Purification and Characterization of a NADPH-Dependent Aldehyde
Reductase from Mung Bean That Detoxifies Eutypine, a
Toxin from Eutypa lata |
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Authors: | Ségolène Colrat Alain Latché Monique Guis Jean-Claude Pech Mondher Bouzayen Jean Fallot and Jean-Paul Roustan |
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Institution: | Ecole Nationale Supérieure Agronomique Unité, Associée, Institut National de la Recherche Agronomique, BP 107, 31326 Castanet-Tolosan cedex, France |
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Abstract: | Eutypine
(4-hydroxy-3-3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin
produced by Eutypa lata, the causal agent of eutypa
dieback in the grapevine (Vitis vinifera). Eutypine is
enzymatically converted by numerous plant tissues into eutypinol
(4-hydroxy-3-3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite
that is nontoxic to grapevine. We report a four-step procedure for the
purification to apparent electrophoretic homogeneity of a
eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna
radiata) hypocotyls. The purified protein is a monomer of 36
kD, uses NADPH as a cofactor, and exhibits a
Km value of 6.3 μm for
eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The
enzyme failed to catalyze the reverse reaction using eutypinol as a
substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2
to 7.5. These data strongly suggest that ERE is an aldehyde reductase
that could probably be classified into the aldo-keto reductase
superfamily. We discuss the possible role of this enzyme in eutypine
detoxification.Many pathogenic bacteria and fungi produce toxins that interfere
with various functions of plant cells and may affect plant defense
mechanisms (Durbin, 1981). Toxin production is commonly associated with
disease severity and can be involved in colonization or systemic
invasion by the pathogen (Schäfer, 1994). Toxin resistance has
been shown in most cases to be based on the ability of the plant to
metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang
and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance
genes that encode proteins involved in detoxification mechanisms have
been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and
Birch, 1997). In many cases a relationship exists between toxin
tolerance and resistance to the disease (Anzai et al., 1989; Meeley et
al., 1992). The availability of toxin-resistance genes will permit a
greater understanding of the mechanisms causing plant disease and will
also set the stage for engineering resistance to plant disease (Keen,
1993).Eutypine (4-hydroxy-3-3-methyl-3-butene-1-ynyl] benzaldehyde) is a
toxin produced by the ascomycete fungus Eutypa lata (Pers.:
Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991).
This disease is responsible for considerable loss in yield and is the
most devastating disease of grapevine (Vitis vinifera) in
many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The
fungus infects the stock through pruning wounds and is present in the
xylem and phloem of the vine trunk and branches (Moller and
Kasamitis, 1978; Duthie et al., 1991). After a long incubation period,
a canker forms around the infected wound. The toxin synthesized by the
fungus in the trunk is believed to be transported by the sap to the
herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates
grapevine cells through passive diffusion and its accumulation in the
cytoplasm has been explained by an ion-trapping mechanism related to
the ionization state of the molecule (Deswarte et al., 1996b). In the
cell the effects of eutypine include reduction of adenylated nucleotide
content, inhibition of succinate dehydrogenase, uncoupling of oxidative
phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to
dwarfed and withered new growth of branches, marginal necrosis of the
leaves, dryness of the inflorescence, and, finally, death of one or
more branches (Moller and Kasamitis, 1981). The toxin appears to be an
important virulence factor involved in symptom development of the
disease (Deswarte et al., 1996a). However, the absence of
toxin-deficient mutants of the fungus and its long incubation period in
the trunk before symptom development have prevented a critical study of
the toxin in vine plants. Determining the gene responsible for eutypine
resistance would therefore be an important critical tool in determining
the role of eutypine toxin in symptom development in the disease; and
it has the potential to confer resistance to transgenic
grapevines.Recently, Colrat et al. (1998) found detoxification to occur in
grapevine cells through the enzymatic reduction of eutypine into its
corresponding alcohol, eutypinol
(4-hydroxy-3-3-methyl-3-butene-1-ynyl] benzyl alcohol). We have
determined that this derivative of the toxin is nontoxic for grapevine
tissues. Furthermore, we have established a relationship between the
susceptibility of grapevine to eutypa dieback and the ability of
tissues to inactivate eutypine, suggesting that the detoxification
mechanism plays an important role in defense reactions. Eutypine is
enzymatically detoxified in numerous plant species and, among them, we
found that the tissues of mung bean (Vigna radiata), a
nonhost plant for the pathogen, exhibit an efficient detoxification
activity. As a prerequisite for demonstrating the involvement of
eutypine toxin in eutypa dieback, we report here the purification to
homogeneity and the characterization of an ERE from etiolated mung bean
hypocotyls. |
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