A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea

Abstract

The plant cuticle composed of cutin, a lipid-derived polyester, and cuticular waxes covers the aerial portions of plants and constitutes a hydrophobic extracellular matrix layer that protects plants against environmental stresses. The botrytis-resistant 1 (bre1) mutant of Arabidopsis reveals that a permeable cuticle does not facilitate the entry of fungal pathogens in general, but surprisingly causes an arrest of invasion by Botrytis. BRE1 was identified to be long-chain acyl-CoA synthetase2 (LACS2) that has previously been shown to be involved in cuticle development and was here found to be essential for cutin biosynthesis. bre1/lacs2 has a five-fold reduction in dicarboxylic acids, the typical monomers of Arabidopsis cutin. Comparison of bre1/lacs2 with the mutants lacerata and hothead revealed that an increased permeability of the cuticle facilitates perception of putative elicitors in potato dextrose broth, leading to the presence of antifungal compound(s) at the surface of Arabidopsis plants that confer resistance to Botrytis and Sclerotinia. Arabidopsis plants with a permeable cuticle have thus an altered perception of their environment and change their physiology accordingly.

Figure 1

Phenotypes of bre1/lacs2-2 and lacs2-3 mutant isolates. (A) Morphology of the rosette of Col-0, lacs2-2, and lacs2-3. (B–D) Macroscopic evaluation of symptoms 3 days after inoculation with B. cinerea of Col-0, bre1/lacs2-2, and lacs2-3. Scale bar: 1 cm. (E–M) Calcofluor white-stained organs of Col-0, bre1/lacs2-2, and lacs2-3, viewed under UV light. Col-0 pictures are shown with the corresponding pictures in bright-field microscopy as inset. Leaves (E–G), young seedlings (H–J), and the tip of a flowering inflorescence (K–M) are shown.

Figure 2

Ultrastructure of the epidermal extracellular matrix of rosette leaves. Lower epidermal extracellular matrix of Col-0 (A) and lacs2-3 (B, C) leaves. Extracellular matrix at the point of fusion between the lower epidermis of one leaf and the upper epidermal cell layer of another leaf in lacs2-3 (D). Scale bar: 500 nm (A) and 800 nm in (B–D).

Figure 3

Composition of aliphatic monomers of leaf polyesters. Large rosette leaves of 5- to 6-week-old plants of Col-0 and lacs2-3 were analyzed. (A) Hydroxylated fatty acids and their derivatives and (B) fatty acids and fatty alcohols. DiOH, dihydroxy carboxylic acid; DiCA, dicarboxylic acid (n=3; ±s.e.; the experiment was repeated once with similar results).

Figure 4

Cuticular transpiration. Loss of water from 5- to 6-week-old rosettes of Col-0 and lacs2-3 plants was measured at the indicated time points (n=4–6; ±s.e.; experiment was repeated twice with similar results).

Figure 5

Permeability of leaf cuticles of mutants having an altered cuticular polyester. (A) Droplets of a toluidine blue solution were incubated on the leaves for 2 h and then washed with water. An overview of typically stained leaves from each genotype is given in the top panel. A microscopic view of the typical staining underneath a droplet that was incubated for 2 h on leaves from each genotype is given below its overview. (B) Toluidine blue droplets were incubated on lacs2-3 leaves for the time points indicated below each leaf and then briefly rinsed with water. Typically stained leaves are presented. A colour version of this figure is available at the EMBO Journal online.

Figure 6

Resistance to Botrytis of mutants having differences in their cuticular polyesters. (A) Macroscopic symptoms 3 days after inoculation with B. cinerea (BMM) on leaves of different genotypes. White arrowheads indicate examples of symptom-free inoculation sites. Scale bar: 1 cm. (B) Number of outgrowing lesions developed 3 days after inoculation with B. cinerea (BMM) in different genotypes (n=4; ±s.e.; different letters indicate significance at P<0.005 using ANOVA). The experiment was repeated twice with similar results.

Figure 7

Analysis of PDB diffusates for the presence of growth-inhibiting activities against B. cinerea. (A) Germination and growth of B. cinerea (BMM) in vitro in the presence of leaf diffusates collected at 18 h (grey bars) and 44 h (black bars) from plants of different genotypes. Three independent experiments were undertaken and six representative pictures were evaluated ±s.d. (B) B. cinerea (BMM) spores were incubated on WT Col-0 leaves in the presence of 1/4 PDB diffusates collected from leaves of different genotypes at indicated time points. Typical symptoms 3 days after inoculation are presented.

Figure 8

Analysis of the activity of different PDB diffusates from lacs2 after enzymatic digestions. Germination and growth of B. cinerea (BMM) in the presence of different 1/4 PDB diffusates (PDB DIF) from lacs2 that had undergone no treatment (control), treatment with lipase from M. miehei at 32 U/μl (lipase), or proteinase K (protease) are shown. Lengths of the germlings grown in treated 1/4 PDB diffusates from lacs2 were normalized to those grown in treated 1/4 PDB (100%). Three to four representative pictures were evaluated ±s.d.

Figure 9

Influence of PDB preincubation on the resistance of lacs2 to Sclerotinia. (A) Macroscopic symptoms 3 days after inoculation with S. sclerotiorum on leaves of Col-0 and lacs2 without (−PDB) and with preincubation (+PDB) with 1/4 PDB for 20 h. (B) A number of outgrowing lesions developed 3 days after inoculation with S. sclerotiorum in Col-0 and lacs2 plants without (−PDB) and with preincubation (+PDB) with 1/4 PDB for 20 h (n=6; ±s.e.; different letters indicate a significance at P<0.005 using ANOVA). The experiment was repeated with equivalent results.