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The life cycle of the budding yeast Saccharomyces cerevisiae . The diagram shows yeast-form cells, which can be induced to undergo different growth responses depending on ploidy and growth condition. Haploid and diploid cells interconvert between the two types by mating and sporulation, respectively. Both haploid and diploid cells can undergo fi lamentous growth, form bio fi lms, or enter stationary phase (quiescence) in response to nutrient (glucose or nitrogen) limitation. Diploid cells also sporulate in response to the limitation of carbon and nitrogen sources. Secreted alcohols act as autoinducers to stimulate fi lamentous growth.
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Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host-cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basi...
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... (Cullen and Sprague 2000). To determine how glucose levels feed into fi lamentous growth regulation, several established nutrient-sensing pathways were examined, which uncovered a role for the protein kinase Snf1 in regulating fi lamentous growth (Cullen and Sprague 2000). Snf1 operates in a separate pathway from Gpr1, by regulating the repressors Nrg1 and Nrg2 at the FLO11 promoter (Kuchin et al. 2002; Vyas et al. 2003), a gene required for fi lamentous growth (see below for further discussion of FLO11 ). Nrg1 and Nrg2 function by recruitment of the Cyc8 – Tup1 complex to promoters. Thus, two different glucose-sensing pathways, Gpr1/Gpa2/Ras2/ PKA and Snf1, regulate fi lamentous growth in yeast. TOR pathway: Initial observations of fi lamentous growth showed that limiting fi xed nitrogen (speci fi cally ammonia) is a trigger of fi lamentous growth (Gimeno et al. 1992). Speci fi cally, mutants defective for ammonium transport were hyper fi lamentous, which suggests that ammonium starvation might be a trigger for fi lamentous growth (Gimeno et al. 1992). In addition, Lorenz and Heitman (1998b) showed that the high-af fi nity ammonium transporter Mep2 is required for fi lamentous growth. The fi lamentation defect of the mep2 mutant arises apparently not from a defect in ammonium transport, as one might expect, but rather from a speci fi c role for that transporter in communi- cating a signal through a small region in its cytosolic domain. The signal may be conveyed through a mechanism that is not well understood via the MAPK pathway (Rutherford et al. 2008). Nitrogen signals have subsequently been shown to be interpreted by the TOR pathway (Crespo et al. 2002), an evolutionarily conserved nutrient-regulatory pathway (Heitman et al. 1991). The serine/threonine protein kinase TOR regulates cellular homeostasis by coordinating metabolic processes with cellular nutrient levels (Sengupta et al. 2010). The TOR pathway regulates the transcription factor Gcn4, which is a regulator of FLO11 expression (Braus et al. 2003; Boeckstaens et al. 2008). The TOR pathway regulates fi lamentous growth in a manner that is apparently independent of the RAS/PKA and MAPK pathways. Evidence for this conclusion comes from the fact that rapamycin inhibits fi lamentous growth under nitrogen-limited conditions, an inhibition that is mediated by the TOR pathway phosphatases Tap42 and Sit4 (Cutler et al. 2001). To summarize, limiting for nitrogen or glucose can induce fi lamentous growth. The fact that the glucose response was fi rst observed in haploid cells (invasive growth, Figure 2, B and C) and the nitrogen limitation response fi rst characterized in diploid cells (pseudohyphal growth, Figure 2D) may have led to the impression that the different cell types respond to different stimuli. In fact, glucose depletion induces fi lamentous growth in both haploid and diploid cells (Cullen and Sprague 2002; Kuchin et al. 2002), and nitrogen limitation also induces fi lamentous growth in both cell types (P. J. Cullen and G. F. Sprague, unpublished observations). How different are haploid and diploid cells with respect to fi lamentous growth? The answer to this question is complicated: relatively few studies directly compare fi lamentous growth in the two cell types, and different assays are used for haploid (Figure 2, C and D) and diploid cells (Figure 2E). A further complication comes from incongruous results. It was initially reported that haploid cells undergo invasive growth better than diploid cells (Roberts and Fink 1994), although we found the opposite to be true (Cullen and Sprague 2002). Nevertheless, the expression of fi lamentation target genes is regulated by different stimuli in haploid compared to diploid cells (Lo and Dranginis 1998), and regulatory pathways Ras2/PKA and MAPK (discussed below) have different roles in regulating the response in haploid and diploid cells (Chen and Thorner 2010). At this point, an important paradox should be discussed. One the one hand, glucose limitation induces fi lamentous growth in both haploid and diploid cells. Indeed, cells grown in nutrient-rich (high glucose) conditions do not produce pseudohyphae. But on the other hand, as stated above, glucose/sucrose is required for fi lamentous growth in a Gpr1-dependent manner. What is the basis for this discrepancy? Although this point has not been explicitly addressed in the literature, there are several possibilities. One is that glucose/sucrose is required for pseusohyphal growth in diploid cells enduring a low-nitrogen stress, the conditions used by the Thevelein group, to establish the requirement. A less interesting alternative is that different strains are sensitized to different nutritional requirements. Diploid cells starved for both nitrogen and glucose undergo sporulation, which raises an important point: how do cells decide whether to undergo fi lamentous growth, enter stationary phase, or sporulate in response to limiting nutrients (Figure 1)? Sporulation has been extensively studied in yeast (Neiman 2011), and many of the signals that trigger meiosis and spore formation are well characterized (Engebrecht 2003). One protein that controls the sporulation/ fi lamentation decision is the repressor of meiosis Rme1 (van Dyk et al. 2003). Rme1p is a zinc- fi nger type transcriptional factor that promotes the mitotic/meiotic decision (Mitchell and Herskowitz 1986). Rme1 binds directly to the FLO11 promoter to induce cell – cell adhesion and invasive growth (van Dyk et al. 2003). Given that Rme1 is not regulated by Ras2 or the MAPK pathway (van Dyk et al. 2003), presumably other pathways regulate Rme1-induced fi lamentous growth. Regulators of the sporulation pathway, Ime1 and Ime2, are also required for fi lamentous growth (Strudwick et al. 2010), although this is true only in the SK1 genetic background. Even in that background, the requirement for Ime2 is extremely weak. Neither protein is required for agar invasion by haploids, but rather only for colony morphology changes shown by diploids (Strudwick et al. 2010). Other sensory pathways: Several other metabolites have been identi fi ed that in fl uence fi lamentous growth. One is alcohol byproducts like 1-butanol (Dickinson 1996; Lorenz et al. 2000a). Response to alcohols has now been identi fi ed as a quorum-sensing behavior. Budding yeast undergo fi lamentous growth in response to cell density using secreted alcohols as a gauge of its population levels (Chen and Fink 2006). Quorum sensing also occurs in C. albicans via sensing different secreted alcohol derivatives (Chen et al. 2004). An intact respiratory pathway, as mediated by a signaling pathway referred to as the retrograde pathway (Butow and Avadhani 2004), also regulates fi lamentous growth (Jin et al. 2008b). Several other metabolites that induce fi lamentous growth have also been identi fi ed, including tetrahydro- folate (vitamin B9). B9 levels feed into FLO11 expression through signaling mechanisms that have not been well characterized (Guldener et al. 2004). External pH may also be sensed in some manner through a signaling pathway that regulates the transcription factor Rim101 (Lamb and Mitchell 2003). Early studies from the Fink lab uncovered two signaling pathways that regulate fi lamentous growth. As discussed above, one major pathway is the Ras2 pathway. The other major pathway is a MAPK pathway composed of kinases that also function in the mating or pheromone response pathway (Figure 4A). The logic underlying testing for a role for the MAPK pathway in fi lamentous growth was that elements of the pheromone response pathway are expressed in diploid cells, even though diploid cells do not mate. What might the pathway ’ s function in diploids be? Liu et al. (1993) reported that four proteins required for mating in haploid cells, the p21-activated (PAK) kinase Ste20, the MAPKKK Ste11, the MAPKK Ste7, and the transcription factor Ste12 (Figure 4A), are also required for fi lamentous growth in diploids. In contrast, the genes encoding the pheromone receptors Ste2/ Ste3, the associated hererotrimeric G protein (Gpa1, Ste4, and Ste18), and the MAPK Fus3 are not required for fi lamentous growth in diploids (or haploids). Thus, it seemed that haploid cells utilize the “ core module ” of Ste20 / Ste11 / Ste7 / Ste12 for mating, whereas diploid cells utilize that same core module for fi lamentous growth regulation (Figure 4A). Although the separation of function by cell type seems a tidy way to establish speci fi city, the tidiness is super fi cial and speci fi city questions loom large. First, the transcription factor Ste12 functions in both pathways. How are different gene sets activated in mating and fi lamentous growth? Second, it soon became apparent that haploid cells execute a similar fi lamentous growth program that requires the same core module (Roberts and Fink 1994). An even more fundamental question therefore is how does the same module direct two distinct physiologic programs in the same cell type? An example of this quandary comes from studies of the global regulatory Rho-family GTPase Cdc42 (Park and Bi 2007). Cdc42 is an essential protein that is required to establish cell polarity (Bender and Pringle 1989; Adams et al. 1990; Shimada et al. 2004; Gao et al. 2007; Tong et al. 2007 and references therein). It has subsequently been shown that Cdc42 functions in the mating pathway and is required for transduction of the signal initiated by the GPCR (Simon et al. 1995). Although it was known that temperature- sensitive mutations in CDC42 were defective for mating (Reid and Hartwell 1977), the assumption was that this resulted from a defect in the overall cell polarity. However, the studies of Simon et al. (1995) suggested a more direct involvement of Cdc42 in the mating pathway. The salient fi nding was that temperature-sensitive versions of Cdc42 and its guanine nucleotide exchange factor (GEF) Cdc24 were defective in ...
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... goes as follows: defects in protein glycosylation or protein folding reduce the glycosylation/stability of the extracellular domain of Msb2 (Yang et al. 2009). Underglycosylated Msb2 mimics the activated form of the protein, because the extracellular domain is inhibitory and activates the MAPK pathway. In protein glycoslyation mutants, Msb2 is under- glycosylated (Cullen et al. 2004; Yang et al. 2009), and the MAPK pathway is active (Cullen et al. 2000). Indeed, most perturbations to Msb2 ’ s mucin homology domain (which is heavily glycosylated in mammalian mucins) (Silverman et al. 2003) results in a hyperactive protein (Cullen et al. 2004). Most intriguingly, protein glycosylation provides a readout of nutrition, because mannosyl substrates are de- rived from glucose-6-phosphate. Therefore, underglycosylation of Msb2 may represent a signal to the MAPK pathway for entry into low-nutrient environments. More generally, the rates of core cellular processes may re fl ect overall nutritional status that becomes sensed and incorporated into the fi lamentation response. Interestingly, the UPR has an inhibitory role in sporulation (Schroder et al. 2000) and mediates its inhibitory effect by recruitment of the HDAC Rpd3 to early meiotic genes through the transcription factor Ume6 (Schroder et al. 2004). In contrast, Rpd3 plays a positive role in fi lamentous growth and is required for the expression of the MSB2 and STE12 genes (Chavel et al. 2010). The reciprocal roles of Rpd3 in promoting fi lamentous growth and dampening meiosis suggest that Rpd3 may be involved in the decision of whether cells should undergo fi lamentous growth or sporulate (Figure 1). The mechanism by which a HDAC, tradi- tionally considered to function as a repressor of gene transcription, promotes the expression of fi lamentation regulatory genes is not clear. Nevertheless, microarray analysis reveals that many genes are downregulated in HDAC mutants, and Rpd3 also positively regulates the HOG pathway (de Nadal et al. 2004). Studies of fi lamentous growth regulation in budding yeast have had at least two major biological impacts. The fi rst is that yeast provides a roadmap to identify and characterize elements of the response that also occurs in other fungal species, particularly fungal pathogens. Many of the genetic pathways that regulate fi lamentous growth in C. albicans and other pathogens have been uncovered through studies in S. cerevisise . As one of many possible examples, Msb2 homologs have recently been identi fi ed in C. albicans and in three plant fungal pathogens. In all cases, Msb2 presides over a MAPK pathway that is important for fi lamentous growth and virulence (Roman et al. 2009; Lanver et al. 2010; Liu et al. 2011; Perez-Nadales and Di Pietro 2011). The second is that fi lamentous growth regulation is a model for understanding eukaryotic cell differentiation. Cell differ- entiation in mammals involves processes that are at present complex and poorly de fi ned. Speci fi cally, the concept of a globally connected network of signaling pathways working in concert, although accepted, is not well understood at the molecular level. Budding yeast provides a working template to understand how signals that initiate from different pathways become routed through common modules to induce a speci fi c behavior. What lies ahead for studies on fi lamentous growth regulation in yeast? Filamentous growth represents a point of convergence between many cellular pathways — the cell cycle, cell polarity, and nutrition — and therefore is an attrac- tive system to understand the connection between different biological processes. Of course, one of the main places where future progress is needed is in further de fi ning the signaling pathways that regulate the response. The Gpr1/ Gpa2/Ras2 pathway is fi lled with controversies that make drawing a coherent picture of that pathway dif fi cult. Para- mount in this regard is resolving the paradox of whether and how carbon sources are sensed and interpreted into the decision of whether or not to undergo fi lamentous growth. Another area in which much progress is needed is in understanding how the MAPK pathway that regulates fi lamentous growth maintains its identity. It could be reasonably argued that all of the components of that pathway (Msb2, Sho1, Cdc42, Ste20, Ste50, Ste11, Ste7, Kss1, and Ste12), with the exception of Tec1, are general components that function in multiple pathways. Solving this identity crisis represents a daunting challenge in the fi eld of cellular signaling. Probably the most important and mysterious aspect of fi lamentous growth regulation involves the integration of signals from multiple pathways into a coherent response. Do the MAPK, TOR, and Ras2 pathways talk to each other, and if so, to what extent? This area in particular is ripe for future investigations. In addition to fi lamentous growth, bakers ’ yeast undergoes other nutrient-limitation – dependent responses (Figure 1). These include entry into a quiescent state, sporulation (in diploids), microbial mat expansion, and quorum sensing. As mentioned above, an important question is how does a cell choose among these different lifestyles? Similarly, is there a relationship between these various responses? For example, C. albicans forms microbial mats or bio fi lms that are composed of multiple cell types including fi lamentous cells and that interface with other communities of micro- organisms (Parsek and Greenberg 2005). Ultimately, the ecology of fi lamentous growth regulation — especially of cell populations in native settings — will be the most fun and challenging to explore. We thank laboratory members for helpful discussions. We apologize to investigators whose work was not cited. G.F.S. is supported by a grant from the U.S. Public Health Service (GM30027) and P.J.C. is supported by grants from the U.S. Public Health Service (GM098629 and DE18425) and the American Cancer Society ...
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... and the immune response (Netea and Marodi 2010; Hajishengallis and Lambris 2011; Kronstad et al. 2011; Moran et al. 2011) and summarize fi ndings not described here. Signal transduction pathways have taken center stage in the effort to understand fi lamentous growth regulation in yeast. Given that many signaling pathways regulate fi lamentous growth, and that some of these pathways are composed of proteins that function in multiple pathways, we will stress issues that relate to signal integration and signal insulation between pathways. We will also address the important question of how signaling pathways accomplish the change in cell type from the yeast mode to the fi lamentous mode. Other review articles have been pub- lished recently on fi lamentous growth regulation (Nobile and Mitchell 2006; Verstrepen and Klis 2006; Whiteway and Bachewich 2007; Zhao et al. 2007; Bruckner and Mosch 2011), nutrient-regulated signaling pathways (Hedbacker and Carlson 2008; Zaman et al. 2008; Sengupta et al. 2010), and mitogen activated protein kinase (MAPK) regulation (Bardwell 2006; Dohlman and Slessareva 2006; Chen and Thorner 2007; Saito 2010), which may offer different perspectives than those described here. Filamentous growth is a fungal-speci fi c growth mode in which cells adopt a unique morphological pattern that allows expansion into new environments. The fi lamentation response is highly variable among species, ranging from mycelial mat or hyphal formation in true fi lamentous fungi to subtle changes in cell shape in yeasts. The biology that attends this response is fascinating and mysterious and ranges from contact-responsive hyphal growth (Kumamoto 2005) to behavior modi fi cation of insect species, such as the erratic behavior exhibited by “ zombie ants ” infected with Ophiocordyceps (Pontoppidan et al. 2009), to the formation of lasso-type structures in Nematode -trapping parasites (Wang et al. 2009a). Some species, like the extensively studied fi ssion yeast Schizosaccharomyces pombe , have only recently be shown to undergo fi lamentous growth as part of their life cycles (Amoah-Buahin et al. 2005). The hyphal growth of fi lamentous fungi is morphologically striking. In Neurospora crassa , hyphal cells are multinucleate (Ramos-Garcia et al. 2009) and grow in bifurcating branches (Ziv et al. 2009) that can undergo cell-to-cell fusion (Steinberg 2007; Aldabbous et al. 2010). Fusion is a dy- namic process that occurs by hyphal-cell recognition through a MAPK-dependent sensing mechanism (Fleissner et al. 2009). Hyphal cells grow rapidly, and cell polarity can be reorganized in response to many different cues. Polarized growth is regulated by a curious structure, the Spitzen- körper (Crampin et al. 2005). Historically, much interest in understanding fi lamentous growth regulation has come from studies in fungal pathogens. Pathogens like Candida albicans and Aspergillis fuma- gatus pose a worldwide threat to human health (Netea et al. 2008; Gastebois et al. 2009). These pathogens are particularly harmful to individuals whose immune system has been compromised by AIDS or by suppression resulting from che- motherapies and other drug treatments (Ben-Ami et al. 2008). Fungal pathogens can also be devastating to plant communities, and harvest loss as a result of damage from fungal species is a serious problem (Rispail et al. 2009). In C. albicans , transition to the fi lamentous cell type is critical for virulence (Lo et al. 1997) and depends on a multitude of extracellular factors including temperature and nutrient availability (Berman 2006). Pathogenicity of C. albicans involves many interrelated processes that include cell-surface variation (Nather and Munro 2008), host – cell adhesion (Latge 2010), bio fi lm formation (d'Enfert 2009), and chro- mosome reorganization (Selmecki et al. 2010). In other fungal pathogens, like Cryptococcus neoformans , fi lamentous growth is not tightly related to pathogenicity, as cells primarily exist in the yeast cell type (Lin 2009). Progress in de fi ning the genetic pathways that regulate fi lamentous growth has bene fi ted from studies in the versatile fungal eukaryote S. cerevisiae . Lessons learned about fi lamentous growth regulation in budding yeast have turned out to be true for many fungal species. Identifying and characterizing the genetic pathways that regulate fi lamentous growth in yeast has contributed to understanding the genetic basis of virulence in fungal pathogens and has provided a model for how eukaryotic cells differentiate into morphologically distinct patterns in response to extrinsic cues. Budding yeast does not undergo true hyphal growth, but rather a pseudohyphal growth pattern in which cells fully separate by cytokinesis — they are not multinucleate — and remain attached to each other by proteins in the cell wall. As for many fungal species, yeast cells can transition between yeast-form growth and fi lamentous-form growth as part of their life cycle (Figure 1). One of the triggers for fi lamentous growth in yeast and many other fungal species is nutrient limitation. Both haploid and diploid yeast cells undergo a version of the response, but the stimuli that trigger it, the underlying genetic machinery, and the resulting morphological changes differ slightly between the two cell types. The term invasive growth has been applied to the fi lamentation phenomenon shown by haploid cells because of their ability to invade agar substrates. The term pseudohyphal growth is sometimes used to describe the response in diploid cells. In this review article, we will use the phrase fi lamentous growth as a general term that applies to both haploid invasive growth and diploid pseudohyphal development. We will not distinguish between these two highly related responses unless it is crucial to do so in some experimental context. Filamentous growth in yeast can be separated into three major changes: an increase in cell length, a reorganization of polarity, and enhanced cell – cell adhesion. Assays to study fi lamentous growth in yeast exist on the macroscopic and microscopic levels. The enhanced cell – cell adhesion of fi lamentous cells is visible by inspecting yeast colonies (Figure 2A). Cells on the underside of the colonies attach to and invade the agar substratum (Figure 2B), and this invasive growth has been used as a tool to determine whether fi lamentous growth occurs (Roberts and Fink 1994) and to screen for mutants that are defective at fi lamentous growth (or are better at it than wild-type cells, e.g. , Palecek et al. 2000). Changes in cell shape are visible by microscopic ex- amination of cells, and speci fi c assays are used to examine the response in haploid (Figure 2C) and diploid cells (Figure 2D). Using these and other assays, many of the genetic pathways that regulate fi lamentous growth have been uncovered. Below, we focus on the signaling pathways that regulate the response. We describe what the stimuli are, how they might be sensed, and how the activated pathways induce fi lamentous growth. In 1992, the Fink lab rejuvenated a little known fi nding that the budding yeast S. cerevisiae undergoes fi lamentous growth as part of its life cycle (Gimeno et al. 1992). Their study drew attention to anecdotal observations about yeast ’ s growth pattern (Gutlliermond 1920; Lodder 1970; Brown and Hough 1965; Eubanks and Beuchat 1982) and made use of genetic and molecular approaches to gain insights about the underlying mechanism of this unexplored behavior. The initial observation was that isolates of S. cerevisiae from a “ wild ” strain background ( S 1278b) form colonies composed of elongated cells that grow in connected chains on low-nutrient medium. This growth pattern resembles the morphology that is exhibited by some species of fi lamentous fungi. Filamentous growth is widely considered to represent a nutritional scavenging response, and the Fink lab connected this morphological behavior to nutrition in two important ways: fi rst, strains defective for ammonium utilization were hyper fi lamentous (Gimeno et al. 1992), suggesting a connection between nitrogen levels and fi lamentous growth. Subsequent studies have shown that the lack of fermentable carbon source can also be a trigger for fi lamentous growth (Cullen and Sprague 2000). Second, and more interesting, the global nutrient regulatory GTPase Ras2 was found to be required for fi lamentous growth regulation. The key experiment used an activated version of Ras2, in which the protein was “ locked ” in its activated (GTP-bound) state, dramatically stimulated the fi lamentous properties of this organism (Gimeno et al. 1992). Altogether, four signaling pathways that regulate fi lamentous growth have been well characterized — rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), target of rapamycin (TOR), and MAPK. We discuss each in turn below but concentrate on the MAPK pathway because it raises intriguing questions regarding signaling speci fi city. receptor Gpr1: The discovery that RAS is involved in fi lamentous growth provided a genetic context for elucidating components of the molecular pathway that plays a role in that growth habit (Figure 3). Yeast encode two RAS genes, RAS1 and RAS2 . The RAS2 gene is expressed at higher levels than RAS1 and is responsible for the majority of Ras function (Kataoka et al. 1984). Ras2 associates with and activates adenylate cyclase, a membrane-associated enzyme that produces the second messenger cyclic adenosine monophosphate (cAMP) (Toda et al. 1985). The Fink lab proposed that the levels of cAMP are critical for the decision of whether or not cells undergo fi lamentous growth (Mosch et al. 1996). Indeed, overexpression of the gene encoding the phosphodies- terase Pde2 dampened fi lamentous growth and suppressed the hyper fi lamentation induced by activated RAS (Ward et al. 1995). As for many eukaryotes, cAMP regulates the activity of a family of ...
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... processes. Comparative genome sequencing between a standard laboratory strain and the S 1278b background coupled with genome- wide deletion analysis of all nonessential genes in the S 1278b background show multiple functional differences between the two genetic backgrounds, and support the notion of a globally regulated cellular response (Dowell et al. 2010). Together these genetic and high-throughput approaches reinforce the idea that the dimorphic transition to fi lamentous growth is a cellular differentiation response that involves the reorganization of many aspects of cellular machinery to produce a speci fi c cell type. How complicated is fi lamentous growth? One way to explore the complexity of the response is to examine the outputs of the signaling pathways that regulate the behavior. A diverse collection of genes is induced by the fi lamentation MAPK pathway. One target encodes the polygalacturonidase Pgu1, an enzyme that metabolizes a component found in plant cell walls (Madhani et al. 1999). Filamentous growth occurs in the grape-producing plant Vitus vinifera (Pitoniak et al. 2009), one environment in which Pgu1 may be required. Another prominent group of transcriptional targets are Ty1 transposons. The fact that transposition is induced by the fi lamentation pathway in response to environmental perturbation may provide a mechanism for adaptive evolution in response to stress (Morillon et al. 2000). In addition to PGU1 and Ty, there are many (hundreds of) targets of the signaling pathways that control fi lamentous growth. What are all of these genes doing? Many of the main targets and highly induced genes do not at present have a clear cellular function. For example, several genes that are considered canonical reporters for the fi lamentation pathway, like YLR042c and SVS1 , have no clear phenotype when deleted and no established cellular function (P. J. Cullen and G. F. Sprague, unpublished data). An existing challenge is to understand at a functional and phenotypic level the roles that the target genes play in fi lamentous growth. One reason for the lack of phenotype could be genetic redundancy. A second reason may be that yeast undergoes critical behaviors for fi lamentous growth that are not obvious under standard laboratory conditions. For example, Pgu1 may be critical for yeast cells to colonize plant tissue but pgu1 mutants would not be expected to show a clear phenotype in laboratory settings. Filamentous growth is also tied into core cellular processes. These include transcription by RNA polymerase II (Singh et al. 2007), protein translation (Strittmatter et al. 2006; Gilbert et al. 2007), tRNA modi fi cation (Murray et al. 1998; Abdullah and Cullen 2009), protein glycosylation (Cullen et al. 2000), the unfolded protein response (UPR) (Schroder et al. 2000, 2004), autophagy (Ma et al. 2007), and the proteasome (Prinz et al. 2004). Together these fi ndings resonate with the current picture of the yeast genetic interaction network, where many cellular processes are connected in some manner to each other (Costanzo et al. 2010). It will be interesting to overlay onto this network the changes in basic cellular machinery that occur during fi lamentous growth. One connection may exist between protein glycosylation, the UPR, and the MAPK pathway. The rationale goes as follows: defects in protein glycosylation or protein folding reduce the glycosylation/stability of the extracellular domain of Msb2 (Yang et al. 2009). Underglycosylated Msb2 mimics the activated form of the protein, because the extracellular domain is inhibitory and activates the MAPK pathway. In protein glycoslyation mutants, Msb2 is under- glycosylated (Cullen et al. 2004; Yang et al. 2009), and the MAPK pathway is active (Cullen et al. 2000). Indeed, most perturbations to Msb2 ’ s mucin homology domain (which is heavily glycosylated in mammalian mucins) (Silverman et al. 2003) results in a hyperactive protein (Cullen et al. 2004). Most intriguingly, protein glycosylation provides a readout of nutrition, because mannosyl substrates are de- rived from glucose-6-phosphate. Therefore, underglycosylation of Msb2 may represent a signal to the MAPK pathway for entry into low-nutrient environments. More generally, the rates of core cellular processes may re fl ect overall nutritional status that becomes sensed and incorporated into the fi lamentation response. Interestingly, the UPR has an inhibitory role in sporulation (Schroder et al. 2000) and mediates its inhibitory effect by recruitment of the HDAC Rpd3 to early meiotic genes through the transcription factor Ume6 (Schroder et al. 2004). In contrast, Rpd3 plays a positive role in fi lamentous growth and is required for the expression of the MSB2 and STE12 genes (Chavel et al. 2010). The reciprocal roles of Rpd3 in promoting fi lamentous growth and dampening meiosis suggest that Rpd3 may be involved in the decision of whether cells should undergo fi lamentous growth or sporulate (Figure 1). The mechanism by which a HDAC, tradi- tionally considered to function as a repressor of gene transcription, promotes the expression of fi lamentation regulatory genes is not clear. Nevertheless, microarray analysis reveals that many genes are downregulated in HDAC mutants, and Rpd3 also positively regulates the HOG pathway (de Nadal et al. 2004). Studies of fi lamentous growth regulation in budding yeast have had at least two major biological impacts. The fi rst is that yeast provides a roadmap to identify and characterize elements of the response that also occurs in other fungal species, particularly fungal pathogens. Many of the genetic pathways that regulate fi lamentous growth in C. albicans and other pathogens have been uncovered through studies in S. cerevisise . As one of many possible examples, Msb2 homologs have recently been identi fi ed in C. albicans and in three plant fungal pathogens. In all cases, Msb2 presides over a MAPK pathway that is important for fi lamentous growth and virulence (Roman et al. 2009; Lanver et al. 2010; Liu et al. 2011; Perez-Nadales and Di Pietro 2011). The second is that fi lamentous growth regulation is a model for understanding eukaryotic cell differentiation. Cell differ- entiation in mammals involves processes that are at present complex and poorly de fi ned. Speci fi cally, the concept of a globally connected network of signaling pathways working in concert, although accepted, is not well understood at the molecular level. Budding yeast provides a working template to understand how signals that initiate from different pathways become routed through common modules to induce a speci fi c behavior. What lies ahead for studies on fi lamentous growth regulation in yeast? Filamentous growth represents a point of convergence between many cellular pathways — the cell cycle, cell polarity, and nutrition — and therefore is an attrac- tive system to understand the connection between different biological processes. Of course, one of the main places where future progress is needed is in further de fi ning the signaling pathways that regulate the response. The Gpr1/ Gpa2/Ras2 pathway is fi lled with controversies that make drawing a coherent picture of that pathway dif fi cult. Para- mount in this regard is resolving the paradox of whether and how carbon sources are sensed and interpreted into the decision of whether or not to undergo fi lamentous growth. Another area in which much progress is needed is in understanding how the MAPK pathway that regulates fi lamentous growth maintains its identity. It could be reasonably argued that all of the components of that pathway (Msb2, Sho1, Cdc42, Ste20, Ste50, Ste11, Ste7, Kss1, and Ste12), with the exception of Tec1, are general components that function in multiple pathways. Solving this identity crisis represents a daunting challenge in the fi eld of cellular signaling. Probably the most important and mysterious aspect of fi lamentous growth regulation involves the integration of signals from multiple pathways into a coherent response. Do the MAPK, TOR, and Ras2 pathways talk to each other, and if so, to what extent? This area in particular is ripe for future investigations. In addition to fi lamentous growth, bakers ’ yeast undergoes other nutrient-limitation – dependent responses (Figure 1). These include entry into a quiescent state, sporulation (in diploids), microbial mat expansion, and quorum sensing. As mentioned above, an important question is how does a cell choose among these different lifestyles? Similarly, is there a relationship between these various responses? For example, C. albicans forms microbial mats or bio fi lms that are composed of multiple cell types including fi lamentous cells and that interface with other communities of micro- organisms (Parsek and Greenberg 2005). Ultimately, the ecology of fi lamentous growth regulation — especially of cell populations in native settings — will be the most fun and challenging to explore. We thank laboratory members for helpful discussions. We apologize to investigators whose work was not cited. G.F.S. is supported by a grant from the U.S. Public Health Service (GM30027) and P.J.C. is supported by grants from the U.S. Public Health Service (GM098629 and DE18425) and the American Cancer Society ...
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Citations
... The multiple hits to the Rim101 pathway are less expected. That pathway is known to have a role in pH sensing (Yan et al., 2020) and regulation of cell size (Shimasawa et al., 2023), and to connect to pathways regulating filamentous growth (Cullen and Sprague, 2012). Yet a role in cell separation during normal physiological growth conditions has not previously been elucidated-perhaps because many lab strains are already well-separated. ...
Haploid and diploid natural isolates of the yeast Saccharomyces cerevisiae were evolved under a daily transfer protocol that selected against clumpy growth and for cell separation. For the haploid populations, whole genome sequencing revealed strong selection to deactivate AMN1, a known regulator of cell separation, recapitulating evolution that likely occurred during the domestication of common lab strains. We also observed multiple mutational hits to the Rim101 pathway, which was not expected. Mutations to both AMN1 and the Rim101 pathway were strongly associated with cell separation. In the diploid populations, we observed the parallel emergence of a large partial deletion of chrIII, the so-called Hawthorn's deletion. We show that this deletion is associated with reduced clumpy growth. The mechanism by which Hawthorne's deletion affects cell separation is unknown but may be related to an effect on budding pattern.
... The budding yeast Saccharomyces cerevisiae induces diverse transcriptional programs via MAPK cascades that allow this microorganism to make appropriate decisions in response to a specific stimulus. As an illustration, in low nutrient conditions, both haploid and diploid cells can alter their growth pattern to form pseudohyphae 9 . Under more drastic nutrient limitations, diploid cells begin to sporulate 10 . ...
Response to extracellular signals via Mitogen-Activated Protein Kinase (MAPK) pathways regulate complex transcriptional programs where hundreds of genes are induced at a desired level with a specific timing. Gene expression regulation is largely encoded in the promoter of the gene, which harbors numerous transcription factor binding sites. In the mating MAPK pathway of Saccharomyces cerevisiae, one major transcription factor, Ste12, controls the chronology of gene expression necessary for the fusion of two haploid cells. Because endogenous promoters encode a wide diversity of Ste12 binding sites (PRE), synthetic promoters were engineered to decipher the rules that dictate mating gene induction. The conformation of PRE dimers that allow efficient gene expression were identified. The strength of binding of Ste12 to the PRE and the distance of the binding sites to the core promoter modulate the level of induction. The speed of activation is ensured by placing a dimer of PRE in a nucleosome depleted region favoring a basal association of Ste12 prior to the stimulus.
... The adenylate cyclase, activated by G protein, triggered the activation of protein kinase A (PKA) in a cAMP-dependent manner. Then PKA phosphorylated the downstream factors regulating growth and morphological transitions (Cullen and Sprague 2012). For instance, PKA might inhibit the transcriptional repressor MpTPR to alter gene expression programs underlying hyphal branching (Adnan et al. 2017) (Fig. 5). ...
Monascus spp. are commercially important fungi due to their ability to produce beneficial secondary metabolites such as the cholesterol-lowering agent lovastatin and natural food colorants azaphilone pigments. Although hyphal branching intensively influenced the production of these secondary metabolites, the pivotal regulators of hyphal development in Monascus spp. remain unclear. To identify these important regulators, we developed an artificial intelligence (AI)–assisted image analysis tool for quantification of hyphae-branching and constructed a random T-DNA insertion library. High-throughput screening revealed that a STE kinase, MpSTE1, was considered as a key regulator of hyphal branching based on the hyphal phenotype. To further validate the role of MpSTE1, we generated an mpSTE1 gene knockout mutant, a complemented mutant, and an overexpression mutant (OE::mpSTE1). Microscopic observations revealed that overexpression of mpSTE1 led to a 63% increase in branch number while deletion of mpSTE1 reduced the hyphal branching by 68% compared to the wild-type strain. In flask cultures, the strain OE::mpSTE1 showed accelerated growth and glucose consumption. More importantly, the strain OE::mpSTE1 produced 9.2 mg/L lovastatin and 17.0 mg/L azaphilone pigments, respectively, 47.0% and 30.1% higher than those of the wild-type strain. Phosphoproteomic analysis revealed that MpSTE1 directly phosphorylated 7 downstream signal proteins involved in cell division, cytoskeletal organization, and signal transduction. To our best knowledge, MpSTE1 is reported as the first characterized regulator for tightly regulating the hyphal branching in Monascus spp. These findings significantly expanded current understanding of the signaling pathway governing the hyphal branching and development in Monascus spp. Furthermore, MpSTE1 and its analogs were demonstrated as promising targets for improving production of valuable secondary metabolites.
Key points
• MpSTE1 is the first characterized regulator for tightly regulating hyphal branching
• Overexpression of mpSTE1 significantly improves secondary metabolite production
• A high-throughput image analysis tool was developed for counting hyphal branching
... The sensing of Phe-OH and Try-OH relies on the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) subunit Tpk2 (Figure 2). It has been reported that the levels of cAMP are critical determinants of cellular filamentous growth (Mosch and Fink, 1997), regulate the activity of a family of protein kinases, referred to as PKA, and Tpk2, the catalytic subunit of PKA, promote filamentous growth Heitman, 1999, 2002;Cullen and Sprague, 2012). Phosphorylation of Flo8p by Tpk2 leads to Flo8p activation (Pan and Heitman, 1999;Guo et al., 2000;Reynolds and Fink, 2001;Chen and Fink, 2006;Cullen and Sprague, 2012), and activated Flo8p, as a transcriptional activator, binds to regions of the FLO11 promoter to promote gene expression (Pan and Heitman, 2002). ...
... It has been reported that the levels of cAMP are critical determinants of cellular filamentous growth (Mosch and Fink, 1997), regulate the activity of a family of protein kinases, referred to as PKA, and Tpk2, the catalytic subunit of PKA, promote filamentous growth Heitman, 1999, 2002;Cullen and Sprague, 2012). Phosphorylation of Flo8p by Tpk2 leads to Flo8p activation (Pan and Heitman, 1999;Guo et al., 2000;Reynolds and Fink, 2001;Chen and Fink, 2006;Cullen and Sprague, 2012), and activated Flo8p, as a transcriptional activator, binds to regions of the FLO11 promoter to promote gene expression (Pan and Heitman, 2002). Flo11p, a product of FLO11, is a glycosylated phosphatidylinositol (GPI)-anchored cell surface flocculating protein that is required for filamentous growth and is involved in pseudohyphal growth, invasive growth, unipolar growth, bipolar growth, cell elongation, and cellular adhesion (Smukalla et al., 2008;Avbelj et al., 2015;Winters et al., 2019). ...
Quorum sensing (QS) is one of the most well-studied cell-to-cell communication mechanisms in microorganisms. This intercellular communication process in Saccharomyces cerevisiae began to attract more and more attention for researchers since 2006, and phenylethanol, tryptophol, and tyrosol have been proven to be the main quorum sensing molecules (QSMs) of S. cerevisiae. In this paper, the research history and hotspots of QS in S. cerevisiae are reviewed, in particular, the QS system of S. cerevisiae is introduced from the aspects of regulation mechanism of QSMs synthesis, influencing factors of QSMs production, and response mechanism of QSMs. Finally, the employment of QS in adaptation to stress, fermentation products increasing, and food preservation in S. cerevisiae was reviewed. This review will be useful for investigating the microbial interactions of S. cerevisiae, will be helpful for the fermentation process in which yeast participates, and will provide an important reference for future research on S. cerevisiae QS.
... In addition to these expected regulators, we identified putative binding sites for several transcription factors that are involved in regulating filamentous growth (e.g., those encoded by ASH1, SIP4, STE12, FKH1, MIG1/MIG2, and NRG1). This category was particularly noteworthy because filamentous growth can be induced in response to glucose depletion as a starvation response, and it requires a haploid-specific gene, TEC1 [27,[98][99][100][101][102][103]. In addition to dimerizing with Ste12p, Tec1p can activate target genes as a monomer in a dosage-dependent fashion [104][105][106], and it has been experimentally mapped to its consensus motif (TEA/ATTS consensus sequence or TCS) in vivo across the genus Saccharomyces [107]. ...
... First, AGT1 is likely to encode a transporter with broad substrate affinity like its S. cerevisiae homolog [64,[113][114][115][116], whereas other phylogenetically distinct maltose transporters tend to have higher specificity [108,117]. It is possible that selection favored placing control of this generalist transporter under a broader transcriptional response to starvation or glucose depletion as part of a scavenging strategy, which the transition to filamentous growth is thought to represent [102]. Indeed, recent work has suggested that maltose may be an unexpected inducer of filamentous growth in S. cerevisiae [118]. ...
Ploidy is an evolutionarily labile trait, and its variation across the tree of life has profound impacts on evolutionary trajectories and life histories. The immediate consequences and molecular causes of ploidy variation on organismal fitness are frequently less clear, although extreme mating type skews in some fungi hint at links between cell type and adaptive traits. Here, we report an unusual recurrent ploidy reduction in replicate populations of the budding yeast Saccharomyces eubayanus experimentally evolved for improvement of a key metabolic trait, the ability to use maltose as a carbon source. We find that haploids have a substantial, but conditional, fitness advantage in the absence of other genetic variation. Using engineered genotypes that decouple the effects of ploidy and cell type, we show that increased fitness is primarily due to the distinct transcriptional program deployed by haploid-like cell types, with a significant but smaller contribution from absolute ploidy. The link between cell-type specification and the carbon metabolism adaptation can be traced to the noncanonical regulation of a maltose transporter by a haploid-specific gene. This study provides novel mechanistic insight into the molecular basis of an environment–cell type fitness interaction and illustrates how selection on traits unexpectedly linked to ploidy states or cell types can drive karyotypic evolution in fungi.
... In other species such as Z. bisporus, Z. mellis, Z. kombuchaensis, Z. machadoi, Z. gambellarensis, Z. parabailii, Z. pseudobailii, and Z. seidelii, the formation of pseudohyphae does not occur (Rosa and Lachance, 2005;James and Stratford, 2011;Torriani et al., 2011;Saksinchai et al., 2012;Solieri et al., 2013;Suh et al., 2013;Cadež et al., 2015;Brysch-Herzberg et al., 2020). Pseudohyphae formation can be triggered by low nitrogen levels and is a form of foraging (Cullen and Sprague, 2012). The location of these Zygosaccharomyces strains in the brood cells may be a determining factor in the expression of pseudohyphae. ...
Symbiotic interactions between microorganisms and social insects have been described as crucial for the maintenance of these multitrophic systems, as observed for the stingless bee Scaptotrigona depilis and the yeast Zygosaccharomyces sp. SDBC30G1. The larvae of S. depilis ingest fungal filaments of Zygosaccharomyces sp. SDBC30G1 to obtain ergosterol, which is the precursor for the biosynthesis of ecdysteroids that modulate insect metamorphosis. In this work, we find a similar insect-microbe interaction in other species of stingless bees. We analyzed brood cell samples from 19 species of stingless bees collected in Brazil. The osmophilic yeast Zygosaccharomyces spp. was isolated from eight bee species, namely Scaptotrigona bipunctata , S. postica , S. tubiba , Tetragona clavipes , Melipona quadrifasciata , M. fasciculata , M. bicolor , and Partamona helleri . These yeasts form pseudohyphae and also accumulate ergosterol in lipid droplets, similar to the pattern observed for S. depilis . The phylogenetic analyses including various Zygosaccharomyces revealed that strains isolated from the brood cells formed a branch separated from the previously described Zygosaccharomyces species, suggesting that they are new species of this genus and reinforcing the symbiotic interaction with the host insects.
... S. cerevisiae has adopted asymmetric cell division as its main mode of proliferation and must therefore establish an axis of polarity once per cell cycle (Martin and Arkowitz, 2014;Chiou et al., 2017). In addition, its polarity network is integrated with several different signaling networks to allow different growth modes in response to environmental cues, such as those that signal cell cycle progression (Yoshida and Pellman, 2008), filamentous growth (Cullen and Sprague, 2012) and the activation of stress response pathways (Saito, 2010;Waltermann and Klipp, 2010). Here, we study whether the polarity network can restore its coupling to signaling networks after this coupling has been lost due to a genetic perturbation and how this restoration depends on selective pressures from the environment. ...
The ability of cells to translate different extracellular cues into different intracellular responses is vital for their survival in unpredictable environments. In Saccharomyces cerevisiae , cell polarity is modulated in response to environmental signals which allows cells to adopt varying morphologies in different external conditions. The responsiveness of cell polarity to extracellular cues depends on the integration of the molecular network that regulates polarity establishment with networks that signal environmental changes. The coupling of molecular networks often leads to pleiotropic interactions that can make it difficult to determine whether the ability to respond to external signals emerges as an evolutionary response to environmental challenges or as a result of pleiotropic interactions between traits. Here, we study how the propensity of the polarity network of S. cerevisiae to evolve toward a state that is responsive to extracellular cues depends on the complexity of the environment. We show that the deletion of two genes, BEM3 and NRP1 , disrupts the ability of the polarity network to respond to cues that signal the onset of the diauxic shift. By combining experimental evolution with whole-genome sequencing, we find that the restoration of the responsiveness to these cues correlates with mutations in genes involved in the sphingolipid synthesis pathway and that these mutations frequently settle in evolving populations irrespective of the complexity of the selective environment. We conclude that pleiotropic interactions make a significant contribution to the evolution of networks that are responsive to extracellular cues.
... Finally, re-evolving a coupling between anaerobic-xylose growth and metabolism in the bcy1Δ parent implicated mutations in PKA subunit TPK1 and the Ino2/4 repressor OPI1 (Table 2), which is known to be directly regulated by PKA phosphorylation [48]. Opi1 has complex roles in regulating phospholipids, including during the switch to invasive growth depending on nutrients [80], a process also regulated by the RAS/PKA pathway [81][82][83][84][85]. While we were unable to elucidate the exact role of these alleles, our results suggest that the OPI1 mutation may alter Opi1 regulation, especially given that the identified mutation in Opi1 occurs at a known CKII kinase site that regulates Opi1 activity [57]. ...
Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phospho-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1Δ strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1Δ parental strain. The evolved strain harbored mutations in PKA subunit TPK1 and lipid regulator OPI1, among other genes, and evolved changes in lipid profiles and gene expression. Deletion of the evolved opi1 gene partially reverted the strain's phenotype to the bcy1Δ parent, with reduced growth and robust xylose fermentation. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.
... Our results showed that triploid strains have higher ability to induce pseudohyphal cells than diploid strains. Similar observations have been reported in other yeast like S. cerevisiae where the ploidy affects the filamentous growth (Cullen and Sprague., 2012;Gancedo J., 2001). Indeed, according to the ploidy, different cell differentiations were observed in S. cerevisiae. ...
Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group.
... The most well-known gene associated with cell adhesion to agar and plastic surfaces is FLO11 [318]. This gene encodes a GPI-anchored cell surface flocculin that contains a specific domain responsible for cellsurface adhesion [316,317] and required for the yeast differentiation into pseudohyphae [327,328]. It is a key protein, involved in the formation of structured colony biofilms [329], flor [330], biofilms [331], and mats [295,332]. ...
Microbes are traditionally regarded as planktonic organisms, individual cells that live independently from each other. Although this is true, microbes in nature mostly live within large multi-species communities forming complex ecosystems. In these communities, microbial cells are held together and organised spatially by an extracellular matrix (ECM). Unlike the ECM from the tissues of higher eukaryotes, microbial ECM, mostly that of yeasts, is still poorly studied. However, microbial biofilms are a serious cause for concern, for being responsible for the development of nosocomial infections by pharmacological drugs-resistant strains of pathogens, or for critically threatening plant health and food security under climate change. Understanding the organization and behaviour of cells in biofilms or other communities is therefore of extreme importance. Within colonies or biofilms, extremely large numbers of individual microbial cells adhere to inert surfaces or living tissues, differentiate, die or multiply and invade adjacent space, often following a 3D architectural programme genetically determined. For all this, cells depend on the production and secretion of ECM, which might, as in higher eukaryotes, actively participate in the regulation of the group behaviour. This work presents an overview of the state-of-the-art on the composition and structure of the ECM produced by yeasts, and the inherent physicochemical properties so often undermined, as well as the available information on its production and delivery pathways.
