Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant

Abstract

Background and aims: We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kβ1β2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kβ1β2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kβ1 and PI4Kβ2.

Methods: To achieve this we used the sid2pi4kβ1β2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kβ1β2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin.

Key results: We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kβ1β2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kβ1β2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner.

Conclusions: Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kβ1β2 triple mutant revealed the role of PI4Kβ1/β2 per se, thus showing the importance of these enzymes in plant defence responses.

Keywords: Arabidopsis thaliana; biotic stress; callose; isochorismate synthase 1; pathogens; phytohormones; pi4kβ1β2/PI4Ks; salicylic acid.

Fig. 1.

Correlation matrix between phytohormone levels. The matrix was built using the Pearson correlation of 27 hormone-related metabolites from 24 independent samples corresponding to six genotypes (WT, sid2, NahG, pi4kβ1β2, sid2pi4kβ1β2 and NahGpi4kβ1β2; four plants per genotype). Positive correlations are displayed in blue and negative correlations in red. Colour intensity and the size of the circles are proportional to the correlation coefficients. Red rectangles highlight the correlation between SA and other hormones.

Fig. 2.

Resistance to biotic stresses of pi4kβ1β2 and sid2pi4kβ1β2 mutants. (A) Resistance to the biotroph Hyaloperonospora arabidopsidis. (B) Resistance to the hemibiotroph Pseudomonas syringae pv. syringae DC3000. Infiltration treatment of 4-week-old-plants, with six independent samples. (C) Resistance to the necrotroph Botrytis cinerea. Four- to 5-week-old A. thaliana were inoculated with a 6-µL drop containing spores of Botrytis cinerea (50 000 spores mL^–1), placed into a plastic box and kept in the dark for 56 h. Statistical differences between the genotypes for B and C were assessed using ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. Different letters indicate a significant difference, Tukey HSD, P  <  0.05, n = 10 for A, n = 7 for B and n = 27 for C. ***Indicate difference from WT, t-test, P < 0.001.

Fig. 3.

Pattern of callose accumulation in pi4kβ1β2 leaves. (A) Aniline blue staining, and fluorescence microscopy. Scale bar = 500 µm. (B) Callose particles accumulated in different ROI. The squares represent the ROI. Data are presented as means ±s.e.m. Statistical differences were assessed using a two-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. Different letters indicate a significant difference, Tukey HSD, P  <  0.05. n = 11.

Fig. 4.

Callose deposition in response to flagellin. (A) Representative images of callose accumulated in leaves of 4-week-old A. thaliana plants by aniline blue staining, 24 h after infiltration with 0.1 µm flg22 or mock infiltration. Scale bar = 1cm. (B) Quantification of callose particles. Values represent an average of five ROI. Data are presented as means +s.d. For each treatment, statistical differences between the genotypes were assessed using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. Different letters indicate a significant difference, Tukey HSD, P  <  0.01, n = 4.

Fig. 5.

Resistance of pi4kβ1β2 to penetration by the non-host pathogen Blumeria graminis f. sp. hordei (Bgh). (A) Four types of interactions counted in the penetration success analysis after trypan blue staining. Scale bar = 5 µm. (B) Data showing penetration success of Bgh 24 hpi in each genotype: the mean number of cells with either haustoria or dead cells, respectively. (C) Data showing mean area of a callose spot per mm² at 24 hpi after interaction with Bgh spores. (D) Data showing mean area of a callose spot per mm² at 24 hpi after interaction with Bgh spores. Data for B, C and D were processed by ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test, the data represent one independent experiment, and the experiment was repeated four times. Letters indicate a significant difference, Tukey HSD, P  <  0.01. (E) Pictures demonstrating callose staining with aniline blue 24 hpi with Bgh. Scale bar = 100 µm.

Fig. 6.

Schematic representation of the effects of the pi4kβ1β2 double mutation on Arabidopsis plants.