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Phytopathologie, Department of Biology, University of Kaiserslautern, 67653 Kaiserslautern, Germany
Correspondence
Matthias Hahn
hahn{at}rhrk.uni-kl.de
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ABSTRACT |
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tps1 mutant was unable to synthesize trehalose, whereas the tre1 mutant showed elevated trehalose levels compared to the wild-type and was unable to mobilize trehalose during conidial germination. Both mutants showed normal vegetative growth and were not affected in plant pathogenicity. Growth of the tps1 mutant was more heat sensitive compared to the wild-type. Similarly, tps1 conidia showed a shorter survival under heat stress, and their viability at moderate temperatures was strongly reduced. In germinating wild-type conidia, rapid trehalose degradation occurred only when germination was induced in the presence of nutrients. In contrast, little trehalose breakdown was observed during germination on hydrophobic surfaces in water. Here, addition of cAMP to conidia induced trehalose mobilization and accelerated the germination process, probably by activation of TRE1. In accordance with these data, both mutants showed germination defects only in the presence of sugars but not on hydrophobic surfaces in the absence of nutrients. The data indicate that in B. cinerea trehalose serves as a stress protectant, and also as a significant but not essential carbon source for germination when external nutrients are low. In addition, evidence was obtained that trehalose 6-phosphate plays a role as a regulator of glycolysis during germination.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-D-glucopyranosyl (1,1)--D-glucopyranoside], a non-reducing disaccharide, is absent in vertebrates, but occurs in nearly all other organisms including bacteria, yeasts, filamentous fungi and plants (Elbein et al., 2003
). In addition to its role as a carbohydrate storage compound, trehalose represents an important stress protectant and is involved in metabolic signalling and regulation of carbohydrate metabolism (Hounsa et al., 1998; Arguelles et al., 2000). The protective properties of trehalose during heat, dehydration and oxidative stresses are explained by its ability to stabilize the native conformation of proteins and membranes, but the molecular basis for this is not well understood (Singer & Lindquist, 1998). Contradictory models have been proposed to explain the stabilizing effects of trehalose, either by replacement of water molecules from the surface of biomolecules, or by entrapment of residual water molecules close to the biomolecule (Thevelein, 1996; Pereira et al., 2004; Skibinsky et al., 2005).
In yeast and Aspergillus nidulans, trehalose synthesis occurs in two steps, catalysed by trehalose-6-phosphate synthase (TPS), which condenses UDP-glucose and glucose 6-phosphate, and by trehalose-6-phosphate phosphatase (Gancedo & Flores, 2004; Fillinger et al., 2001). Trehalose breakdown is catalysed by trehalases. Originally, two types of trehalases were described in fungi: so-called acid trehalases, with a low pH optimum, which are required for utilization of trehalose as a carbon source, and neutral trehalases, which mobilize intracellular trehalose (Thevelein, 1996; Amaral et al., 1997). Magnaporthe grisea, which does not possess an acid trehalase, contains a neutral trehalase (NTH1), and a new type of fungal trehalase (TRE1) that has properties of both neutral and acid trehalases (Foster et al., 2003). The phenotypes of mutants defective in trehalose metabolism have supported the role of trehalose as a protectant against heat and other stresses in yeasts and filamentous fungi (d'Enfert et al., 1999; Fillinger et al., 2001). In addition, trehalose has been shown to contribute to the energy requirements during conidial germination in A. nidulans (d'Enfert et al., 1999). In the plant-pathogenic fungus M. grisea, trehalose synthesis and metabolism have been found to be required at different stages of plant infection (Foster et al., 2003).
Botrytis cinerea, the causal agent of grey mould, infects more than 230 described host plant species (Jarvis, 1977). Its ubiquitous occurrence and its lifestyle as a facultative necrotrophic pathogen imply that it has to cope with a variety of biotic and abiotic stresses. In fact, its ability to produce and to withstand high concentrations of reactive oxygen species during pathogenesis has been well documented (Prins et al., 2000; Rolke et al., 2004). In addition, the grey mould fungus is equipped with a set of membrane transport proteins including several ABC and MFS transporters which confer resistance to a variety of toxic compounds (de Waard et al., 2006; Hayashi et al., 2002).
We have previously shown that germination of B. cinerea conidia occurs in response to either chemical or physical stimuli (Doehlemann et al., 2005, 2006). Germination in response to the presence of carbon sources (e.g. glucose) was found to be dependent on a signalling pathway involving the 3 subunit of a heterotrimeric G protein (BCG3), cAMP and the mitogen-activated protein (MAP) kinase BMP1. In contrast, germination in response to hydrophobic surfaces – in the absence of nutrients – was not controlled by BCG3 and cAMP, but by another signalling pathway that was completely dependent on BMP1. Germination on full media, which does not require any of the above-mentioned signalling components, might be controlled by a third, so far poorly characterized signalling pathway (Doehlemann et al., 2006).
In order to study the role of trehalose in B. cinerea, we have cloned and disrupted the genes encoding a neutral trehalase (Bctre1) and a trehalose-6-phosphate synthase (Bctps1). Characterization of the mutants and physiological studies revealed that trehalose is dispensable for pathogenesis, but important as a heat protectant. Furthermore, trehalose metabolism was shown to play a significant role during carbon source-induced germination, but not during germination induced on hydrophobic surfaces in the absence of external nutrients.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For selective growth of transformants, HA agar (1 % malt extract, 0.4 % glucose, 0.4 % yeast extract, 1.5 % agar, pH 5.5) supplemented with hygromycin (Duchefa; 100 µg ml–1) was used. After single-spore isolation, transformants were cultivated without selection on tomato malt agar.
Germination, plant infection and growth tests.
Conidial germination assays, penetration assays on onion epidermis layers and plant infection tests were performed as described by Doehlemann et al. (2006). Growth tests providing osmotic or oxidative stresses were performed on plates containing minimal medium (Gamborgs B5 basal salt mixture; Duchefa), 0.5 % (w/v) glucose and 1.5 % agar. To provide oxidative stress, 2–5 mM H2O2 was added. Alternatively, the herbicide paraquat (Gramoxone; Syngenta) was added in concentrations of 5–10 mM. Osmotic stresses were provided by adding NaCl (0.5–1.5 M) or sorbitol (0.5–1.0 M). To test the growth of B. cinerea on different carbon sources in minimal medium, glucose was replaced by equal concentrations of the tested carbon source (fructose, sucrose, trehalose, sodium acetate, glycerol, yeast extract). To study the growth behaviour at different temperatures, B. cinerea was grown on HA agar.
Cloning and disruption of the tps1 and tre1 genes from B. cinerea.
Starting out from EST sequences available for B. cinerea (tps1, AL110892; tre1, AL114821) and known sequences from the corresponding genes in Aspergillus nidulans, Metarhizium anisopliae, Neurospora crassa and Magnaporthe grisea, major regions of the tps1 and tre1 genes were amplified by PCR with combinations of gene-specific and degenerate primers (Table 1), and the products (tps1, 1567 bp; tre1, 2628 bp) were cloned into the vector pBS(+). For targeted disruption of the genes, a hygromycin resistance cassette was introduced by an inverse PCR strategy (see Fig. 2). For tps1 disruption, inverse PCR was performed using primers TPS1-8 and TPS1-9. The amplification product, containing pBS(+) plus 0.6 kb and 0.7 kb, respectively, of flanking tps1 sequences, was digested with NotI and ligated with a hygromycin resistance cassette which had been amplified from pLOB1 using primers HYG1 and HYG2 and digested with NotI (Doehlemann et al., 2006). From the resulting plasmid, the tps1 knock-out construct was amplified with PCR, using primers TPS1-KO1 and TPS1-KO2, to yield the linear DNA which was used to transform B. cinerea. For disruption of tre1, inverse PCR was performed using primers TRE1-KO3 and TRE1-KO4, resulting in an amplification product containing pBS(+) plus 0.55 kb and 0.5 kb, respectively, of flanking tre1 sequences. The product was digested with NotI and ligated with the hygromycin resistance cassette as described above. The knock-out construct was amplified from the resulting plasmid using primers TRE1-KO1 and TRE1-KO2 (Fig. 2).
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. Semi-quantitative RT-PCR was performed as described by Doehlemann et al. (2005), using the B. cinerea actin gene actA as a constitutive control. Protein sequences were aligned by using the CLUSTAL W algorithm (Thompson et al., 1994), with manual corrections at the N-termini. For creating the dendrogram, Align Plus 4 (Scientific & Educational Software) was used to perform exhaustive multi-way pairwise alignments of all sequences, using the BLOSUM62 scoring matrix, followed by progressive assembly of the alignments using the neighbour-joining method.
Measurement of intracellular trehalose in germinating conidia.
Conidia were harvested from 10-day-old tomato malt agar cultures of B. cinerea and washed as described by Doehlemann et al. (2006). A total of 107 conidia per experiment were used. They were germinated either in liquid HA medium [107 conidia (30 ml)–1], or in Gamborg B5 minimal medium supplemented with a carbon source on glass surfaces (105 conidia ml–1), or in water on polypropylene surfaces (105 conidia ml–1). After incubation, the germinating conidia were harvested from the surfaces with a cell scraper. Conidia were centrifuged at 3200 g and resuspended in 100 µl H2O. The suspension was boiled for 5 min to eliminate enzyme activity and to release soluble sugars and polyols. After centrifugation at 11 000 g for 5 min, the supernatant was collected. Two 20 µl aliquots of each sample were each added to 20 µl 0.1 M sodium citrate buffer. To one of the aliquots, 4 µl porcine kidney acid trehalase (Sigma) was added, and the samples were incubated overnight at 37 °C. The concentration of glucose in each sample was assayed photometrically with a glucose assay kit (Sigma).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Later, the complete tps1 genomic sequence was made available to us by CNRS-Genoscope, which has recently completed the B. cinerea genome project. The tps1 gene (accession no. DQ632610) contains a 1689 bp coding region, including two introns. The predicted TPS1 protein is 523 amino acids long, and has similarities of 78 and 74 % to TPS proteins from Magnaporthe grisea and Aspergillus nidulans, respectively. A sequence encoding a putative trehalose-6-phosphate phosphatase was also identified in the as yet not annotated B. cinerea genome, indicating that the trehalose synthesis pathway is identical to that in yeast and A. nidulans.
Searching the publicly accessible B. cinerea EST clones for trehalase sequences led to the identification of a clone encoding parts of a neutral trehalase (TRE1). A 3.4 kb DNA fragment covering the tre1 gene was amplified from genomic B. cinerea B05.10 DNA, based on sequence data available from the website of the Broad Institute (http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/home.html). The 2413 bp tre1 coding region contains four introns and encodes a predicted TRE1 protein with 727 amino acids. Sequence comparisons with other fungal trehalases revealed that TRE1 clusters with neutral trehalases from A. nidulans (TreB) and N. crassa (TreB), as well as with NTH1 from M. grisea (70–77 % similarity). Lower similarities (20–21 %) were found to the TRE1 trehalase of M. grisea and to predicted proteins from N. crassa and Gibberella zeae. A hardly detectable similarity exists between neutral trehalases and acid trehalases (Fig. 1; Amaral et al., 1997).
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). Nevertheless, on minimal medium containing 50 mM trehalose, B. cinerea's growth rate reached only about 20 % compared to that on other carbon sources such as glucose, fructose, sucrose and glycerol, indicating that trehalose is not a preferred carbon source of B. cinerea (not shown). To monitor the activity of tps1 and tre1, semi-quantitative RT-PCR experiments were performed. Similar transcript levels of both genes were found in ungerminated and germinating conidia (not shown).
Construction, growth and pathogenicity of tps1 and tre1 mutants
In order to examine the role of trehalose and trehalose mobilization in B. cinerea, disruption mutants of both tps1 and tre1 were created. The gene disruption strategies are shown in Fig. 2a, b). For both genes, three knock-out isolates (tps1 and tre1, respectively) were confirmed by PCR (Fig. 1c, d). Similar phenotypes for the knock-out isolates of each gene were observed in all experiments shown in this paper. As described below (see Fig. 4), the tps1 mutant contained only traces of trehalose, while increased trehalose levels compared to the wild-type were measured in the tre1 mutant. This confirmed that TPS1 is required for synthesis, and TRE1 for intracellular mobilization of trehalose. Both mutants showed normal vegetative growth on various full and minimal media. Sporulation of the tps1 mutant reached wild-type levels, whereas the tre1 mutant produced only about 40 % of the number of wild-type conidia on tomato malt agar. To check the pathogenic performance of the mutants, penetration assays on onion epidermis layers (not shown) and infection tests on detached tomato leaves were perfomed. Both mutants showed a similar virulence to the wild-type strain (Fig. 3).
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). As already described for A. nidulans and N. crassa conidia (d'Enfert & Fontaine, 1997), trehalose mobilization was observed significantly before germ tubes were formed, which did not occur until 2–3 h after addition of medium (not shown). In the tps1 mutant, trehalose was virtually absent in conidia, germlings and mycelium, proving that TPS1 is required for trehalose synthesis in B. cinerea (Fig. 4 and data not shown). In conidia of the tre1 mutant, the trehalose content was increased by 20 % as compared to the wild-type. In contrast to the wild-type, no mobilization of trehalose occurred during germination in complete medium (Fig. 4). These data indicate that TRE1 is responsible for trehalose degradation during germination of B. cinerea conidia.
When B. cinerea wild-type germlings were exposed to a temperature shift from 20 to 30 °C, the trehalose content rapidly increased about tenfold. In a heat-shocked tre1 mutant, trehalose accumulated to even higher levels than the wild-type, whereas the tps1 mutant did not accumulate any trehalose (Fig. 4). At 20 °C, mycelial growth on HA complete medium was identical in all strains. At 30 °C, however, different degrees of growth inhibition were observed: radial growth of the tps mutant was only about 50 % compared to the wild-type. Remarkably, the tre1 mutant showed a slightly, but significantly, faster growth rate than the wild-type under heat stress (Fig. 5). In contrast, treatments with oxidative (H2O2 or paraquat) or osmotic stresses (NaCl or sorbitol) did not reveal significant differences in mycelial growth between the wild-type and the mutant strains (data not shown).
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tps1 conidia were less able to survive heat stress, and lost viability more rapidly during heat treatment than wild-type conidia. In contrast, the heat tolerance of tre1 conidia was not significantly different from that of wild-type conidia (Fig. 6). In a TPS-deficient mutant of A. nidulans, conidia had a reduced viability during storage (Fillinger et al., 2001). A similar phenotype was observed for the tps1 mutant. After 2 weeks storage at 20 °C, the tps1 conidia had almost completely lost their viability, while wild-type conidia still showed a germination rate of about 85 % (Fig. 7b). Microscopic investigation revealed that 2-week-old tps1 conidia were severely affected in their cell integrity. After suspension in water, aggregation of the cytoplasm was observed in the majority of spores (data not shown). After treatment with 0.1 % Tween 20, most tps1 conidia were found to be collapsed whereas wild-type spores remained unaffected by this treatment (Fig. 7a). These findings strongly support the role of trehalose as a protective agent increasing the survival of conidia at both moderate and high temperatures.
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, rapid mobilization of trehalose occurred in conidia germinated in full medium. When germination was induced by 10 mM fructose on a glass surface, trehalose was also quickly degraded prior to visible germination. After 2 h, trehalose concentration had dropped to approximately 20 % of the concentration in ungerminated conida, and it stayed at this level during germ tube outgrowth. In contrast, spores that had germinated on a hydrophobic surface in water showed a very delayed trehalose breakdown. During the first 4 h, trehalose did not seem to be degraded at all. After 8 h, when 20 % of the conidia had germinated, the trehalose contents had decreased by only about 30 %, whereas at the same germination rate in fructose-supplemented medium the trehalose concentration had already reached its lowest level (Fig. 8). In yeast and A. nidulans, neutral trehalases are known to be activated by cAMP (d'Enfert et al., 1999; Amaral et al., 1997; de Almeida et al., 1997). In B. cinerea, however, cAMP was found to be involved only in carbon-source-induced germination, but not in germination on hydrophobic surfaces (Doehlemann et al., 2006). We therefore reasoned that TRE1 is not activated during germination in the absence of nutrients due to the lack of cAMP. To test this idea, wild-type conidia were germinated on a polypropylene surface in water, supplemented with the membrane-permeable cAMP derivative CPT-cAMP. Indeed, this treatment resulted in a more rapid mobilization of trehalose, concomitantly with a strongly accelerated germination (Fig. 8).
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tre1 and tps1 mutants show specific germination defectstre1 and tps1 mutants and the wild-type. In full medium, rapid germination of conidia occurred, with no difference between the wild-type and the mutants (data not shown). When conidia were germinated in minimal medium containing low amounts of sugars, clear differences between the strains were evident. In the presence of 10 mM glucose or fructose, the tre1 mutant showed a slightly delayed germination when compared to the wild-type (Fig. 9a). This delay was more pronounced in the presence of only 1 mM glucose or fructose; nevertheless, after 24 h, almost full germination of tre1 conidia had occurred (Fig. 9b). Since carbon-source-induced germination was found to be mediated by cAMP-dependent signalling (Doehlemann et al., 2006), these data are in agreement with the stimulating effect of cAMP on trehalose mobilization and germination of wild-type conidia on hydrophobic surfaces. Our data also indicate that trehalose mobilization accelerates germination at low external nutrient concentrations. When conidia were germinated on a hydrophobic surface in the absence of external nutrients (conditions that do not induce trehalose mobilization; cf. Fig. 8), both tre1 and tps1 mutants showed germination kinetics similar to the wild-type (Fig. 9c).
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tps1 conidia (Fig. 9d). This phenotype cannot be explained just by the lack of trehalose as an energy source, considering the comparatively minor germination defect of the tre1 mutant. Yeast and M. grisea mutants lacking TPS have been described to be seriously handicapped in their growth on glucose or fructose as sole carbon sources (Bell et al., 1998; Foster et al., 2003). This was explained by the lack of trehalose 6-phosphate (T6P) which acts as a negative regulator of hexokinase activity and which is required for the control of glycolysis (Thevelein & Hohmann, 1995; Bell et al., 1998; Foster et al., 2003). We did not observe any defect in mycelial growth of the tps1 mutant in minimal media containing glucose or fructose. Nevertheless, germination of the tps1 mutant in the presence of galactose (which is phosphorylated in the cell not by hexokinase but by galactokinase) was similar to the wild-type germination after 24 h (Fig. 9d). We therefore suggest that the germination defect of the tps1 mutant in the presence of glucose or fructose is mainly due to the lack of T6P as a regulator of carbohydrate metabolism. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Our results indicate an important role of trehalose as a protectant concerning both heat stress tolerance and general conidial survival. Trehalose metabolism plays a role in conidial germination in two aspects: first, trehalose provides a significant source of energy for the germinating spore when external nutrient sources are limiting. Second, the trehalose precursor T6P seems to play a role in regulation of the glycolytic flux, which is also important for induction of germination.
To analyse the role of trehalose synthesis in B. cinerea, mutants defective in TPS and a neutral trehalase were created. The absence of trehalose from tps1 conidia confirmed that TPS is essential for trehalose synthesis. The phenotype of the tre1 mutant showed that the neutral trehalase TRE1 is required for breakdown of intracellular trehalose, similar to the situation in other fungi.
Regarding their growth phenotype, both mutants showed no difference from the wild-type when growing on minimal or full media, except for a moderate reduction of sporulation efficiency of the tre1 mutant. Disruption of the neutral trehalase led also to reduced sporulation in M. grisea but not in A. nidulans (Foster et al., 2003; d'Enfert et al., 1999). Similar to B. cinerea, a TPS-deficient mutant of A. nidulans showed normal vegetative growth (Fillinger et al., 2001). In contrast, TPS mutants of yeast and M. grisea were unable to grow in minimal media containing glucose or fructose (Foster et al., 2003; Thevelein & Hohmann, 1995). This was explained by the absence of T6P and will be discussed further below.
In the rice blast pathogen M. grisea, TPS-deficient mutants did not synthesize trehalose and formed appressoria which were defective in penetration, whereas mutants defective in the neutral trehalase NTH1 showed successful penetration but a decreased ability to colonize plant tissue (Foster et al., 2003). In contrast, neither tps1 nor tre1 mutants of B. cinerea showed any defects in pathogenicity. The turgor pressure in the morphologically often poorly differentiated appressoria of B. cinerea is unknown, but probably much lower than the pressure in mature melanized appressoria of M. grisea, which has been estimated to be up to 80 bar (de Jong et al., 1997). Thus, in B. cinerea germlings, the mobilization of trehalose does not seem to be essential for subsequent plant cell penetration.
As in other fungi, trehalose was found to be an important heat protectant in B. cinerea. After heat-shock treatment, the trehalose concentration in wild-type germlings quickly increased almost tenfold, to about twofold storage levels of conidia. Growth of the tps1 mutant was much more inhibited than that of the wild-type at 30 °C. Interestingly, the tre1 mutant was found to be more heat resistant than the wild-type, showing about 20 % increase in radial growth, presumably due to its increased trehalose levels. Protection by trehalose was also evident in the resting stage of B. cinerea: survival of tps1 conidia at 45 °C was lowered, with a half-life reduction of about 25 % compared to the wild-type and the tre1 mutant. Furthermore, tps1 conidia displayed a drastically reduced survival at normal temperature. In A. nidulans, lack of trehalose due to disruption of the tpsA gene also led to increased sensitivity of growth to high temperatures as well as oxidative stress, and also to a rapid loss of viability of conidia at ambient temperature. However, tpsA conidia showed similar sensitivity to heat treatment as wild-type conidia (Fillinger et al., 2001). No data regarding heat stress tolerance have been reported for trehalose-deficient mutants in M. grisea (Foster et al., 2003). In baker's yeast, trehalose has also been described to contribute to osmotic stress tolerance (Hounsa et al., 1998). In contrast, we did not find evidence for this in B. cinerea, nor for its role in protection against other stress factors. Thus, trehalose appears to play similar but not identical roles in cellular protection in different fungi.
Substantial amounts of trehalose have been found in conidia of various fungi. For instance, N. crassa and A. nidulans contain up to 10 and 4 % trehalose (per fresh weight), respectively (Schmitt & Brody, 1976; Ruijter et al., 2003). The trehalose content of B. cinerea conidia was estimated to be about 1.5 %. Trehalose breakdown has been described in A. nidulans and N. crassa as the first detectable biochemical event following germination induction, occurring much earlier than germ tube appearance (d'Enfert, 1997). Similarly, in B. cinerea conidia which were induced to germinate in the presence of full media or 10 mM fructose, trehalose degradation started to occur after 30 min, while germination was not observed until after 3–4 h. In contrast, trehalose degradation was strongly delayed and did not precede germination in conidia that were induced to germinate on a hydrophobic surface, in the absence of nutrients. However, addition of cAMP to these conidia resulted in significant mobilization of trehalose and accelerated germination. In Saccharomyces cerevisiae, trehalose breakdown requires activation of the neutral trehalase, mediated by cAMP-dependent protein kinase A and possibly Ca2+ (Souza et al., 2002; d'Enfert 1997; de Almeida et al., 1997). The inability of adenylate-cyclase-deficient N. crassa mutants to mobilize trehalose during germination also indicates that cAMP signalling is required for activation of neutral trehalase (de Pinho et al., 2001). We therefore hypothesize that TRE1 activity in B. cinerea is dependent on cAMP signalling as well. Further support for this came from the observation that a B. cinerea bcg3 mutant, which is defective in cAMP signalling, does not show any degradation of trehalose during germination (Doehlemann et al., 2006; data not shown). Similarly, it has been demonstrated recently that G-protein-mediated cAMP signalling, activated by hexoses, is necessary for trehalase activity in A. nidulans (Lafon et al., 2005). This is supported by the fact that the predicted TRE1 protein contains the typical N-terminal RRXS motive, which has been described as a cAMP-dependent protein kinase phosphorylation site (Kopp et al., 1993; Rittenhouse et al., 1986). Other typical features such as a Ca2+-binding domain are also conserved in the TRE1 sequence (Amaral et al., 1997).
The rapid mobilization of trehalose in germinating conidia indicated that it is an important energy source for germination. A. nidulans conidia lacking the neutral trehalase TreB showed a delay in germination when incubated under carbon limitation (d'Enfert et al., 1999). The germination phenotype of the B. cinerea mutants was found to be dependent on the signal which induced germination. Both mutants germinated in a similar way to the wild-type in rich media or on hydrophobic surfaces in water. However, a delayed germination was observed for the B. cinerea tre1 mutant in the presence of sugars at low concentrations, suggesting an energetic role of trehalose in the initial phase of germination. An even stronger germination defect was observed in the presence of glucose or fructose in the tps1 mutant. This was different from A. nidulans and M. grisea TPS mutants, which were not reported to be defective in germination (Fillinger et al., 2001; Foster et al., 2003). On the other hand, disruption of TPS activity in M. grisea resulted in the inhibition of growth on several carbon sources, similar to what has been described for yeast (Thevelein & Hohmann, 1995). This growth disturbance of TPS-deficient mutants has been explained by the deregulation of glycolytic flux due to the absence of T6P (Gancedo & Flores, 2004; Eastmond & Graham, 2003; Thevelein, 1996). A similar explanation for the impaired germination of the tps1 mutant in the presence of fructose or glucose was supported by the observation of a high sensitivity of B. cinerea hexokinase to T6P. In extracts prepared from a B. cinerea mutant expressing only hexokinase but not glucokinase activity, addition of 1 mM T6P resulted in 90 % reduction of hexose phosphorylation acivity (O. Rui & M. Hahn, unpublished). This strongly supports the idea that glycolytic deregulation in the tps1 mutant disturbs germination in the presence of glucose or fructose, but not with galactose.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Arguelles, J. C. (2000). Physiological roles of trehalose in bacteria and yeasts: a comparative analysis. Arch Microbiol 174, 217–224.[CrossRef][Medline]
Bell, W., Sun, W., Hohmann, S., Wera, S., Reinders, A., De Virgilio, C., Wiemken, A. & Thevelein, J. M. (1998). Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex. J Biol Chem 273, 33311–33319.
de Almeida, F. M., Lucio, A. K., Polizeli, M. L., Jorge, J. A. & Terenzi, H. F. (1997). Function and regulation of the acid and neutral trehalases of Mucor rouxii. FEMS Microbiol Lett 155, 73–77.[Medline]
de Jong, J. C., McCormack, B. J., Smirnoff, N. & Talbot, N. J. (1997). Glycerol generates turgor in rice blast. Nature 389, 244–245.[CrossRef]
d'Enfert, C. (1997). Fungal spore germination: insights from the molecular genetics of Aspergillus nidulans and Neurospora crassa. Fungal Genet Biol 21, 163–172.[CrossRef]
d'Enfert, C. & Fontaine, T. (1997). Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth and trehalose. Mol Microbiol 24, 203–216.[CrossRef][Medline]
d'Enfert, C., Bonini, B. M., Zapella, P. D. A., Fontaine, T., da Silva, A. M. & Terenzi, H. F. (1999). Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa. Mol Microbiol 32, 471–483.[CrossRef][Medline]
de Pinho, C. A., de Lourdes, M., Polizeli, T. M., Jorge, J. A. & Terenzi, H. F. (2001). Mobilisation of trehalose in mutants of the cyclic AMP signalling pathway, cr-1 (CRISP-1) and mcb (microcycle conidiation), of Neurospora crassa. FEMS Microbiol Lett 199, 85–89.[Medline]
de Waard, M. A., Andrade, A. C., Hayashi, K., Schoonbeek, H., Stergiopoulus, I. & Zwiers, L.-H. (2006). Impact of fungal transporters on fungicide sensitivity, multidrug resistance and virulence. Pest Man Sci 62, 195–207.[CrossRef][Medline]
Doehlemann, G., Molitor, F. & Hahn, M. (2005). Molecular and functional characterization of a fructose specific transporter from the gray mould fungus Botrytis cinerea. Fungal Genet Biol 42, 601–610.[CrossRef][Medline]
Doehlemann, G., Berndt, P. & Hahn, M. (2006). Different signalling pathways involving a G protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia. Mol Microbiol 59, 821–835.[CrossRef][Medline]
Eastmond, P. J. & Graham, A. (2003). Trehalose metabolism: a regulatory role for trehalose-6-phosphate? Curr Opin Plant Biol 6, 231–235.[CrossRef][Medline]
Elbein, A. D., Pan, Y. T., Pastuszak, I. & Carrol, D. (2003). New insights on trehalose: a multifunctional molecule. Glycobiology 12, 17R–27R.
Fillinger, S., Chaveroche, M.-K., van Dijck, P., de Vries, R., Ruijter, G., Thevelein, J. & d'Enfert, C. (2001). Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans. Microbiology 147, 1851–1862.
Foster, A. J., Jenkinson, J. M. & Talbot, N. J. (2003). Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J 22, 225–235.[CrossRef][Medline]
Gancedo, C. & Flores, C. (2004). The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi. FEMS Yeast Res 4, 351–359.[CrossRef][Medline]
Hayashi, K., Schoonbeck, H.-J. & de Waard, M. A. (2002). Bcmfs1, a novel major facilitator superfamily transporter from Botryits cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides. Appl Environ Microbiol 68, 4996–5004.
Hounsa, C. G., Brandt, E. V., Thevelein, J., Hohmann, S. & Prior, B. A. (1998). Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress. Microbiology 144, 671–680.
Jarvis, W. R. (1977). Botryotinia and Botrytis species. Taxonomy and pathogenicity. A guide to the literature. Monograph no. 14. Ottawa: Research Branch, Canada Department of Agriculture.
Kopp, M., Müller, H. & Holzer, H. (1993). Molecular analysis of the neutral trehalase gene from Saccharomyces cerevisiae. J Biol Chem 268, 4766–4774.
Lafon, A., Seo, J. A., Han, K. H., Yu, J. H. & d'Enfert, C. (2005). The heterotrimeric G-protein GanB ()-SfaD (
)-GpgA(
) is a carbon source sensor involved in early cAMP-dependent germination in Aspergillus nidulans. Genetics 171, 71–80.
Möller, E. M., Bahnweg, G., Sandermann, H. & Geiger, H. H. (1992). A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissue. Nucleic Acids Res 20, 6115–6116.
Pereira, C., Lins, R. D., Chandrasekhar, I., Freitas, L. C. & Hünenberger, P. H. (2004). Interaction of the disaccharide trehalose with a phospholipid bilayer: a molecular dynamics study. Biophys J 86, 2273–2285.[Medline]
Prins, T. W., Tudzynski, P., von Tiedemann, A., Tudzynski, B., ten Have, A., Hansen, M., Tenberge, K. & van Kan, J. A. L. (2000). Infection strategies of Botrytis cinerea and related necrotrophic pathogens. In Fungal Pathology, pp. 33–64. Edited by J. W. Kronstad. Dordrecht: Kluwer Academic Press.
Reis, H., Pfiffi, S. & Hahn, M. (2005). Molecular and functional characterization of a secreted lipase from Botrytis cinerea. Mol Plant Pathol 6, 257–267.[CrossRef]
Rittenhouse, J., Harrsch, P. B., Kim, J. N. & Markus, F. (1986). Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. J Biol Chem 261, 3939–3943.
Rolke, Y., Liu, S., Quidde, T. & 7 other authors (2004). Functional analysis of H2O2-generating systems in Botrytis cinerea: the major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol Plant Pathol 5, 17–27.[CrossRef]
Ruijter, G. J. G., Bax, M., Patel, H., Flitter, S. J., van de Vondervoort, P. J. I., de Vries, R. P., van Kuyk, P. A. & Visser, J. (2003). Mannitol is required for stress tolerance in Aspergillus niger conidiospores. Eukaryot Cell 2, 690–698.
Schmitt, J. C. & Brody, S. (1976). Biochemical genetics of Neurospora crassa conidial germination. Bacteriol Rev 40, 1–41.
Skibinsky, A., Venable, R. M. & Pastor, R. W. (2005). A molecular dynamics study of the response of lipid bilayers and monolayers to trehalose. Biophys J 89, 4111–4121.[Medline]
Singer, M. A. & Lindquist, S. (1998). Thermotolerance in Saccharomyces cerevisiae: the yin and yang of trehalose. Trends Biotechnol 16, 460–468.[CrossRef][Medline]
Souza, A. C., De Mesquita, J. F., Panek, A. D., Silva, J. T. & Paschoalin, V. M. F. (2002). Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae. Braz J Med Biol Res 35, 11–16.[Medline]
Thevelein, J. M. (1996). Regulation of trehalose metabolism and its relevance to cell growth and function. In The Mycota, vol. 3, Biochemistry and Molecular Biology, pp. 395–420. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Thevelein, J. M. & Hohmann, S. (1995). Trehalose synthase: guard to the gate of glycolysis in yeast? Trends Biol Sci 20, 3–9.[CrossRef]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Received 7 April 2006;
revised 18 May 2006;
accepted 23 May 2006.
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, Wild-type; 
, 
, 
), on polypropylene in water (
), and on polypropylene in water supplemented with CPT-cAMP (100 µg ml–1; 
). The corresponding germination rates are indicated with solid symbols. Values are the means±SE of two independent experiments with at least two replicates.