P BERTO¹, P COMMÉNIL², L BELINGHERI² and B DEHORTER²

¹Unité de Phytopathologie, FUSAGX, 2, Passage des Déportés, B-5030 Gembloux, Belgium; ²Laboratoire de Physiologie des Parois Végétales, USTL, SN2 2eme étage, F-59655 Villeneuve d'Ascq Cedex, France

Background and objectives
Fungal pathogens can penetrate host tissues through undamaged plant cuticles, and electron microscopic studies of this process indicate that enzyme hydrolysis of the cuticle may be involved. So far, cutinases are the only enzymes which have been implicated in enzymatic penetration of the cuticle. However, recent data on fungal cutinase activity and pathogenicity are contradictory and indicate that other esterases than cutinase may perform a role in plant/fungus interactions. When Botrytis cinerea and Alternaria brassicicola were grown in a medium containing a fatty acid ester as an inducer, they produced extracellular lipases of 60 kDa [1] and of 80 kDa [2], respectively. These purified enzymes have been found to degrade unsaturated long-chain fatty acid esters reported to be components of cutin and waxes, indicating a possible involvement in interactions between conidia and cuticle. The role of these enzymes was investigated during the early phases of plant infection.

Results and conclusions
Fungal growth of B. cinerea in liquid medium with apple cutin as the sole carbon source was accompanied with the secretion of a cutinase (18 kDa) and a lipase (60 kDa). The lack of lipase production in dextrose-grown cultures and its synthesis in the presence of purified cutin suggest that lipase is cutin-induced rather than constitutive. The purified lipase showed cutinolytic activity using [³H] cutin, and possessed a higher specific activity than cutinase for low enzyme concentrations. Polyclonal antibodies raised against the B. cinerea lipase were purified and immunological specificity was demonstrated by immunoblotting. In vitro, lipase activity was inhibited by anti-lipase antibodies.

The lipase and the cutinase produced by B. cinerea were found to have different biochemical and serological properties. Purified polyclonal antibodies raised against the lipase specifically reacted with the lipase, and not with the cutinase. In vivo, the addition of antibodies to spore suspensions of B. cinerea completely suppressed lesion formation on the tomato leaves. Although B. cinerea conidia germinated in the presence of the anti-lipase antibody, the spore germ tubes did not penetrate the leaf cuticle. Thus, specific anti-lipase antibodies applied with an inoculum protect the leaves against attack.

These results suggest that B. cinerea lipase activity is required in the infection of tomato leaves. An 80-kDa lipase was detected on ungerminated spores of A. brassicicola. This lipolytic enzyme showed in vitro cutinolytic activity. Antibodies raised against the B. cinerea lipase cross-react with the 80-kDa lipase produced by A. brassicicola. No immunological cross- reactivity with anti-lipase antibodies was detected between cutinases produced when apple cutin was used as the sole carbon source. In vitro, lipase activity was inhibited by the antibody solution. In vivo, the addition of anti-lipase antibodies in spore suspensions of A. brassicicola delayed but did not prevent lesion formation on cauliflower leaves. However, the effect of the antibody was overcome when leaves were previously wiped with chloroform-soaked cotton wool to remove epicuticular waxes. In situ microscopic observations revealed that spore germination was not inhibited by anti-lipase antibodies. These results again suggest that A. brassicicola lipase could be involved in the first interactions with the plant cuticle.

Lipase appears as a novel enzyme associated in the early steps of plant infection. Athough its role in pathogenicity is not yet clear, it seems to differ according to plant-fungal pathogen system.

References
1. Comménil P, Belingheri L, Sancholle M, Dehorter B, 1995. Lipids 30, 351-356.
2. Berto P, Belingheri L, Dehorter, B, 1997. Biotechnology Letters 19, 533-536.