<em>In planta</em> sequential hydroxylation and glycosylation of a fungal phytotoxin: Avoiding cell death and overcoming the fungal invader

Abstract

The interaction of plants with their pathogens involves an array of remarkably simple chemical reactions, some of which transform highly bioactive secondary metabolites (e.g., antimicrobial or phytotoxic produced by the plant or by the pathogen) into mutually harmless products (i.e., detoxification). For example, one of the most significant fungal pathogens [Phoma lingam (Tode ex Fries) Desmarzières, perfect stage Leptosphaeria maculans (Desmarzières) Ces. et de Notaris] of rapeseed (Brassica napus and Brassica rapa, Cruciferae family) is able to overcome the plant's natural chemical defenses, phytoalexins, through various detoxification steps (1). On the other hand, to facilitate tissue colonization, some pathogenic fungi produce phytotoxic metabolites which selectively damage host-plants [host-selective toxins (HSTs)], or a wider range of plants (nonselective toxins; ref. 2). Plants may be resistant to a particular pathogen if they produce an enzyme(s) that catalyzes detoxification of the pathogen's toxin(s) (3, 4). Consequently, such detoxification traits are desirable and of much importance in strategic plant breeding or in engineering disease resistance. Whereas a number of phytoalexin detoxifying reactions have been shown to occur in very diverse plant pathogenic fungi (1, 5, 6), detoxification of phytotoxins has been reported in only a few plant species (7).

Three remarkable examples help to illustrate the significance of phytotoxin detoxification in plants. The first plant disease resistance gene to be cloned encoded an enzyme able to detoxify HC-toxin, a host-selective toxin (HST) produced by a maize fungal pathogen (8). Detoxification of HC-toxin, a cyclic tetrapeptide, resulted from a simple reduction of a carbonyl on the side chain of one of the constituent amino acids (9, 10). In another example, the first transgenic tobacco plants resistant to wildfire (a bacterial disease of tobacco) were obtained by introducing the tabtoxin detoxifying gene (3). Tabtoxin is a nonselective toxin produced by the wildfire bacterium. Genetically engineered tobacco plants resistant to wildfire disease were generated by introducing the gene encoding an acetyltransferase, which catalyzed detoxification of tabtoxin. Interestingly, this gene was cloned from the phytopathogenic wildfire bacterium, which is self-resistant to its own toxin because of production of this detoxifying enzyme. A more recent example reported that transgenic sugar cane plants expressing a phytotoxin detoxifying gene were more resistant to leaf scald disease than wild-type plants (11). In this example, although the chemical structure of albicidin, the toxin produced by the leaf scald bacterium, has not been elucidated, the detoxifying gene appears to encode an esterase. The albicidin esterase gene was cloned from a biocontrol agent that provides protection against leaf scald disease (12). From these examples, it is apparent that phytotoxin detoxifying enzymes play an important role in protecting plants from the phytotoxin producing microorganism, whether bacterial or fungal.

Alternaria blackspot is one of the most damaging and widespread fungal diseases of rapeseed (B. napus and B. rapa), an oilseed of great economic importance worldwide (13). Alternaria brassicae (Berk.) Sacc., the blackspot causing fungus, produces the cyclic depsipeptide toxins destruxin B and homodestruxin B (1 and 2 in Fig. ​Fig.1),1), both in vitro (14) and in planta (15, 16). Ultrastructural studies have shown that destruxin B causes tissue damage similar to that observed in plants naturally infected with A. brassicae (17). Nonetheless, the role of destruxins in the development of blackspot disease is not clearly established, partly because the sexual stage of A. brassicae is not known, and no pathotypes or non-destruxin-producing strains are available. A variety of assays to establish the phytotoxicity of destruxin B to whole plants (18), seedlings (19), excised leaves (14, 15, 18, 19), pollen grains (20), protoplast, and cell cultures (18, 19), suggest that destruxin B phytotoxicity is host-selective. This apparent selectivity might be due to detoxification reactions occurring in tissues of plant species resistant to A. brassicae. Considering that no sources of Alternaria blackspot resistance are known within Brassica species, such information is of enormous importance in generating blackspot-resistant plant lines.

Figure 1

Chemical structures of destruxin B (1), homodestruxin B (2), hydroxydestruxin B (3), hydroxyhomodestruxin B (4), β-d-glucosyl hydroxydestruxin B (5), α-d-glucosyl hydroxydestruxin B (6), and β-d-tetracetylglucosyl hydroxydestruxin B (7).

Toward this end, we became interested in investigating the metabolism of destruxins in plants susceptible and resistant to A. brassicae. Sources of blackspot resistance within the family Cruciferae (syn. Brassicaceae) include Sinapis alba, a white mustard used as condiment and vegetable, and some weedy species. In a preliminary communication we reported that hydroxylation of destruxin B (1), and homodestruxin B (2), correlated with resistance and susceptibility to A. brassicae of two different species (21). Here we demonstrate that complete detoxification of destruxin B (1) in plants resistant to A. brassicae is a two-step process involving sequential hydroxylation and glucosylation. The chemical structure of the nontoxic destruxin B product, β-d-glucosyl hydroxydestruxin B (5, Fig. ​Fig.1),1), was determined by a combination of spectroscopic analyses and chemical synthesis of both the β and α glucosides (5 and 6, Fig. ​Fig.1)1) as well as the tetracetyl derivative 7 (Fig. ​(Fig.1).1). Results of the phytotoxicity assays of hydroxydestruxin B, hydroxyhomodestruxin B, and β-d-glucosyl hydroxydestruxin B (Fig. ​(Fig.1, 1, compounds 3-5) are described. Interestingly, although these hydroxylation and glucosylation reactions occur in both resistant and susceptible species, hydroxylation was the rate limiting step in the susceptible species, whereas glucosylation was the rate limiting step in the resistant species. Remarkably, it was established that hydroxydestruxin B (3) induced the biosynthesis of phytoalexins in resistant but not in susceptible species. Taken together, our studies suggest that S. alba can overcome the fungal invader through detoxification of destruxin B coupled with production of phytoalexins.

Results are the means of six independent experiments ± standard error.

Results are the means of three independent experiments ± standard error.

Acknowledgments

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