan moiety. The sites where the RRA1–RRA3 and XEG113 enzymes are involved and the type of linkage are indicated. XEG113, xyloendoglucanase 113; RRA1–3, reduced residual arabinose 1–3; Ser, serine; Hyp, hydroxyproline; Galp, galactopyranose; Araf, arabinofuranose. This figure is adapted with permission from Velasquez et al. (2012).

Fig. 8.

Model illustrating the impact of extensin arabinosylation in Arabidposis thaliana root colonization by the oomycete Phytopthora parasitica. Due to their correct arabinosylation, extensins are already cross-linked (to a certain extent) within the cell wall in non-infected roots. Root inoculation with P. parasitica zoospores activates various defence responses. We suggest that these include a significant increase in extensin cross-linking forming a strong 3-D network that reinforces the cell wall and limits pathogen colonization. However, in the case of an incomplete arabinosylation of extensins, very few (or no) cross-links would be formed even after oomycete inoculation, modifying the cell wall organization and strength. This would result in increased zoospore binding to root cells. leading to amplified colonization by the pathogen.