The Arabidopsis thaliana LysM-containing Receptor-Like Kinase 2 is required for elicitor-induced resistance to pathogens

Abstract

In Arabidopsis thaliana, perception of chitin from fungal cell walls is mediated by three LysM-containing Receptor-Like Kinases (LYKs): CERK1, which is absolutely required for chitin perception, and LYK4 and LYK5, which act redundantly. The role in plant innate immunity of a fourth LYK protein, LYK2, is currently not known. Here we show that CERK1, LYK2 and LYK5 are dispensable for basal susceptibility to B. cinerea but are necessary for chitin-induced resistance to this pathogen. LYK2 is dispensable for chitin perception and early signalling events, though it contributes to callose deposition induced by this elicitor. Notably, LYK2 is also necessary for enhanced resistance to B. cinerea and Pseudomonas syringae induced by flagellin and for elicitor-induced priming of defence gene expression during fungal infection. Consistently, overexpression of LYK2 enhances resistance to B. cinerea and P. syringae and results in increased expression of defence-related genes during fungal infection. LYK2 appears to be required to establish a primed state in plants exposed to biotic elicitors, ensuring a robust resistance to subsequent pathogen infections.

Keywords: Botrytis cinerea; chitin; plant innate immunity; priming.

Figure 1

Impact of lyk mutations on basal and elicitor‐induced resistance to B. cinerea. Leaves of four‐week‐old WT, lyk2‐1, lyk2‐2, lyk5‐2 and cerk1‐2 plants were sprayed with water or with 100 μg ml⁻¹ chitin (a), 200 μg ml⁻¹ OG or 1 μM flg22 (b). After 24 h, leaves were inoculated with a B. cinerea spore suspension (5 × 10⁵ spores ml⁻¹). (c) Leaves of four‐week‐old WT, lyk2‐1, 35S:LYK2 1.1 and 15.5 (OE 1.1 and OE 5.15) plants were inoculated with B. cinerea. Lesion areas were measured 48 hours after inoculation. Data are means ± SE (n = 12); asterisks indicate statistically significant differences between WT and mutants, according to Student's t‐test (**, p > .01; ***, p < .001). These experiments were repeated three times with similar results

Figure 2

Early chitin‐induced responses are unaffected in lyk2 mutants. (a–b) 10‐day‐old WT, lyk2‐1 and lyk2‐2 seedlings were treated for 5 (a) and 10 min (b) with water (W) or chitin at the concentration of 10 μg ml⁻¹ (C10) or 25 μg ml⁻¹ (C25). Phosphorylated MPK3, MPK4, MPK6 and MPK11 were detected by immunoblot using an anti‐P44/P42 antibody. Total MPK3 and MPK6 were detected by immunoblot using an anti‐MPK3 and anti‐MPK6 antibodies. Arrows indicate the molecular weight (in kDa) of the marker bands. Equal loading was evaluated by Ponceau‐S Red staining and using an anti‐Actin antibody. (c–f) Leaf discs of four‐week‐old WT, lyk2‐1 and lyk2‐2 plants were treated for the indicated times with 100 μg ml⁻¹ chitin (c,d) or 100 nM flg22 (e,f). H₂O₂ production was measured with a luminol‐based assay and expressed in relative light units (RLU s⁻¹). Data points represent the average of at least 12 discs ± SE. Bars in (d) and (f) represent the average of total H₂O₂ production ± SE. Differences between total RLUs in WT and lyk2‐1 or lyk2‐2 were not significant (ns), according to Student's t‐test (p > .05). (g, h) FRK1 (g) and PAD3 (h) expression in WT and lyk2 seedlings treated with water or chitin (5 and 25 μg ml⁻¹) for 1 (g) and 3 h (h) was analysed by qRT‐PCR using UBQ5 as control. Data are means ± SE (n = 3 biological replicates). Asterisks indicate statistically significant differences according to Student's t‐test (***, p < .001) These experiments were repeated three times with similar results [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Chitin‐induced callose deposition is reduced in lyk2 mutants. (a,b) Rosette leaves of four‐week‐old plants of WT or lyk2‐1 and lyk2‐2 lines were infiltrated with water, chitin (100 μg ml⁻¹) or flg22 (100 nM) and stained with aniline blue 24 hr after infiltration. (a) Representative images for each treatment. Scale bars = 100 nm. (b) Callose deposits were quantified as fluorescence intensity per unit of the infiltrated leaf surface. Values represent means + SE of six different leaf samples from at least five independent plants (four microscopic fields of 0.1 mm² for each leaf). Asterisks indicate statistically significant differences between mutant lines and WT according to Student's t‐test (***, p < .001). This experiment was repeated twice with similar results [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Chitin‐triggered MAPK activation in lyk2 mutants pre‐treated with elicitors. 10‐day‐old WT, lyk2‐1 and lyk2‐2 seedlings were pre‐treated with water or flg22 (10 nM) for 24 hr and subsequently treated with water or chitin (25 μg ml⁻¹) for 10 (a) or 20 min (b). Phosphorylated MPK3, MPK4, MPK6 and MPK11 were detected by immunoblot using an anti‐P44/P42 antibody. Total MPK3 and MPK6 were detected by immunoblot using anti‐MPK3 and anti‐MPK6 antibodies. Antibodies against actin were used as controls. The arrows indicate the molecular weight of marker bands (in kDa). This experiment was repeated three times with similar results

Figure 5

Priming of chitin‐induced gene expression in lyk mutants. 10‐day‐old WT, lyk2‐1, lyk2‐2, lyk5‐2 and cerk1‐2 seedlings were pre‐treated with water or 10 nM flg22 for 24 h and subsequently treated with water or chitin (25 μg ml⁻¹) for 1 h. FRK1 and PAD3 expression was measured by qRT‐PCR and normalized using UBQ5. (a) Bars represent mean expression ± SE (n = 3 biological replicates), asterisks indicate statistically significant differences between WT and mutants, according to Student's t‐test (*, p < .05; **, p < .01; ***, p < .001). (b) Expression of FRK1 and PAD3, relative to WT seedlings pre‐treated with water and then treated with water, of the same samples as in (a). Bars represent mean fold‐change ± SE (n = 3 biological replicates). Different letters indicate statistically significant differences, according to one‐way ANOVA followed by Tukey's HSD test (p < .01). This experiment was repeated three times with similar results

Figure 6

Priming of pathogen‐induced PAD3 and PR‐1 expression in lyk mutants. Four‐week‐old WT, lyk2‐1, lyk2‐2, lyk5‐2 and cerk1‐2 plants were sprayed with water, 200 μg ml⁻¹ OG or 1 μM flg22 and inoculated after 24 hr with a B. cinerea spore suspension. PAD3 and PR‐1 expression was measured 24 hr after inoculation by qRT‐PCR and normalized using UBQ5. (a) Bars represent mean expression ± SE (n = 3 biological replicates); asterisks indicate statistically significant differences between WT and mutants, according to Student's t‐test (*, p < .05; **, p < .01; ***, p < .001). (b) Mean expression fold‐change (± SE, n = 3 biological replicates), relative to water‐treated WT plants, of the same plants as in (a). Asterisks indicate statistically significant differences between WT and mutants, according to Student's t‐test (*, p < .05; **, p < .01; ***, p < .001). Different letters indicate statistically significant differences, according to one‐way ANOVA followed by Tukey's HSD test (p < .01). This experiment was repeated three times with similar results

Figure 7

Overexpression of LYK2 increases resistance to B. cinerea and expression of defence genes in responses to chitin and infection. (a) PAD3 expression in WT, 35S:LYK2 1.1 and 15.1 seedlings, treated for 3 hr with chitin at the indicated concentrations, was determined by qRT‐PCR. UBQ5 was used for normalization. Data are means (± SE, n = 3 biological replicates). (b) Four‐week‐old WT and 35S:LYK2 line 1.1 and line 15.5 seedlings were treated with water or chitin (25 μg ml⁻¹) for the indicated time. Phosphorylated MPK3, MPK4, MPK6 and MPK11 were detected by immunoblot using an anti‐P44/P42 antibody. Total MPK3 and MPK6 were detected using anti‐MPK3 and anti‐MPK6 antibodies. Antibodies against actin were used as controls. The arrows indicate the molecular weight of marker bands (in kDa). (c, d) WT and 35S:LYK2 Line 1.1 four‐week‐old plants were sprayed with water, 200 μg ml⁻¹ OG or 1 μM flg22 and inoculated after 24 hr with a B. cinerea spore suspension. PAD3 (c) and PR1 (d) expression, at the indicated times, was measured by qRT‐PCR and normalized using UBQ5. Data are means (± SE, n = 3 biological replicates). Asterisks indicate statistically significant differences between WT and 35S:LYK2 line 1.1 plants, according to Student's t‐test (*, p < .05; **, p < .01; ***, p < .001). The results are representative of three (a, c, and d) or two (b) independent experiments

Figure 8

Role of LYK2 in resistance to Pseudomonas syringae. (a) Rosette leaves of four‐week‐old WT, lyk2‐1, lyk2‐2 and cerk1‐2 plants were sprayed with water (H₂O) or flg22 and, after 24 hr, infiltrated with Pst DC3000. Bacterial growth was measured at the indicated times (days postinfection, dpi). (b) Rosette leaves of four‐week‐old WT and 35S:LYK2 lines 1.1 and 15.5 plants were infiltrated with Pst DC3000, and bacterial growth was measured at the indicated times. Bars indicate mean log₁₀ of colony‐forming units (CFUs) per cm⁻² (± SE, n = 12). For each time point, statistically significant differences, according to Student's t‐test, between similarly treated WT and mutants are indicated by asterisks (*, p < .05; ***, p < .01); in (a), differences between water‐ and flg22‐treated plants of the same genotype are indicated by pound signs (###, p < .01). The results are representative of three independent experiments

Figure 9

LYK2 constitutively interacts with LYK5. (a,b), LYK2‐GFP and LYK5‐myc (a) or CERK1‐myc (b) were transiently co‐expressed in Nicotiana benthamiana. LYK2‐GFP and CERK1‐myc were immunoprecipitated (IP) with anti‐GFP (a) and anti‐myc (b) beads, respectively, and immunoblot (IB) experiments were performed with anti‐GFP and anti‐myc antibodies. Left and right panels are cropped from the same gel. (c), LYK2 fused to the N‐terminal part of YFP (LYK2‐NYFP) and LYK5 fused to the C‐terminal part of YFP (LYK5‐CYFP) were transiently co‐expressed in Nicotiana tabacum by Agrobacterium‐mediated transformation. Images were taken 2 days after agroinfiltration by confocal laser scanning microscopy. Left panels, YFP; middle panels, bright field; right panels, merge of YFP and bright field. Bar = 20 μm [Colour figure can be viewed at wileyonlinelibrary.com]