The Arabidopsis thaliana lectin receptor kinase LecRK-I.9 is required for full resistance to Pseudomonas syringae and affects jasmonate signalling

Abstract

On microbial attack, plants can detect invaders and activate plant innate immunity. For the detection of pathogen molecules or cell wall damage, plants employ receptors that trigger the activation of defence responses. Cell surface proteins that belong to large families of lectin receptor kinases are candidates to function as immune receptors. Here, the function of LecRK-I.9 (At5g60300), a legume-type lectin receptor kinase involved in cell wall-plasma membrane contacts and in extracellular ATP (eATP) perception, was studied through biochemical, gene expression and reverse genetics approaches. In Arabidopsis thaliana, LecRK-I.9 expression is rapidly, highly and locally induced on inoculation with avirulent strains of Pseudomonas syringae pv. tomato (Pst). Two allelic lecrk-I.9 knock-out mutants showed decreased resistance to Pst. Conversely, over-expression of LecRK-I.9 led to increased resistance to Pst. The analysis of defence gene expression suggests an alteration of both the salicylic acid (SA) and jasmonic acid (JA) signalling pathways. In particular, LecRK-I.9 expression during plant-pathogen interaction was dependent on COI1 (CORONATINE INSENSITIVE 1) and JAR1 (JASMONATE RESISTANT 1) components, and JA-responsive transcription factors (TFs) showed altered levels of expression in plants over-expressing LecRK-I.9. A similar misregulation of these TFs was obtained by JA treatment. This study identified LecRK-I.9 as necessary for full resistance to Pst and demonstrated its involvement in the control of defence against pathogens through a regulation of JA signalling components. The role of LecRK-I.9 is discussed with regard to the potential molecular mechanisms linking JA signalling to cell wall damage and/or eATP perception.

Keywords: Arabidopsis; Pseudomonas; jasmonic acid; lectin; plant-pathogen interactions; receptor-like kinases.

Figure 1

Expression of lectin receptor kinase (LecRK) clade I genes in response to infection. (A) The phylogenetic tree of the LecRK clade I subfamily was generated using the neighbour‐joining method after alignment of the amino acid sequences of kinase domains of the subfamily members with ClustalW. (B) The expression of five LecRK genes was determined by quantitative real‐time polymerase chain reaction (Q‐RT‐PCR) with cDNA generated from leaves of 4‐week‐old Col‐0 plants inoculated with Pseudomonas syringae pv. tomato (Pst) DC3000 (black circles) and Pst DC3000 avrRpm1 (open circles) at 10⁷ colony‐forming units (cfu)/mL, or treated with water (squares). Six members of clade I were not significantly altered and were below the detection limit. The expression values of each gene were normalized using the expression level of the housekeeping gene At2g28390 as an internal standard. The mean ± standard deviation of three biological replicates is shown. Asterisks denote statistically significant differences according to one‐way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test (P < 0.01). In the panels showing LecRK‐I.4, LecRK‐I.5 and LecRK‐I.8, significant differences are between plants inoculated with Pst DC3000 avrRpm1 or Pst DC3000 and plants infiltrated with water (control). In the panel showing LecRK‐I.9, significant differences are between plants inoculated with Pst DC3000 avrRpm1 and plants inoculated with Pst DC3000, as well as plants infiltrated with water (control).

Figure 2

Histochemical analysis of β‐glucuronidase (GUS) activity in transgenic Arabidopsis thaliana plants containing a P_LecRK‐I.9GUS transgene. (A) Activation of the LecRK‐I.9 promoter in response to infiltration with Pseudomonas syringae pv. tomato (Pst). Leaves of 5‐week‐old transgenic plants were infiltrated with water, the avirulent strain Pst DC3000 avrRpm1 or the virulent strain Pst DC3000 at 2.5 × 10⁵ colony‐forming units (cfu)/mL. Leaves were stained for GUS activity at 0, 6, 9, 12, 24 and 34 h after infiltration. (B) Activation of the LecRK‐I.9 promoter in response to wounding. Healthy leaves of transgenic plants stained for GUS (1) were compared with similar leaves that were treated with tweezers (2) or cut with a razor blade (3). GUS staining was performed 4 h after wounding. Arrowheads indicate wounding. Bars are 1 mm.

Figure 3

Phenotypes of LecRK‐I.9 mutant and over‐expressing lines in response to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst). (A) Leaves of the over‐expressing (Ox) line and wild‐type (WT) 5‐week‐old plants were inoculated with 5 × 10⁶ colony‐forming units (cfu)/mL of Pst DC3000 or Pst DC3000 avrRpm1. Representative leaves are shown at 48 h post‐inoculation (hpi) (Pst DC3000 avrRpm1) and 72 hpi (Pst DC3000). The leaves were inoculated on the opposite site to the arrowheads. (B) Leaves of two lecrk‐I.9 null mutants (I.9‐1 and I.9‐2) and wild‐type (WT) 5‐week‐old plants were inoculated with 7 × 10⁵ cfu/mL of Pst strain DC3000 or Pst strain DC3000 avrRpm1. (C) In planta growth of Pst DC3000 and Pst DC3000 avrRpm1 in 5‐week‐old Arabidopsis thaliana plants of the indicated genotypes. Leaves were infiltrated with a suspension of Pst at 5 × 10⁵ cfu/mL. For each genotype, 20 leaf discs from five plants were harvested at time 0 (white bars) and at 3 days after infiltration (black bars). The mean bacterial counts are presented as cfu/mL, and represent the averages of three independent experiments. Samples with different letters are significantly different at P < 0.01 by one‐way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test of comparisons at day 3 after infiltration between the different bacterial strains on individual plant genotypes.

Figure 4

Expression of defence and cell death‐related genes in response to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst). Transcript levels were evaluated by quantitative real‐time polymerase chain reaction (Q‐RT‐PCR) at three or six time points: 0, 24 and 48 h post‐inoculation (hpi) for PR1, PR4, HSR3 and VPE and 0, 4, 8, 12, 24 and 48 hpi for VSP1 and LOX2. Leaves of 4‐week‐old plants [wild‐type (WT), grey bars; lecrk‐I.9‐1, open bars; lecrk‐I.9‐2, open bars; 35S::lecrk‐I.9 (Ox), black bars] were inoculated with Pst DC3000 avrRpm1 (A) or Pst DC3000 (B) at 10⁷ colony‐forming units (cfu)/mL. The expression values of each gene were normalized using the expression level of the housekeeping gene At2g28390 as an internal standard. The mean ± standard deviation of three biological replicates is shown. Asterisks denote statistically significant differences according to one‐way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test (P < 0.01) at a given time point between the mutant or over‐expressing (Ox) lines and the wild‐type plants inoculated with either Pst DC3000 avrRpm1 or Pst DC3000.

Figure 5

Role of jasmonic acid (JA) in LecRK‐I.9 expression and LecRK‐I.9‐mediated defence. (A) LecRK‐I.9 gene expression in response to treatment with signalling molecules. Transcript levels were quantified by quantitative real‐time polymerase chain reaction (Q‐RT‐PCR) at 0, 6, 12, 24 and 48 h post‐inoculation (hpi) (bars from white to black). ACC, 1‐aminocyclopropane‐1‐carboxylic acid; SA, salicylic acid. (B) LecRK‐I.9 gene expression in wild‐type (Col‐0) and defence signalling mutants after inoculation with Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpm1 at 10⁷ colony‐forming units (cfu)/mL. Transcript levels were quantified by Q‐RT‐PCR at 0, 4, 8, 12, 24 and 48 hpi (bars as in A). The expression values of LecRK‐I.9 were normalized using the expression level of the housekeeping gene At2g28390 as an internal standard. The mean ± standard deviation of three biological replicates is shown. Asterisks denote statistically significant differences according to one‐way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test (P < 0.01) at a given time between treated and control plants (A) and between mutant lines and wild‐type (WT) plants (B).

Figure 6

Expression of genes related to the jasmonate pathways in the lecrk‐I.9 mutant and over‐expressing lines after inoculation with Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpm1 and jasmonic acid (JA) treatment. Transcript levels were quantified by quantitative real‐time polymerase chain reaction (Q‐RT‐PCR) in leaves of wild‐type plants (squares, green lines), lecrk‐I.9 mutant lines (circles, red lines) and over‐expressing lines (diamonds, blue lines) at 0, 4, 8, 12, 24 and 48 h post‐inoculation (hpi) with Pst DC3000 avrRpm1 (A) or 50 µm JA (B). The expression values of each gene were normalized using the expression level of the housekeeping gene At2g28390 as an internal standard. The mean ± standard deviation of three biological replicates is shown. Asterisks denote statistically significant differences according to one‐way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test (P < 0.01) at a given time between over‐expressing lines and wild‐type plants.