Intronic T-DNA insertion renders Arabidopsis opr3 a conditional jasmonic acid-producing mutant

Abstract

Jasmonic acid and its derived metabolites (JAs) orchestrate plant defense against insects and fungi. 12-Oxo-phytodienoic acid (OPDA), a JA precursor, has also been implicated in plant defense. We sought to define JAs and OPDA functions through comparative defense susceptibility characteristics of three Arabidopsis (Arabidopsis thaliana) genotypes: aos, lacking JAs and OPDA; opda reductase3 (opr3), deficient in JA production but can accumulate OPDA; and transgenics that overexpress OPR3. opr3, like aos, is susceptible to cabbage loopers (Trichoplusia ni) but, relative to aos, opr3 has enhanced resistance to a necrotrophic fungus. Gas chromatography-mass spectrometry reveals that opr3 produces OPDA but no detectable JAs following wounding and looper infestation; unexpectedly, substantial levels of JAs accumulate in opr3 upon fungal infection. Full-length OPR3 transcripts accumulate in fungal-infected opr3, potentially through splicing of the T-DNA containing intron. Fungal resistance correlates with levels of JAs not OPDA; therefore, opr3 resistance to some pests is likely due to JA accumulation, and signaling activities ascribed to OPDA should be reassessed because opr3 can produce JAs. Together these data (1) reinforce the primary role JAs play in plant defense against insects and necrotrophic fungi, (2) argue for a reassessment of signaling activities ascribed to OPDA, and (3) provide evidence that mutants with intron insertions can retain gene function.

Figure 1.

JA rescue of male fertility, and accumulation of JAs and OPDA in mechanically wounded Arabidopsis. A, Male fertility is restored in aos and opr3 by the exogenous application of JA. B, Levels of JAs in untreated leaves (CR) and wounded leaves 2 h after mechanical damage (W). C, Levels of OPDA in untreated leaves (CR) and wounded leaves 2 h after mechanical damage (W). Means ± sd are shown. n = 3. [See online article for color version of this figure.]

Figure 2.

opr3 has increased susceptibility to cabbage loopers. A, Looper weights 12 d after release of a newly hatched larva in an arena containing five plants of indicated genotype. Means ± sem of two independent experiments, each with approximately 15 arenas per genotype, are shown. B, Representative photos of gl-1, opr3, and aos showing tissue damage. C, Levels of JAs in control (CR) and looper-infested (L) leaves 72 h post infestation. Means ± sd are shown. n = 3. D, Levels of OPDA in control (CR) and looper-infested (L) leaves 72 h post infestation. Means ± sd are shown. n = 3. [See online article for color version of this figure.]

Figure 3.

opr3 has increased resistance to B. cinerea relative to aos and accumulates camalexin and JAs. A, aos, opr3, and gl-1 leaf lesion diameters at 48 and 72 hpi with fungal conidia. Mean lesion diameters ± sd are shown. n = 40. Photographs of representative leaves of each genotype 72 hpi. Bar = 1 cm. B, Leaf camalexin levels at 72 hpi. Means ± sd. n = 30. C, Levels of leaf JAs at 48 and 72 hpi with fungal conidia. Means ± sd. n = 8. D, Levels of leaf OPDA at 48 and 72 hpi with fungal conidia. Means ± sd. n = 8. Within any given treatment, letters indicate significant differences (P < 0.005, Tukey’s test).

Figure 4.

OPR3 transcript levels. A, Semiquantitative RT-PCR of full-length OPR3 and ACTIN transcripts in gl-1 and opr3 that were not treated (CR), wounded (W), looper infested for 72 h (L), or B. cinerea infected 72 hpi (B). Primers used to generate the full-length OPR3 transcript amplified the full open reading frame from the start codon to the stop codon. B, Quantitative RT-PCR of OPR3 transcripts relative to TUB4 transcripts in gl-1 and opr3 that were not treated (CR), wounded (W), looper infested for 72 h (L), or B. cinerea infected 72 hpi (B). Means ± sd. n = 6. C, Quantitative RT-PCR analysis of OPR3 transcripts relative to TUB4 transcripts in gl-1, opr3, and aos at 48 and 72 hpi with B. cinerea. Means ± sd. n = 6.

Figure 5.

JAs are required for defense against B. cinerea. A, Leaf lesion diameters of Col-0 and aos sprayed with indicated JA concentrations at 72 hpi with fungal conidia. Mean lesion diameters ± sd are shown. n = 30. B, Representative leaves of each genotype 72 hpi. Bar = 1 cm. C, OPDA levels in aos and Col-0 leaves with no JA treatment (Ctrl) and with 0.5 mm JA (JA) 72 hpi with fungal conidia. Letters above bars indicate statistical significance; bars not sharing letters represent significant mean differences at P < 0.05, Tukey’s test.

Figure 6.

OPR3-OE plants are more resistant to B. cinerea. A, Leaf lesion diameters of OPR3-OE and Col-0 48 hpi and 72 hpi with fungal conidia. Representative leaves of each genotype at 72 hpi. Bar = 1 cm. B, Levels of JAs in Col-0 (WT) and OPR3-OE leaves 72 hpi with fungal conidia. C, OPDA levels in Col-0 (WT) and OPR3-OE leaves 72 hpi with fungal conidia. Mean lesion diameters ± sd. n = 40. Bars with different letters indicate significant differences (P < 0.005, Tukey’s test) within a treatment.