Mutations in LACS2, a long-chain acyl-coenzyme A synthetase, enhance susceptibility to avirulent Pseudomonas syringae but confer resistance to Botrytis cinerea in Arabidopsis

Abstract

We identified an Arabidopsis (Arabidopsis thaliana) mutant, sma4 (symptoms to multiple avr genotypes4), that displays severe disease symptoms when inoculated with avirulent strains of Pseudomonas syringae pv tomato, although bacterial growth is only moderately enhanced compared to wild-type plants. The sma4 mutant showed a normal susceptible phenotype to the biotrophic fungal pathogen Erysiphe cichoracearum. Significantly, the sma4 mutant was highly resistant to a necrotrophic fungal pathogen, Botrytis cinerea. Germination of B. cinerea spores on sma4 mutant leaves was inhibited, and penetration by those that did germinate was rare. The sma4 mutant also showed several pleiotropic phenotypes, including increased sensitivity to lower humidity and salt stress. Isolation of SMA4 by positional cloning revealed that it encodes LACS2, a member of the long-chain acyl-CoA synthetases. LACS2 has previously been shown to be involved in cutin biosynthesis. We therefore tested three additional cutin-defective mutants for resistance to B. cinerea: att1 (for aberrant induction of type three genes), bodyguard, and lacerata. All three displayed an enhanced resistance to B. cinerea. Our results indicate that plant cutin or cuticle structure may play a crucial role in tolerance to biotic and abiotic stress and in the pathogenesis of B. cinerea.

Figure 1.

The sma4 mutant displays enhanced susceptibility to Pst DC3000 expressing avrRpt2 or avrB. A and B, Six-week-old sma4 plants were dip inoculated with Pst DC3000(avrRpt2) (A) or Pst DC3000(avrB) (B) and photographed 3 d later. C and D, Bacterial growth for Pst DC3000(avrRpt2) (C) or Pst DC3000(avrB) (D) was monitored over a 4-d time course. Each data point represents the mean and se of three samples.

Figure 2.

The sma4 mutant is highly sensitive to lowered humidity. Wild-type Col-0 and sma4 plants were grown in soil for 10 d after germination under a clear plastic dome to maintain near 100% relative humidity. A, Seedlings photographed immediately after removing the dome. B, Seedlings photographed 2 h after the dome was removed.

Figure 3.

Ion leakage from sma4 mutant leaves is enhanced. A, Col-0 and sma4 ion leakage in response to PsgR4(avrRpt2). PsgR4(avrRpt2) was infiltrated into single leaves at a concentration of 5 × 10⁷ cfu/mL. Five leaf discs were floated abaxial side down in deionized water for 4 h, after which conductivity of the solution was measured in micro-ohms. Each data point is the mean ± se of three samples. B, sma4 leaves leak ions faster than wild-type Col-0 leaves after salt exposure. Ion leakage from sma4 and Col-0 leaves was measured after vacuum infiltration with MgCl₂ or MgSO₄ solution.

Figure 4.

The sma4 mutant displays enhanced disease resistance to B. cinerea. A, Leaves from 6-week-old Col-0 and sma4 plants were detached and placed in petri dishes and inoculated with B. cinerea. Leaves were photographed 5 d after inoculation. B, Lesion size induced by B. cinerea. Lesion size was determined by measuring the major axis of the necrotic area at 5 d after inoculation. Bars represent the mean and sd from eight samples. C and D, Leaves from Col-0 and sma4 plants were stained with trypan blue and photographed 3 d after infection with B. cinerea.

Figure 5.

Resistance to B. cinerea does not require COI1 or EIN2. A, Leaves from 6-week-old plants were detached and placed in petri dishes and inoculated with B. cinerea. Representative leaves were photographed 3 d after inoculation. B, Lesion size induced by B. cinerea was measured 3 d after inoculation. Bars represent the mean and sd from eight samples. C, Six-week-old plants were dip inoculated with Pst DC3000(avrRpt2). Plants were photographed 3 d after inoculation.

Figure 6.

Structure of the SMA4 gene and complementation of the sma4 mutation. A, Exon and intron structure of SMA4. The exon regions are indicated with rectangles and intron regions with lines. The sma4 mutation is a point mutation in intron 4. The letters show nucleotide sequences from the beginning of intron 4. The T-to-A mutation in sma4 is indicated by an uppercase letter. B and C, Complementation of the sma4 mutation by transformation. Plants were dip inoculated with DC3000(avrRpt2) and photographed 3 d after inoculation (B), or inoculated with B. cinerea and photographed 5 d after inoculation (C).

Figure 7.

The cutin-defective mutants att1, bdg, and lcr display enhanced resistance to B. cinerea. A, Leaves from 5-week-old plants were detached and placed in petri dishes and inoculated with B. cinerea. Leaves were photographed 3 d after inoculation. B, Lesion size induced by B. cinerea. Lesion size was determined by measuring the major axis of the necrotic area at 3 d after inoculation. Bars represent the mean and sd from eight samples.