Microbial Ecology

Microbial Ecology Interactions Between Yeasts and Grapevines: Filamentous Growth, Endopolygalacturonase and Phytopathogenicity of Colonizing Yeasts Sabine Gognies1, Essaı¨d Ait Barka2, Ange´lique Gainvors-Claisse1 and Abdel Belarbi1 (1) Laboratoire de Microbiologie Geńe´rale et Molećulaire, Universite´ de Reims, UFR Sciences, URVVC, UPRES EA 2069, B.P. 1039, 51687 Reims Cedex 2, France (2) Laboratoire de Stress, Defeńses et Reproduction des Plantes, Universite´ de Reims, UFR Sciences, URVVC, UPRES EA 2069, B.P. 1039, 51687 Reims Cedex 2, France Received: 20 May 2005 / Accepted: 7 September 2005 / Online publication: 11 January 2006

Abstract

It has been clearly established that phytopathogenic fungi, bacteria, and viruses exert biotic stresses on plants. Much less is known, however, about the interactions between enological species of yeast and their host plants. In a previous study, we described how Saccharomyces cerevisiae, the most common enological yeast, can act as a grapevine (Vitis vinifera L.) pathogen, causing growth retardation or plant death. In the present in vitro study on 11 strains of yeast belonging to different genera, which often occur on the surfaces of vineyard grapes and V. vinifera, a link was found to exist between strain phytopathogenecity and pseudohyphal growth habits and/or endopolygalacturonase activity. The results obtained here are consistent with earlier findings showing that the phytopathogenicity of yeast strains depends on the filamentous growth process, and show that endopolygalacturonase alone is not responsible for the invasion of plants tissues. The mechanisms observed here may be of significant ecological importance and may help to explain the long periods of yeast survival found to occur in vineyards.

Introduction

Saccharomyces cerevisiae is by far the best-known yeast strain colonizing ripe fruits under natural conditions. The natural pattern of distribution of the populations of this vineyard yeast has not yet been clearly established, although many authors have stated that this species Correspondence to: Abdel Belarbi; E-mail: [email protected] DOI: 10.1007/s00248-005-0098-y

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forms extremely small populations that have rarely been isolated from intact berries [25, 27]. Other authors have suggested that S. cerevisiae coexists initially with many other yeasts on the surface of grape berries, and becomes more predominant as the fermentation proceeds [31, 36]. These other yeasts belong to various genera: Hanseniaspora, Kloeckera, Metschnikowia, Pichia, Brettanomyces, Kluyveromyces, Cryptococcus, Schizosaccharomyces, Debaryomyces, and Rhodotorula. An ecological study has shown that these yeast flora remain unchanged during successive years in the same vineyard, but vary from one region to another [10]. Gimeno et al. have reported that some S. cerevisiae can show a markedly different pattern of cell and colony morphology. In the diploid S. cerevisiae, for instance, nitrogen starvation causes a transition to occur from vegetative growth to pseudohyphal growth. During this process, the cells become elongated, and at the same time, a unipolar budding process occurs, and unseparated buds produce chains of cells known as pseudohyphae [13]. On given solid media under depleted glucose conditions, some haploid strains of S. cerevisiae also undergo morphological changes [5]. This process has been called invasive growth because the filaments penetrate into the agar below the colony [34]. Despite this definition, the authors suggested that some diploid cells may be invasive and that this process may be more marked than in the case of diploid than haploid cells [34]. We therefore propose to restrict the term pseudohyphal growth to the context of microscopic observations on either haploid or diploid cells, whereas invasive growth is more appropriate when dealing with macroscopic observations carried out after rinsing colonies with distilled water.

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Endopolygalacturonases, which belong to the polysaccharidase family, are important pathogenic factors, not only because they are involved in cell wall degradation, as plant cell walls are mainly composed of polysaccharides, but also because they indirectly elicit plant defense reactions via the oligosaccharides they release [4]. Some studies have shown that pathogenicity on plants or animals of fungal species depends on the filamentous process [1, 35], because the virulence of fungal pathogens depends mainly on the ability of these pathogens to undergo the dimorphic transition process. Recent studies on pseudohyphal differentiation in S. cerevisiae have provided substantial insights into the molecular mechanisms governing filamentous growth, which are also to be found in many other fungi [2, 12]. Many genes have been found to be involved in the control of pseudohyphal growth, the direct and indirect effects of which are difficult to distinguish [24, 26]. Because some of these genes induce the filamentation process, we suggested that they might somehow contribute to the pathogenecity of the fungus. FLO11 and PGL1 were the first genes of this kind thought to possibly play a downstream role in the cascade controlling filamentous growth [14, 20]. We recently reported on the potential virulence of S. cerevisiae toward Vitis vinifera [14, 15] and established that yeast strains showing filamentous growth as well as endoPG activity triggered necrosis of the host plantlets, whereas yeast devoid of both endoPG activity and pseudohyphal growth did not affect the plantlet viability. These observations were confirmed by the use of isogenic S. cerevisiae strains [14]. In addition, filamentous growth may be controlled by the PGL1 gene [14]. Because filamentous growth and endopolygalacturonase activity contribute so importantly to the penetration of V. vinifera by S. cerevisiae, it was proposed to study these two processes in other yeasts commonly occurring on grapes reported in Table 1. The aim of the present study was therefore to show the importance of each process (endopolygalacturonase activity and pseudohyphal development) in the phytopathogenicity of these yeast species toward V. vinifera. Material and Methods

Diseasefree plantlets of V. vinifera L. belonging to the cultivar BChardonnay^ were obtained by growing nodal explants on Murashige and Skoog medium in 25-mm test tubes [29]. Plantlets were grown under 200 mE m s white fluorescent light with a 16/8-h photoperiod and a constant day/night temperature of 25-C. Plant Material and in Vitro Growth Conditions.

Microorganisms, Growth Media, and Conditions. The yeast strains used in this study are described in Table 1. Some of these strains were certified strains obtained from

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the Collections at the Pasteur Institute (CIP) and the American Type Culture Collection (ATCC). The S. cerevisiae (S.c) SCPP strain and the S.c DJI 10 strain were characterized using biochemical, molecular biology, and genetic methods in our laboratory. The microorganisms were maintained on YPD medium (1% yeast extract, 2% bactopeptone, 2% dextrose, 2% bacto agar). Nitrogenlimiting (SLAD) medium [13] and amended-pectin medium [14] were used to test the ability of strains to undergo the pseudohyphal growth and invasive processes. Detection of Endopolygalacturonase Activity. Cell cultures of the various yeast strains in concentrations of 105 cells mL–1 were dropped onto pectin-solid medium (1% pectin from apples, degree of esterification G5% (Sigma, St Quentin Fallavier, France), 0.67% yeast nitrogen base, 1% dextrose, 0.6% agarose, and 50 mM phosphate buffer, pH 5.5). After being incubated for 3 days at 30-C, the polygalacturonase activity was monitored on agarose plates using ruthenium red solution (0.1%) [11, 26]. Plates were immersed in 0.2% NaOH solution for 2 min to increase the staining contrast. Photomicroscopy. Whole colony photographs were taken directly on agar plates with an optical microscope (Nikon, Tokyo, Japan) fitted with a 35-mm camera (Sony, Tokyo, Japan). Single-cell pictures were taken with an optical microscope (Nikon) at 400 magnification. Plant Inoculation. The yeast strains were collected by centrifugation (3000 g, 15 min) and washed twice with phosphate-buffered saline (PBS). The pellets were suspended in PBS, the yeast concentration was adjusted

Table 1. Yeast strains used in this study

Strains

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Saccharomyces cerevisiae X2180-1B MATa Saccharomyces cerevisiae S1278-B Saccharomyces cerevisiae strain SCPP Saccharomyces cerevisiae strain DJI10 Schizosaccharomyces pombe Candida tropicalis Debaryomyces hansenii Pichia membranifaciens Saccharomyces pastorianus Kloeckera apiculata Saccharomyces cerevisiae var. ellipsoideus

ATCC26787 ATCC42800 Wild-type strain isolated in our laboratory [10] Wild-type strain isolated in our laboratory [14] CIP1402-82 CIP1035-71 CIP641-65 CIP631-65 CIP2046-92/ATCC9080 CIP893-65 CIP635-66/ATCC16664

CIP: Pasteur Institute Collection; ATCC: American Type Culture Collection.

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Table 2. Study of filamentous characteristics and endoPG activity in the 11 yeast strains tested on SAD and amended-pectin media

Invasion Strains Saccharomyces cerevisiae X2180-1B MATa Saccharomyces cerevisiae ~1278-B Saccharomyces cerevisiae strain SCPP Saccharomyces cerevisiae strain DJI10 Schizosaccharomyces pombe Candida tropicalis Debaryomyces hansenii Pichia membranifaciens Saccharomyces pastorianus Kloeckera apiculata Saccharomyces cerevisiae var. ellipsoideus

SLAD medium _ _ + + _ T T + T + _

Pseudohyphae growth

Pectin medium _

PG activity _

T + + _

T + + _ _ _

T _ + T + T

T _ _ +

SLAD medium _ _ + + _ _ _ + _ + _

Pectin medium _ T + + _ _ _ + T + _

–: No invasion or no pseudohyphae or no endoPG activity; +: invasion or pseudohyphae or endoPG activity; T: slight invasion or more elongated cells or basal endoPG activity.

Figure 1. Some cell culture views: images on pectin medium after 6 days. (A) Growth, (B) invasive growth, (C) microscopic observations. Kloeckera apiculata and Saccharomyces cerevisiae DJI10 strain underwent a dimorphic switch and differentiated from the unicellular form. S. cerevisiae var. ellipsoideus and S. cerevisiae X2180-1B strains did not switch in this way to the filamentous form.

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to 103 or 106 cells mL–1, and the suspensions were used as an inoculum [14, 15]. The roots of 2-week-old plantlets were immersed in the inoculum for 1 min and blotted with sterile filter paper, and the plantlets were transplanted into culture tubes. Noninoculated controls were dipped in PBS only [14, 15]. Inoculated plantlets during 3 weeks were stored in a growth chamber as previously described [14, 15]. Six plantlets were used per strain and per cell concentrations. Microscopic preparations were performed as described previously by Gognies et al. [15]. Briefly, internodal sections were cut into 1-mm pieces from six plantlets treated as described above. The samples were immersed in cold fixative solution containing 8% glutaraldehyde and 2% paraformaldehyde in 0.2 M potassium buffer (pH 7.24), vacuum-filtered for 20 min, and immersed in fresh fixative solution for 20 h. Samples were subsequently washed with 0.2 M potassium buffer (pH 7.24), postfixed in 2% osmium tetroxide prepared in the same buffer, and dehydrated in a graded ethanol series. The specimens were then washed with an acetone series and embedded in Araldite (Fluka, St Quentin Fallavier, France). Sections were stained with bromophenol blue and examined under an optical microscope (Olympus model BH-2, Japan). Microscopic Preparation.


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of this study showed that there were no differences between the invasive behavior of strains growing on SLAD medium and that of the strains inoculated on the pectin-amended one. We also determined the endopolygalacturonase activity occurring in all the yeast strains. Only the S.c SCPP strain, S.c DJI10 strain, and S.c var. ellipsoideus strains were able to produce this activity (Table 2). Involvement of Filamentous Growth and/or Endopolygalacturonase Activity in the Phytopathogenic ProTwo strains were selected and tested to cess.

establish whether pseudohyphal growth and/or endopolygalacturonase activity are involved in the phytopathogenic process, namely S. cerevisiae ancient terminology

Results Correlations Between Filamentous Development and Some yeast species Endopolygalacturonase Activity.

showed pseudohyphal development and/or invasive growth, depending on the medium used. In strains grown on SLAD medium at 30-C for 6 days, for example [Saccharomyces cerevisiae (S.c) SCPP strain, S.c DJI10 strain, Pichia membranifaciens, CIP631-65, and Kloeckera apiculata CIP893-65), invasive behavior occurred, whereas in other strains (Candida tropicalis CIP1035-71, Debaryomyces hansenii CIP 641-65, and S. pastorianus CIP2046-92/ATCC9080], only slightly invasive behavior or none at all (S. cerevisiae var. ellipsoideus CIP635-66/ ATCC16664, S.c X2180-1B MATa ATCC26787, S.c S1278B ATCC42800, and Schizosaccharomyces pombe CIP1402-82) was detected (Table 2). In all the strains showing strong invasive growth (Fig. 1B), pseudohyphae were present (Fig. 1C). Pectins, which are one of the main components of the plant cell wall, provide a substrate for endoPG. Because starch-based medium induces pseudohyphal growth as well as invasive growth [19, 20], the ability of the various strains studied to invade the agar plates and to switch from the unicellular to filamentous form were tested on pectin amended medium (Table 2). The results

Figure 2. Effects after 3 weeks of yeast inoculum exposure on 2-week-old Vitis vinifera L. plantlets, using (A) Kloeckera apibulata and (B) Saccharomyces cerevisiae var. ellipsoideus. 1: Inoculation at – a concentration of 103 cells mL 1; 2: inoculation at a concentration –1 6 of 10 cells mL ; 3: without any inoculation (control). Arrows indicate necrosis of the leaf surface.

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[32] S.c var. ellipsoideus, which showed endoPG activity but no pseudohyphal growth, and K. apiculata, which was found to be devoid of endoPG activity but to have acquired the pseudohyphal form. The phytopathogenic process was assessed in 2week-old plantlets inoculated with either of the above two strains at two concentrations (103 and 106 cells mL–1). Results show that the presence of yeasts in the vicinity of the plantlets either delayed the growth or induced necrosis of the plantlets, or both, depending on the yeast strain used. To demonstrate whether K. apiculata can survive inside plantlets, inoculated plants were taken out of their culture medium. The upper part of the stem was cut and the surface was washed and blotted onto sterile filter paper. Stems were then cut into small sections and placed on Petri dishes containing YPD medium. After 2 days, sections from inoculated plantlets exhibited yeast growth around the sectioned stems, proving that yeasts were indeed alive inside the plantlet’s stems (data not shown). We have previously performed this type of experiment to demonstrate the presence of S. cerevisiae inside the plantlets [14, 15]. With K. apiculata, regardless of the concentration, an obvious delay in growth was observed in plantlets after 3 weeks of exposure, which did not occur in the untreated control plants (Fig. 2A1–3). No necrosis was

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observed under these conditions. On the other hand, when plantlets were inoculated with S.c var. ellipsoideus, delayed plant growth was also observed (Fig. 2B1–3) and necrosis occurred, in plantlets treated with 103 cells mL–1 (Fig. 2B1), and even more conspicuously in those exposed to a concentration of 106 cells mL–1 (Fig. 2B2). The incidence of necrosis ranged between 64% and 75% in the case of the former concentration level and reached almost 98% in that of the latter. The growth delay can be explained by the presence of yeast in the rhizosphere, which prevents contact from occurring between tissues and medium and leads to the starvation of the plant tissues. We suppose that yeasts form a physical barrier between the root and the medium, prevent the nutrients uptake by the plantlets and therefore leading to a growth delay of them. To determine whether the yeasts had penetrated into the plantlets within 3 weeks, histological studies were performed. Cross-sections of plantlets inoculated with K. apiculata looked quite healthy, resembling the control material (Fig. 3C), when the yeast was applied at low concentrations (Fig. 3A1). However, when applied at high concentrations, K. apiculata was observed within the tissues (Fig. 3A2). The tissues of plantlets inoculated with low concentrations of S.c var. ellipsoideus were only

Figure 3. Microscopic histological observations on cross-sections of Vitis vinifera L. plantlets exposed to: (A) Kloeckera apiculata strain at – a concentration of 103 cells mL 1 –1 6 (1) or 10 cells mL (2); (B) Saccharomyces cerevisiae var. ellipsoideus strain at a – concentration of 103 cells mL 1L –1 6 (1) or 10 cells mL (2); (C) plantlets without any inoculation (control material). In plants exposed to the yeast Kloeckera apiculata, the arrows show the presence of the yeast within the stem tissues (A2), and in those exposed to Saccharomyces cerevisiae var. ellipsoideus, the arrows indicate the disorganized plantlet tissues (B1–2). Scale bar = 20 mm.

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slightly disorganized, as shown in Fig. 3B1. Maximum disorganization occurred when this yeast was applied at high concentrations (Fig. 3B2). No yeast was detected within the tissues, however, regardless of the level of S.c var. ellipsoideus to which they were exposed (Fig. 3B1–2).

Discussion

In this work, yeast strains were tested to determine their ability to acquire the invasive phenotype (macroscopic observations) and/or to develop pseudohyphae (microscopic observations) on SLAD medium, which has been reported to promote the formation of pseudohyphae in many S. cerevisiae strains [13]. Yeast strains showed similar patterns of development to those described here when grown on pectin-amended medium. This finding supports the previous suggestion that pseudohyphal differentiation occurs in response to specific nutrients [5, 7, 19, 21,37] and not only in response to nitrogen starvation. It is worth discussing the case of K. apiculata, which has been reported by Barnett et al. [3] to form no pseudohyphae on a medium described by Kurtzman and Fell [18]. However, under our laboratory conditions, K. apiculata developed pseudohyphae on the amendedpectin medium used to test whether pseudohyphal growth and invasive processes occur in yeast strains (Fig. 1C). This finding confirms the importance of the culture medium used to induce pseudohyphal growth. It would therefore be interesting to determine whether any other strains that have not been found so far to develop pseudohyphae may in fact do so if they are exposed to more appropriate media. To successfully attack a host cell, a pathogen must cross the outer barrier of the plant cell, which is mainly composed of polysaccharides. To overcome this obstacle, pathogens induce the degradation of the cell wall via a battery of secreted polysaccharidases such as endopolygalacturonase. We previously reported that endoPG activity was correlated with pseudohyphal growth or invasive growth in S. cerevisiae strains [14, 15]. However, this result was not obtained in the case of the strains tested in the present study. This indicates that these two processes were not in fact necessarily linked to endoPG activity, because some yeast strains acquire the ability to form filaments via other mechanisms. Many data have been published that support this idea, as the pseudohyphal growth of S. cerevisiae requires the cooperation of diverse signaling pathways such as a MAP kinase cascade and a cAMP-dependent pathway [2, 8, 22, 23]. The present microscopic observations showed that K. apiculata, a yeast capable of acquiring the filamentous form but devoid of endoPG activity, was able to

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penetrate plantlet tissues but did not induce necrosis of the host plantlets. However, S.c var. ellipsoideus (a yeast strain not capable of filamentous growth but showing endoPG activity) did not enter the plantlet tissues but induced their necrosis. Maximum structural disorganization was observed when S.c var. ellipsoideus was applied at high concentrations. Based on these results, it can be concluded that filamentous differentiation is necessary for yeasts to be able to enter plant tissues. In yeasts capable of filamentous growth, the presence of the endoPG enzyme enhances the ability to invade the host tissues. When endoPG activity occurs without the occurrence of the filamentation process, endoPG activity induces the maceration of plantlet tissues [6, 30], as we observed in the case of S.c var. ellipsoideus. Based on the results of the present study, pseudohyphal growth seems to be necessary for these fungi to colonize plant tissues, and endoPG is certainly not the only factor involved in this particular growth. The latter finding is consistent with data obtained on S. cerevisiae by Madhani et al. [24] and Mosch and Fink [28] suggesting that other genes may affect pseudohyphal growth. In our study, we have used only 2 strains. Thus we are aware that not enough yeast strains (possessing the same characteristics as K. apiculata or S.c var. ellipsoideus strains), have been studied to confirm that filamentous growth is an important parameter in phytopathogenicity. Moreover, others parameters occur in phytopathogenicity [17], and more studies will be able to give us more information. Pretorius [31] have described the coexistence of yeasts in vineyards depending on many chemical, physical, and biotic factors. The available data suggest that fermentative yeast species occur in extremely small populations on healthy, undamaged grapes, but they have only rarely been isolated from intact berries and vineyard soils [25, 27]. Pretorius [31] has discussed the controversy focusing on the origin of various yeast species. According to the first school of thought, the main origin of yeasts is the vineyard, and their presence or absence varies between each plant and grape cluster [33, 36]. Other authors have opted in favor of a direct association with artificial, man-made environments such as wineries and fermentation plants, and claim that these yeasts are not of natural origin. The results of the present study show that yeasts taking the filamentous growth pathway can penetrate the plant tissues and may even survive their hosts. These findings tend to support the first school of thought. However, we have to be careful about our conclusions because our study was carried out on plantlets in vitro, and different results may be obtained if the same procedure is applied to vineyard grapes or old grapevine plants, as their anatomical structure and physiological functions are so different [16]. On the other hand, the

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yeast concentrations to which the plantlets were exposed here were certainly not representative of those occurring naturally, although some authors have detected yeast concentrations of about 105 to 106 CFU berry–1 [9]. The concentrations used here were adopted because their effects have been established in our previous studies [14, 15]. Additionally, it is worth noting that the present study was carried out on a single strain per genus, and that the results obtained therefore cannot be taken to apply to the whole species. However, the experimental pair S. cerevisiae/V. vinifera used in the present study should provide an appropriate tool for investigating the interactions between fungi and their host plants in future studies.

Acknowledgments

The authors would like to thank Mrs Jessica Blanc for her careful reading of the manuscript. This research was supported by a grant from Europol’agro.

References 1. Banuett, F (1995) Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize. Annu Rev Genet Rev 29: 179–208 2. Banuett, F (1998) Signaling in the yeasts: an informational cascade with links to the filamentous fungi. Microbial Mol Biol Rev 62: 249–274 3. Barnett, JA, Payne, RW, Yarrow, W (2000) In: Cambridge University Press (Ed.) Yeast: Characteristics and Identification, 3rd ed. University Press, Cambridge, pp 1–1138 4. Cervone, F, De Lorenzo, G, Aracri, B, Bellincampi, D, Caprari, C, Clark, AJ, Desiderio, A, Devoto, A, Leckie, F, Mattei, B, Nuss, L, Salvi, G (1996) The role of polygalacturonase, PGIP and pectin oligomers in fungal infection. In: Visser, J, Voragen, AGL (Eds.) Progress in Biotechnology: Pectins and Pectinases. Elsevier, Amsterdam, pp 191–205 5. Cullen, PJ, Sprague Jr, GF (2000) Glucose depletion causes haploid invasive: growth in yeast. Proc Natl Acad Sci 97: 13619–13624 6. DeBary, A (1886) Veber Einige Sclerotinien und SclerotienKrankheiten. Bot Ztg 44: 376–488 7. Dickinson, JR (1996) FFuel_ alcohols induce hyphal-like extensions and pseudohyphal formation in yeasts. Microbiology 142: 1391– 1407 8. D’Souza, CA, Heitman, J (2001) Conserved cAMP signaling cascades regulate fungal development and virulence. FEMS Microbial Rev 25: 349–364 9. Fleet, GH (2002) The yeast ecology of wine grapes. In: Ciani, M (Eds.) Biodiversity and Biotechnology of Wine Yeasts. Research Signpost, Trivandrum, India, pp l–19 10. Fre´zier, V, Dubourdieu, D (1992) Ecology of yeast strains Saccharomyces cerevisiae during spontaneous fermentation in a Bordeaux winery. Am J Enol Vitic 43: 375–380 11. Gainvors, A, Fre´zier, V, Lemaresquier, H, Lequart, C, Aigle, M, Belarbi, A (1994) Detection of polygalacturonase, pectin-lyase and pectin-esterase activities in a Saccharomyces cerevisiae strain. Yeast 10: 1311–1319 12. Gancedo, JM (2001) Control of pseudohyphae formation in Saccharomyces cerevisiae. FEMS Microbial Rev 25: 107–123

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13. Gimeno, CJ, Ljungdahi, PO, Styles, CA, Fink, GR (1992) Unipolar cell division in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68: 1077–1099 14. Gognies, S, Belarbi, A (2002) Endopolygalacturonase of Saccharomyces cerevisiae: involvement in pseudohyphae development of haploids and in pathogenicity on Vitis vinifera. Plant Sci 163: 759– 769 15. Gognies, S, Belarbi, A, Ait Barka, E (2002) Saccharomyces cerevisiae, a potential pathogen towards grapevine, Vitis vinifera. FEMS Microbiol Ecol 37: 143–150 16. Hartmann, C, Joseph, C, Millet, B (1998) In: Nathan (Ed.) Biologie et Physiologie de la plante. 17. Kang, Z, Buchenauer, H (2002) Studies on the infection process of Fusarium culmorum in wheat spikes. Degradation of host cell wall components and localization of trichothecene toxins in infected tissues. Eur J Plant Phyto 108: 653–660 18. Kurtzman, CP, Fell, JW (1998) In: Elsevier (Ed.) The Yeasts, a Taxonomic Study, 4th ed. Amsterdam 19. Lambrechts, MG, Bauer, FF, Marmur, J, Pretorius, IS (1996) Muc1, a mucin-like protein which is regulated by Mss10, is critical for pseudohyphal differentiation in yeast. Proc Natl Acad Sci U S A 93: 8419–8424 20. Lo, WS, Dranginis, AM (1998) The cell surface flocculin FLO11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae. Mol Biol Cell 9: 161–171 21. Lorenz, MC, Cutler, NS, Heitman, J (2000) Characterization of alcohol-induced filamentous growth in Saccharomyces cerevisiae. Mol Biol Cell 11: 183–199 22. Madhani, HD, Fink, GR (1997) Combinatorial control required for the specificity of yeast MAPK signalling. Science 275: 1314– 1317 23. Madhani, HD, Fink, GR (1998) The control of filamentous differentiation and virulence in fungi. Trends Cell Biol 8: 348–353 24. Madhani, HD, Galitski, T, Lander, ES, Fink, GR (1999) Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants. Proc Natl Acad Sci 96: 12530–12535 25. Martini, A (1993) Origin and domestication of the wine yeast Saccharomyces cerevisiae. J Wine Res 4: 165–176 26. McKay, AM (1988) A plate assay method for the detection of fungal polygalacturonase secretion. FEMS Microbiol Lett 56: 355– 358 27. Mortimer, R, Polsinelli, M (1999) On the origins of wine yeast. Res Microbiol 150(3): 199–204 28. Mosch, HU, Fink, GR (1997) Dissection of filamentous growth by transposon mutagenesis in Saccharomyces cerevisiae. Genetics 145(3): 671–684 29. Murashige, T, Skoog, F (1962) A revised medium for rapid growth and biomass assays with tobacco tissue cultures. Physiol Plant 15: 473–497 30. Prade, RA, Zhan, D, Ayoudi, P, Mort, AJ (1999) Pectins, pectinases and plant–microbe interactions. Biotechnol Genet Eng Rev 16: 361–391 31. Pretorius, IS (2000) Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16: 675– 729 32. Rainieri, S, Zambonelli, C, Kaneko, Y (2003) Saccharomyces sensu stricto: systematics, genetic diversity and evolution. J Biosci Bioeng 96(1): 1–9 33. Rementeria, A, Rodriguez, JA, Cadaval, A, Amenabar, R, Muguruza, JR, Hernando, FL, Sevilla, MJ (2003) Yeast associated with spontaneous fermentations of white wines from the BTxakoli de Bizkaia^ region (Basque Country, North Spain). Int J Food Microbiol 1;86(1–2): 201–207 34. Roberts, RL, Fink, GR (1994) Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental

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programs in the same cell type: mating and invasive growth. Genes Dev 8: 2974–2985 35. Scherer, S, Magee, PT (1990) Genetics of Candida albicans. Microbiol Rev 54: 226–241 36. Schuller, D, Alves, H, Dequin, S, Casal, M (2005) Ecological survey of Saccharomyces cerevisiae strains from vineyards in the

S. GOGNIES


BETWEEN YEASTS

AND

GRAPEVINES

Vinho Verde Region of Portugal. FEMS Microbiol Ecol 51: 167– 177 37. VanDyk, D, Pretorius, IS, Bauer, FF (2005) Mss11p is a central element of the regulatory network that controls FLO11 expression and invasive growth in Saccharomyces cerevisiae. Genetics 169(1): 91–106