Impaired Cuticle Functionality and Robust Resistance to Botrytis cinerea in Arabidopsis thaliana Plants With Altered Homogalacturonan Integrity Are Dependent on the Class III Peroxidase AtPRX71

Abstract

Pectin is a major cell wall component that plays important roles in plant development and response to environmental stresses. Arabidopsis thaliana plants expressing a fungal polygalacturonase (PG plants) that degrades homogalacturonan (HG), a major pectin component, as well as loss-of-function mutants for QUASIMODO2 (QUA2), encoding a putative pectin methyltransferase important for HG biosynthesis, show accumulation of reactive oxygen species (ROS), reduced growth and almost complete resistance to the fungal pathogen Botrytis cinerea. Both PG and qua2 plants show increased expression of the class III peroxidase AtPRX71 that contributes to their elevated ROS levels and reduced growth. In this work, we show that leaves of PG and qua2 plants display greatly increased cuticle permeability. Both increased cuticle permeability and resistance to B. cinerea in qua2 are suppressed by loss of AtPRX71. Increased cuticle permeability in qua2, rather than on defects in cuticle ultrastructure or cutin composition, appears to be dependent on reduced epidermal cell adhesion, which is exacerbated by AtPRX71, and is suppressed by the esmeralda1 mutation, which also reverts the adhesion defect and the resistant phenotype. Increased cuticle permeability, accumulation of ROS, and resistance to B. cinerea are also observed in mutants lacking a functional FERONIA, a receptor-like kinase thought to monitor pectin integrity. In contrast, mutants with defects in other structural components of primary cell wall do not have a defective cuticle and are normally susceptible to the fungus. Our results suggest that disrupted cuticle integrity, mediated by peroxidase-dependent ROS accumulation, plays a major role in the robust resistance to B. cinerea of plants with altered HG integrity.

Keywords: Botrytis cinerea; cell wall; cuticle; pectin; peroxidase; plant immunity; plant-microbe interactions.

Figure 1

Plants with altered HG have increased leaf cuticle permeability that is suppressed by exogenous ABA. (A,B), WT, PG, and qua2 five-week-old plants were sprayed with 0.01% MeOH or 100μm ABA and, after 24h, rosette leaves were collected. (A), Leaf disks were stained with droplets of toluidine blue and washed with water. (B), Leaves were incubated for the indicated times with 80% EtOH and the percentage of total chlorophyll released in the medium was spectrophotometrically measured. Bars indicate average percentage of total chlorophyll leaked at the indicated time of incubation ± SD (n = 3). For each time point, letters indicate statistically significant differences, according to two-way ANOVA followed by Tukey’s significance test (p < 0.05). These experiments were repeated three times with similar results.

Figure 2

Resistance to Botrytis cinerea conferred by altered HG is suppressed by exogenous ABA. Rosette leaves of five-week-old WT, PG, qua2, and tsd2-1 plants were treated with 0.01% MeOH (white bars) or 100μm ABA (grey bars) and, after 24h, inoculated with a B. cinerea spore suspension. Lesion area was measured 2days after inoculation (n > 10 lesions for each genotype). Bars indicate average lesion area ± SE. Different letters indicate statistically significant differences, according to two-way ANOVA followed by Tukey’s significance test (p < 0.05). This experiment was repeated three times with similar results.

Figure 3

Cutin deposition and cuticle thickness are enhanced in qua2 mutant leaves. (A), Five-week-old plants were sprayed with 0.01% MeOH (control) or 100μm ABA and leaves collected after 24h. Quantification of transesterified aliphatic and aromatic cutin monomers reveals an increase of main cutin components in qua2 which are not further increased after ABA treatment. The graph shows the analysis of the principal cutin monomers and the inset shows the sum of the indicated monomer classes per genotype and treatment. Data are mean ± SD; n = 4 replicates. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s significance test (p < 0.05). DW, dry weight; DCA, dicarboxylic acid; ω-OH, ω-hydroxy acid; and FA, fatty acid. (B), TEM images of the cuticle of epidermal cells of the adaxial face of rosette leaves of five-week-old WT (top panel) and qua2 (middle panel) plants. Pictures are representative of 15 (WT) and 13 (qua2) images. Bars, 125nm. Bottom panel measurement of cuticle thickness from images taken as above described.

Figure 4

Increased cuticle permeability of qua2 leaves is partially dependent on AtPRX71. Permeability of the cuticle of fully expanded rosette leaves (A) of five-week-old Col-0 (WT), qua2, atprx71, qua2 atprx71, and 35S::AtPRX71 #22 (35SPRX71) plants was determined by toluidine blue staining. (B) The amount of chlorophyll leached from rosette leaves as in (A) was measured after incubation in 80% EtOH at the indicated times. Bars indicate average concentration ± SD (n = 3). For each time point, asterisks indicate significant differences, according to Student’s t-test between WT and mutants (^*p < 0.05; ^***p < 0.01). These experiments were repeated three times with similar results. (C) Toluidine blue staining of the entire rosette of five-week-old plants. Representative images of at least five plants per genotype are shown.

Figure 5

Cell-adhesion defects in qua2 leaves are suppressed by loss of AtPRX71. Rosette leaves of five-week-old Col-0 (WT) (A), qua2 (B), atprx71 (C), qua2 atprx71 (D), and 35S::AtPRX71 #22 (35SPRX71) (E) plants were stained with ruthenium red and epidermal cells of the adaxial face were photographed. Asterisks indicate gaps between adjacent cells. (A–E), Representative images of at least five images per genotype. (F), For each experiment, total area of the gaps between adjacent cells per image was analyzed (total area of images = 60,000μm²). Bars indicate average gap area ± SD (n = 3). Asterisks indicate statistically significant difference between qua2 and qua2 atprx71, according to Student’s t-test (^***p < 0.01).

Figure 6

Resistance to B. cinerea in qua2 is partially dependent on AtPRX71. Rosette leaves of five-week-old WT plants, qua2, atprx71, and qua2 atprx71 plants, and of two independent transgenic lines overexpressing AtPRX71 (35SPRX71 #22 and #24) were inoculated with a B. cinerea spore suspension, and lesion area was measured 2days after inoculation (n > 10 for each genotype). Bars indicate average lesion area ± SE. Different letters indicate statistically significant differences, according to one-way ANOVA followed by Tukey’s significance test (p < 0.01).

Figure 7

Cuticle permeability and ROS accumulation in mutants with impaired cell wall integrity. (A,B), Rosette leaves of five-week-old Col-0 (WT), mur1, mur4, prc1, irx1-2, and fer-4 plants were stained with toluidine blue (A) to assay cuticle permeability or (B) incubated for the indicated times with 80% EtOH, and the concentration of chlorophyll leaked in the medium and of total chlorophyll was spectrophotometrically measured. Bars in (B) indicate average percentage of total chlorophyll leaked at the indicated time of incubation ± SD (n = 3). For each time point, asterisks indicate statistically significant differences with the control-treated WT samples, according to Student’s t-test (^***p < 0.01; ^*p < 0.01). (C), rosette leaves as in (A,B) were stained with DAB for ROS accumulation. These experiments were repeated three times with similar results.

Figure 8

Resistance to B. cinerea in cell wall mutants. Rosette leaves of five-week-old WT plants and of prc1, ixr1-2, mur1, mur4, qua2, and fer-4 mutant plants were inoculated with a B. cinerea spore suspension, and lesion area was measured 2days after inoculation (n > 12 lesions for each genotype). Bars indicate average lesion area ± SE. Different letters indicate statistically significant differences, according to one-way ANOVA followed by Tukey’s significance test (p < 0.01).