Molecular and functional characterization of a fructose specific transporter from the gray mold fungus Botrytis cinerea

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Abstract

In the gray mold fungus Botrytis cinerea, spore germination and plant infection are stimulated in the presence of nutrients, in particular sugars. Applied at micromolar concentrations, fructose is a more potent inducer of germination than glucose. To test whether preferred fructose uptake is responsible for this effect, and to study the mechanism of fructose transport in B. cinerea, a gene (frt1) encoding a fructose transporter was cloned. FRT1 is highly similar to recently identified fructose transporters of yeasts, but much less to other fungal hexose transporters characterized so far. By using a hexose uptake deficient yeast strain for expression, FRT1 was found to be a high affinity proton coupled symporter specific for fructose but not for glucose. B. cinerea frt1 disruption mutants were created and showed normal vegetative growth and plant infection, but a delay in fructose-induced germination when compared to wild-type. Sugar uptake experiments with both wild-type and mutant conidia showed a higher affinity for glucose than for fructose. Thus, we propose that the specific effect of fructose on germination is not due to transport but rather to an as yet unknown intracellular sensing.

Introduction

Botrytis cinerea, the causal agent of gray mold, is a facultative necrotrophic plant pathogen that is able to infect flowers, fruits, stems and leaves either through wounds or directly through the intact cuticle. It has long been known that germination of conidia and infection through intact plant surfaces is greatly stimulated by the availability of nutrients (Kosuge and Hewitt, 1964, Orellana and Thomas, 1962). On inert artificial surfaces, various amino acids and sugars efficiently induced germination of conidia, while mineral salts such as ammonium and phosphate were effective only in the presence of low concentrations of sugars (Blakeman, 1975). On cuticular surfaces, however, dry-inoculated conidia are also able to germinate at high humidity in the absence of liquid water (Prins et al., 2000). The requirement of nutrients for germination is also known for several saprotrophic fungi such as Aspergillus nidulans and Neurospora crassa (Osherov and May, 2001) while spores of the more specialized hemibiotrophic and biotrophic plant pathogenic fungi, such as Magnaporthe grisea, Colletotrichum lindemuthianum, the rusts and the mildews are usually able to germinate in pure water.

Of the sugars, fructose has been pointed out as the best inducer of germination in B. cinerea, being more effective than glucose and other hexoses or disaccharides (Blakeman, 1975). This is surprising since glucose is usually the most efficient hexose not only as a nutrient, but also as a signalling compound. One explanation for the particular activity of fructose in conidia could be that this sugar is preferentially taken up by a fructose-specific transport system. Fructose-specific uptake in fungi is not common, but has been observed in N. crassa when the mycelium was subjected to carbon starvation (Rand and Tatum, 1980), and in the fructophilic yeast Zygosaccharomyces bailii harbouring a fructose-specific sugar uptake system (Sousa-Dias et al., 1996). Generally, however, hexose uptake by filamentous fungi such as A. nidulans (Mark and Romano, 1971) and Pyrenopeziza brassicae (Walters et al., 1996) or by baker’s yeast (Chang et al., 2004) usually occurs with higher affinity to glucose than to fructose. Up to now, all characterized hexose transporters like from the ascomyetes Trichoderma harzianum (Cirillo, 1968) and Aspergillus niger (van Kuyk et al., 2004), and from the basidiomycetes Amanita muscaria (Wiese et al., 2000) and Uromyces fabae (Voegele et al., 2001), display preferential uptake of glucose compared to fructose.

Recently, a novel high-affinity fructose-specific transporter (FSY1) has been identified in the yeast Saccharomyces pastorianus (Gonçalves et al., 2000). By searching in the gene databases, we have identified partial cDNA sequences from B. cinerea which encoded a transporter named FRT1 with substantial structural similarities to FSY1. In this paper, we present the cloning and a structural and functional analysis of this transporter and demonstrate that it is highly specific for fructose. Sugar uptake experiments with B. cinerea wild-type and frt1 knock-out strains showed that FRT1 contributes to fructose-induced germination, but the high sensitivity of B. cinerea conidia to fructose could not be explained by preferential uptake of fructose.

Section snippets

Fungal growth

The haploid Botrytis cinerea strain B05.10 was provided by P. Tudzynski (University of Münster, Germany). The fungus was grown on tomato malt agar (1.5% malt extract with 250 g of homogenized tomato leaves per liter, 1.5% agar) and incubated for 5–6 days at 20 °C. Conidia were harvested by scraping them from sporulating plates with 10 ml sterile distilled H2O, and washed twice with sterile water before further use. For selective growth of transformants, HA agar (1% malt extract, 0.4% glucose, 0.4%

Cloning of frt1

Among the more than 6000 B. cinerea EST sequences which are deposited in the public data bases, two ESTs were identified that encoded partial amino acid sequences with substantial similarity to the fructose specific transporter FSY1 from S. pastorianus (Gonçalves et al., 2000). Based on these sequences, a 287 bp PCR fragment containing part of the coding region of the putative fructose transporter gene (frt1) of B. cinerea was labeled with digoxigenin and used as a probe for the screening of a

Discussion

In this paper, we describe the cloning and functional characterization of a new type of fructose-specific transporters from a filamentous fungus. Belonging to the sugar porter family of MFS transporters, it is structurally similar to fructose transporters of the yeasts S. pastorianus and Kluyveromyces lactis, and even more to predicted proteins from filamentous ascomycetes such as M. grisea, A. nidulans, and F. graminearum. Alignment of their sequences allowed us to define two highly conserved

Acknowledgments

We are grateful to Paul Tudzynski (University of Münster) and Jan van Kan (University of Wageningen), for providing materials and helpful advice. We thank E. Neuhaus and R.T. Voegele for critical reading of the manuscript. G.D. and this work were funded by a graduate program of the Deutsche Forschungsgemeinschaft (GRK 845/1).

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