Involvement of the caleosin/peroxygenase RD20 in the control of cell death during Arabidopsis responses to pathogens

Abstract

Caleosins, mostly found in lipid droplets of seeds and leaves, are believed to play physiological roles through their enzymatic capacities to produce oxylipins. We recently identified the caleosin RD20 as a peroxygenase reducing endogenous fatty acid hydroperoxides into their corresponding alcohols. Such oxylipins confer tolerance to oxidative stress by decreasing reactive oxygen species accumulation and by minimizing cell death. RD20 expression being induced by pathogens, we have examined the mode of action of this caleosin in response to biotic stress. Plants overexpressing RD20 exhibited an alteration of their leaf cuticle wax components and an increased resistance to the fungus Alternaria brassicicola. Conversely, silencing RD20 led to an enhanced propagation of the fungus and to reduced severity of the damages caused by the inoculation of the bacteria Pseudomonas syringae pv tomato. We discuss these findings and propose that the major function of RD20 is to generate oxylipins modulating oxidative status and cell death.

Keywords: Alternaria; Pseudomonas; RD20; caleosin; cell death; defense; oxylipins; reactive oxygen species.

Figure 1.

Overexpression of RD20 is accompanied by an alteration of lipid homeostasis. (A) The fatty acid contents of the seeds overexpressing RD20 and wild type were measured by gas-chromatography and expressed as chain length ratio (the ratio between the total amounts of C20 to C24 fatty acids and the total amounts of C16 to C18 fatty acids). Each point was made in triplicate and the experience was repeated 3 times. (B) Modification of the cuticular waxes composition of the transgenic lines was estimated by measuring by gas-chromatography their relative component contents vs. the respective control. Measurements were made in triplicate. In the 2 panels, the transgenic lines were significantly different from the control when **P < 0.05 (t-test).

Figure 2.

Alteration of RD20 expression affects disease symptoms of Arabidopsis plants after Alternaria brassicicola and Pseudomonas syringae inoculations. The severity of the symptoms caused by A. brassicicola was monitored after 5 μl (5 × 10⁵ spores/ml) of fungal spores were deposited on each half leaf of 3 fully expended leaves of 8 of 2 mo old transgenic plants (n = 64). (A) Lesions observed 10 d after infection on plants where RD20 was silenced (rd20s) or overexpressed (RD20-OE); (B) and after staining by Trypan blue. Bars represent 1 mm. (C) Three weeks after inoculation, the pathogen was restricted within inoculated area in RD20-OE and control leaves whereas it largely spread over leaf of silenced plants. The avirulent bacteria Pst avrRpm1 (D) and the virulent strain Pst (E) were infiltrated in 3 fully expanded leaves of 20 of 2 mo old transgenic plants (n = 60). Bacterial lesions were evaluated 2 and 3 d after infiltration and the magnitude of the damages was rated on a 4-point scale designed as I, II, III and IV according to the degree of severity: Stage I, Stage II, Stage III, Stage IV. The damages were significantly different from those of the control when **P < 0.05 (t-test).