Role of Mitochondrial Retrograde Pathway in Regulating Ethanol-Inducible Filamentous Growth in Yeast

In yeast, ethanol is produced as a by-product of fermentation through glycolysis. Ethanol also stimulates a developmental foraging response called filamentous growth and is thought to act as a quorum-sensing molecule. Ethanol-inducible filamentous growth was examined in a small collection of wine/European strains, which validated ethanol as an inducer of filamentous growth. Wine strains also showed variability in their filamentation responses, which illustrates the striking phenotypic differences that can occur among individuals. Ethanol-inducible filamentous growth in Σ1278b strains was independent of several of the major filamentation regulatory pathways [including fMAPK, RAS-cAMP, Snf1, Rpd3(L), and Rim101] but required the mitochondrial retrograde (RTG) pathway, an inter-organellar signaling pathway that controls the nuclear response to defects in mitochondrial function. The RTG pathway regulated ethanol-dependent filamentous growth by maintaining flux through the TCA cycle. The ethanol-dependent invasive growth response required the polarisome and transcriptional induction of the cell adhesion molecule Flo11p. Our results validate established stimuli that trigger filamentous growth and show how stimuli can trigger highly specific responses among individuals. Our results also connect an inter-organellar pathway to a quorum sensing response in fungi.


INTRODUCTION
Fungal species represent a diverse group of microorganisms. Most fungal species exist in the wild. Other species live in commensal or pathogenic relationships with host organisms, while others still have been domesticated for food and technological benefits. Saccharomyces sensu stricto represents a group of highly related yeasts (Borneman and Pretorius, 2015). Saccharomyces cerevisiae and its relatives are commonly used in research laboratories and a variety of industrial processes. The ability of Saccharomyces to produce ethanol from several sugar sources makes it an essential component of the brewing and wine-making industries. Yeast not only produces ethanol as the major by-product of the alcoholic fermentation of sugars but also produces minor compounds such as aromatic (or fusel) alcohols that impart flavor and bouquet to wines. These properties have been studied to improve ethanol production and to understand the molecular basis of nutrient sensing and regulatory mechanisms in eukaryotes (Fleet and Heard, 1993;Ribéreau-Gayon et al., 2000;Beltran et al., 2004Beltran et al., , 2008Alper et al., 2006;Zaman et al., 2008).
Many fungal species, including yeasts, can undergo filamentous growth. Filamentous growth in yeast is a developmental foraging response, where cells become elongated and grow in connected chains (Gimeno et al., 1992;Kron et al., 1994). In some settings, cells can penetrate surfaces, which is known as invasive growth (Roberts and Fink, 1994). Some fungal species grow as multinucleate hyphae. Other species, like S. cerevisiae, produce pseudohyphae where cells undergo cytokinesis at each cell division. Filamentous growth has been extensively studied in yeast and other species, particularly pathogens, which require filamentous growth for virulence (Madhani and Fink, 1998;Lengeler et al., 2000;Polvi et al., 2015). Such studies have led to insights into the triggers, signaling pathways and transcriptional targets that control developmental responses in fungi and other eukaryotes.
Fungal species also utilize small molecules to interpret information about their environment. Like many other microbial species (Miller and Bassler, 2001;Parsek and Greenberg, 2005;Rumbaugh et al., 2009), S. cerevisiae exhibits quorum-sensing responses (Hlavacek et al., 2009;Prunuske et al., 2012). Yeast can sense and respond to ammonia (Palkova et al., 1997), aromatic (fusel) alcohols (Chen and Fink, 2006), and ethanol (Dickinson, 1994(Dickinson, , 1996Lorenz et al., 2000). By products of the Ehrlich reactions (Hazelwood et al., 2008), fusel alcohols are formed by conversion of several amino acids into glutamate as a nitrogen source under nitrogen-limiting conditions (Ljungdahl and Daignan-Fornier, 2012). Fusel alcohols are produced at higher levels in nitrogen-limiting medium and sensed in a densitydependent manner by a PKA-dependent mechanism to regulate filamentous growth (Chen and Fink, 2006). Multiple fungal species produce and sense a variety of aromatic alcohols, which may impart selectivity in this type of cellular communication Chen and Fink, 2006;Sprague and Winans, 2006;Kruppa, 2008;Langford et al., 2013). Recent efforts have expanded the diversity alcohols that can be sensed and measured their impact on fungal behavioral responses (Ghosh et al., 2008;Wuster and Babu, 2010;Sharma and Prasad, 2011;Albuquerque and Casadevall, 2012;Bojsen et al., 2012;Avbelj et al., 2015;Williams et al., 2015). An open question has been to identify the regulatory pathways that control alcoholmediated morphogenesis and understand how cells detect and respond to these stimuli. Addressing this problem has a practical benefit, as industrial manipulation of yeast may be accelerated by understanding density-dependent growth and behavioral responses (Westman and Franzen, 2015).
To better understand common and unique elements of the filamentous growth response, a diverse collection of strains was examined from the "wine/European" group (Goffeau et al., 1996;Wei et al., 2007;Borneman et al., 2008Borneman et al., , 2011Argueso et al., 2009;Liti et al., 2009;Novo et al., 2009). Most strains tested underwent filamentous growth in response to limiting glucose, limiting nitrogen, or the presence of ethanol or fusel alcohols. A specific role for the mitochondrial retrograde (RTG) pathway, which controls the response to compromised mitochondrial function (Liu and Butow, 2006) and is known to regulate filamentous growth (Jin et al., 2008;Chavel et al., 2010Chavel et al., , 2014Aun et al., 2013;Starovoytova et al., 2013), was identified as a specific regulator of ethanol-inducible invasive growth. RTG regulated TCA cycle flux in response to ethanol to modulate filamentous growth. Thus, the study connects an inter-organellar signaling pathway to a quorum-sensing morphogenetic response in fungi.

Yeast Strains, Media, and Growth Conditions
Yeast strains are described in Table 1. Standard media was used (Rose et al., 1990). Yeast strains were generated by polymerase chain reaction (PCR)-based homologous recombination techniques using auxotrophic or antibiotic resistant markers (Goldstein and McCusker, 1999 (Gimeno et al., 1992)]. Media was supplemented with uracil for auxotrophic mutants. For some experiments, SD and SLAD media were supplemented with 500 µM tryptophol, tyrosol, or phenylethanol and 2%(v/v) ethanol. The CIT2-lacZ plasmid has been described (Liu and Butow, 1999) and was provided by Dr. Zhengchang Liu (Louisiana State University, New Orleans). Beta-galactosidase assays were performed as described (Chavel et al., 2014).

Pseudohyphal Growth Assays
Examination of pseudohyphae was determined as described (Gimeno et al., 1992). Strains were grown for 16 h at 28 • C in MM and harvested by centrifugation (1,000 rpm for 3 min). To obtain single colonies, cells were diluted by a factor of 10 6 in sterile water, and 100 µL of cells were spread onto media (SAD, SALG, and SLAD). Plates were incubated at 28 • C and observed daily for 10 d by microscopy for colony morphology.

Invasive Growth Assays
Strains were grown for 16 h at 30 • C in MM, harvested by centrifugation (10,000 rpm for 3 min) at an optical density (O.D. A 600 ) of 2.0, washed once in sterile water and resuspended in sterile water. Ten microliters of cells were spotted on semisolid agar media. Plates were incubated at 28 • C. Invasive growth was determined by the plate-washing assay (Roberts and Fink, 1994). Colonies were photographed before and after washing over a 10 days period. Plates were washed in a stream of water (soft wash) and colonies were rubbed from the surface with a gloved finger (hard wash). ImageJ (http://rsb.info.nih. gov/ij/) was used to quantitate invasive growth (Zupan and Raspor, 2008). Background intensity was determined for each spot and subtracted from the densitometry of the area of invaded cells. Densitometric analysis was performed on invasive patches over multiple days. Tukey's t-test was used to determine statistical significance and generate p-values. The Shapiro-Wilk and Jarque-Bera normality tests showed that the data fit a normal distribution. A non-parametric statistics test (Wilcoxon test) showed the same results as the Tukey's t-test.

Quantitative Polymerase Chain Reaction (qPCR) Analysis
Quantitative PCR was performed as described (Beltran et al., 2004). Ethanol addition stimulated the expression of FLO11 at all-time points except 24 h. Strains were grown in MM for 24 h at 28 • C, washed with MiliQ sterile water (Millipore Q-PODTM Advantage A10) and resuspended in the indicated media at an O.D. A 600 of 2.0. Cells were inoculated in SLAD media and in SAD media, and samples were taken at 2 h. To study the effect of nitrogen concentration in FLO11 expression, strains were grown in MM for 24 h at 28 • C, washed with MiliQ sterile water (Millipore Q-PODTM Advantage A10) and resuspended in SAD and SLAD media at an O.D. A 600 of 2.0. Samples were taken at 2 h to analyze the FLO11 expression. To study the effect of ethanol in FLO11 expression, cells were inoculated at an O.D. A 600 of 2.0 in SLAD medium with or without ethanol (2% v/v) Samples were taken at 45 min, 2, 8, and 24 h. RNA extraction was performed using an RNeasy Mini Kit (Qiagen). RNA concentration was adjusted to 320 ng/µL. Reverse transcription was performed using SuperScript R III Reverse Transcriptase (Invitrogen) and Oligo (dt) 20 Primer (Invitrogen). qPCR was performed using an Applied Biosystems 7300 Fast Real-Time PCR System (Applied Biosystems, USA). SyberGreen master mix was used according to the manufacturer's instructions (Applied Biosystems, USA). Reactions contained 25 µL sample (5 µL cDNA, 1 µM each primer, 10 µL SyberGreen master mix, H 2 0 q.s.p. 25 µL). The starting quantity of genes was normalized with ACT1 (Chavel et al., 2010). Relative gene expression was calculated using the 2 − Ct formula, where Ct is defined as the cycle at which fluorescence was determined to be statistically significant above background; Ct is the difference in Ct of the FLO11 gene and housekeeping gene (ACT1). The primers used were FLO11 forward (5 ′ -CACTTTTGAAGTTTATGCCACACAAG-3 ′ ) and FLO11 reverse (5 ′ -CTTGCATATTGAGCGGCACTAC-3 ′ ) based on Chen and Fink (2006), and ACT1 forward (5 ′ -TGGATTCCGGTGATGGTGTT-3 ′ ) and ACT1 reverse (5 ′ -CGGCCAAATCGATTCTCAA-3 ′ ).

Microscopy
Differential-interference-contrast (DIC) and bright-field microscopy was performed using an Axioplan 2 fluorescent microscope (Zeiss) with a PLAN-APOCHROMAT 100X/1.4 (oil) objective (N.A. 0.17). Digital images were obtained with the Axiocam MRm camera (Zeiss). Axiovision 4.4 software (Zeiss) was used for image acquisition and analysis and for rendering 3D Z-stack images. Images were further analyzed in Adobe Photoshop, where adjustments of brightness and contrast were made.

Exploring Filamentous Growth in a Collection of Wild and Industrial Yeast Strains
To understand the common and unique features of filamentous growth in yeast, a collection of wild and industrial yeast strains used in wine making was examined ( Table 1). Strains were compared to 1278b, a well-characterized strain background that undergoes filamentous growth (Gimeno et al., 1992), and S288c, which is commonly used in research laboratories (Mortimer and Johnston, 1986) but has acquired mutations due to genetic manipulation that render it unable to undergo filamentous growth (Liu et al., 1996;Dowell et al., 2010;Chin et al., 2012).
One aspect of filamentous growth is invasive growth, which can be assessed by the plate-washing assay (PWA), and which measures penetration of filamentous cells into surfaces (Roberts and Fink, 1994). Invasive growth in nutrient-rich (SAG) conditions was compared to conditions that induce filamentous growth, nitrogen limitation (SLAD; Gimeno et al., 1992) and glucose limitation (SALG; Cullen and Sprague, 2000) as shown in Figure 1A. The results were quantitated by densitometric analysis ( Figure 1B). As expected, S288c did not undergo invasive growth, and 1278b underwent invasive growth that was higher in media lacking glucose or nitrogen (Figure 1A, washed and Figure 1B). Most wine strains underwent invasive growth, which was stimulated in nitrogen-and glucose-limited medium (including VIN7, W27, QA23, T73, SB, and S1; Figures 1A,B). Three strains showed a different trend: P5 invaded equally well in glucose-rich and glucose-limiting media, P24 did not invade nitrogen-limiting medium, and Nsa showed constitutive invasion. Moreover, the pattern of invasive growth varied widely among strains ( Figure 1A).
Another aspect of filamentous growth is pseudohyphal growth, which can be measured by microscopic examination of colony peripheries (Gimeno et al., 1992). As expected, S288c did not form pseudohyphae, and 1278b formed pseudohyphae in nitrogen-limiting medium (Figure 1C, SLAD). Most strains formed pseudohyhae in nitrogen-limiting media ( Figure 1C, including VIN7, W27, QA23, T73, SB, P5, S1, and Nsa), except SB, which did not form pseudohyphae until day 16 (for Figure 1C, day 5 is shown) and P24, which did not form pseudohyphae by day 20 when the experiment was terminated. The pattern of pseudohyphae varied among strains. With the exception of Nsa, which formed pseudohyphae in glucose-(Figure S1A, Nsa SALG, arrow) and nitrogen-limiting media, all other strains formed pseudohyphae exclusively under nitrogen-limitng conditions. Invasive and pseudohyphal growth require cell adhesion mediated by the flocculin Flo11p (Lambrechts et al., 1996;Lo and Dranginis, 1996;Guo et al., 2000). FLO11 expression is induced during filamentous growth (Rupp et al., 1999a). A subset of wine strains that were tested all showed induction of FLO11 expression under nitrogen-limiting conditions ( Figure S1B). Therefore, above results agree with the widely accepted notion that glucose and nitrogen limitation are general inducers of filamentous growth.
Ethanol also stimulates filamentous growth (Dickinson, 1994(Dickinson, , 1996Lorenz et al., 2000). Ethanol induced filamentous growth specifically in nitrogen-limiting medium ( Figure S2A) and showed a maximal effect at a concentration of 2% ( Figure S2B). At this concentration, ethanol did not impact growth ( Figure  S2C; yeast can survive in 12% ethanol; Lleixà et al., 2016). Thus, tests were performed at 2% ethanol in nitrogen-limiting media. As expected, S288c did not show invasive growth by the addition of ethanol (Figures 2A-C), and 1278b showed ethanol-inducible invasive growth (Figures 2A,B). In particular, cells invaded the agar more robustly (Figures 2A,B), and pseudohyphae formed at earlier time points (Figure 2C, colonies were grown for 2 days compared to 5 days in Figure 1C). With the exception of P24 and Nsa, most strains showed increased invasive growth in response to ethanol (Figures 2A-C including VIN7, W27, QA23, T73, SB, S1, and P5). By these criteria, ethanol can also be viewed as a general inducer of filamentous growth. The fusel alcohol tryptophol stimulates filamentous growth in 1278b strains ( Figure S3; Chen and Fink, 2006). Tryptophol stimulated invasive growth of most wine strains in nitrogenrich (SAD) but not nitrogen-limiting (SLAD) medium ( Figure  S3, including VIN7, W27, QA23, T73, and S1). Thus, in line with previous studies, fusel alcohols like tryptophol are general inducers of filamentous growth.

Major Filamentation Regulatory Pathways are not Required for Ethanol-Inducible Filamentous Growth
We focused on ethanol-inducible filamentous growth because ethanol was a stronger inducer of filamentous growth than fusel alcohols. How ethanol is sensed and triggers filamentous growth has not been extensively studied. The ethanol response occurred in diploid (Figure 2) and haploid (Figure 3) strains of the 1278b background, which facilitated genetic analysis of the response.
Signaling pathways known to regulate filamentous growth were tested for a role in regulating ethanol-inducible filamentous growth. Specifically, mutants were tested that lack key regulators of fMAPK (ste11 ; Ste11p is the MAPKKK), Ras2p-cAMP-PKA (ras2 ) and PKA (Tpk in yeast) subunits Tpk1p, Tpk2p, and Tpk3p (tpk1 , tpk2 , and tpk3 ), Snf1p (snf1 ), Rim101p (rim101 ), Rpd3p(L) (sin3 ), Elongator (elp2 ), and Pho85p (pho85 ). Surprisingly, all of the mutants showed enhanced invasive growth in media containing ethanol (Figures 3A,B). The examination of colony perimeters generally bore this out, either showing enhanced filament formation or clumpiness ( Figure 3C, arrows), which is indicative of elevated cell-cell adhesion. Colony perimeters did not show a change for the ste11 and rim101 mutants. Thus, fMAPK and Rim101 pathways may play some role in mediating ethanol-dependent filamentous growth. In summary these results show that ethanol exerts its effect on filamentous growth independent of several of the major regulatory pathways that control filamentous growth.
Unexpectedly, several mutants did not show an invasive growth defect in SLAD media. Specifically, the rim101 , sin3 , snf1 , elp2 , and pho85 mutants invaded the agar as well as or better then wild-type cells [Figures 3A,B; tpk3 is not defective for invasive growth (Robertson and Fink, 1998a;Robertson et al., 2000;Chavel et al., 2010)]. We have previously shown that the rim101 (Chavel et al., 2014), sin3 (Chavel et al., 2010), snf1 (Cullen and Sprague, 2000), elp2 (Abdullah and Cullen, 2009), and pho85 (Chavel et al., 2014) mutants have an invasive growth defect on rich media, and we verified that phenotype here ( Figure S4A; YPD). Thus, there may be differences in the roles these pathways play in regulating   invasive growth depending on growth on YPD or SLAD. This hypothesis is consistent with the fact that mutants scored for pseudohyphal and invasive growth do not completely overlap in a genome-wide screen  and with the fact that several pathways, like Snf1p, play different roles in response to carbon and nitrogen limitation (Orlova et al., 2010).

Mitochondrial Retrograde Pathway is Required for Ethanol-Inducible Invasive Growth
Other proteins and pathways regulate filamentous growth than those tested above . A broader collection of genes implicated in filamentous growth regulation was examined. One of these is the mitochondrial retrograde pathway (or RTG pathway; Sekito et al., 2002;Liu et al., 2003;Liu and Butow, 2006;Kleine and Leister, 2016), which senses changes in metabolic respiration (Aun et al., 2013) to regulate filamentous growth. The RTG pathway has recently been shown to regulate the filamentation response to the alcohol butanol (Starovoytova et al., 2013). Rtg2p is a positive regulator of the retrograde pathway (Ferreira Junior et al., 2005). The rtg2 mutant was defective for ethanol-dependent invasive growth (Figures 4A-C). The RTG pathway is composed of two other regulators, the basic helixloop-helix leucine zipper transcription factors Rtg1p and Rtg3p, which hetero-dimerize to regulate transcription (Jia et al., 1997). The rtg1 and rtg3 mutants were also defective for ethanoldependent invasive growth (Figures 4A-C).
The RTG pathway controls expression of genes that function to ameliorate defects in mitochondrial function (Epstein et al., 2001). The activity of the RTG pathway can be assessed by examining the expression of the CIT2 gene, which is a target of the retrograde pathway (Liao and Butow, 1993;Chelstowska and Butow, 1995;Kos et al., 1995;Jia et al., 1997;Liu and Butow, 1999) that encodes peroxisome citrate synthase (Kim et al., 1986). Ethanol stimulated the activity of a CIT2-lacZ transcriptional reporter ( Figure 4D) in a manner that was dependent on Rtg2p ( Figure 4D). Interestingly, the data indicates that ethanol induces the RTG pathway. One possibility is that nitrogen and ethanol both activate the RTG pathway. The addition of ethanol to cells grown in nitrogen-limiting media showed an additional stimulation ( Figure 4D). Thus, nitrogen limitation and ethanol both contribute to RTG pathway activity. Therefore, the mitochondrial retrograde pathway regulates ethanol-inducible filamentous growth.

Mitochondrial Retrograde Pathway Regulates Ethanol-Inducible Filamentous Growth Independent of fMAPK, TOR, and HOG Pathways
To define how the RTG pathway connects to the ethanol response, known regulators of that pathway were examined. Mks1p is a negative regulator of multiple pathways, including Rtg2p in the mitochondrial retrograde pathway (Dilova et al., 2004;Ferreira Junior et al., 2005). Mks1p was not required for invasive growth in response to ethanol (Figures 5A-C), which indicates that another negative regulator of the pathway might function in this context. The RTG pathway can regulate the fMAPK pathway (Chavel et al., 2010), as part of a highly coordinated transcriptional sensing and signaling circuit among the pathways that regulate filamentous growth (Borneman et al., 2006;Bharucha et al., 2008;Chavel et al., 2014). We tested whether cells with an up-regulated RTG pathway functioned through fMAPK. An mks1 ste11 double mutant, which has an up-regulated retrograde pathway and lacks the MAPKKK for the fMAPK pathway (Ste11p), showed ethanol-inducible invasive growth. This result aligns with the abovementioned results that fMAPK does not regulate ethanol-dependent filamentous growth and indicates that the mitochondrial retrograde pathway does not control filamentation through fMAPK (Figures 5A,B). As shown above, the mks1 ste11 double mutant did not show an increase in filamentation at colony peripheries ( Figure 5C).
Another major regulator of the mitochondrial retrograde pathway is the TOR pathway, which is a ubiquitous nutrientregulatory pathway in eukaryotes (Bar-Peled and Sabatini, 2014). TOR plays an important role in nutrient-regulated responses in yeast (Heitman et al., 1991) and is a master regulator of nitrogen control (Beck and Hall, 1999;Cardenas et al., 1999;Bruckner et al., 2011;Kingsbury et al., 2015). TOR signaling also links nitrogen quality to the activity of the Rtg1p and Rtg3p transcription factors (Komeili et al., 2000). TOR specifically regulates the expression of genes encoding RTG pathway components (Crespo et al., 2002;Dilova et al., 2004). We found that the TOR pathway was not required for ethanol-inducible filamentous growth (Figures 5A-C; tor1 , tco89 ). In addition, the AGC-type kinase Sch9p, which is phosphorylated by and is a major target of TORC1, and which contributes to TORC1-mediated regulation of ribosome biogenesis (Urban et al., 2007;Wei and Zheng, 2009), was not required for ethanol-dependent invasion (sch9 Figures 5A,B, although it was required for filamentation at colony perimeters Figure 5C). These results may not be entirely surprising, because although TOR and the mitochondrial retrograde pathway are functionally connected, the retrograde response to mitochondrial dysfunction is not dependent on TOR1-dependent regulation of retrograde gene expression (Giannattasio et al., 2005). Therefore, the mitochondrial retrograde pathway controls ethanol-inducible filamentous growth independent of TOR and at least partly independently of Sch9p.
In addition to TOR, the SAP-or p38-type high osmolarity glycerol response (HOG) MAP kinase pathway, which controls the response to osmotic and other stresses (Westfall et al., 2004;Saito, 2010), also regulates the RTG pathway (Ruiz-Roig et al., 2012). The HOG pathway was not required for ethanol-inducible filamentous growth (Figures 5A-C, pbs2 ). Another function of the RTG pathway is to stimulate peroxisome biogenesis in periods of mitochondrial stress (Liao and Butow, 1993;Chelstowska and Butow, 1995;Kos et al., 1995;Epstein et al., 2001). Peroxisomes, which control elements of metabolism and can be regulated by the RTG pathway (Chelstowska and Butow, 1995), may impact ethanol-dependent filamentous Beta-galactosidase activity of the CIT2-lacZ reporter in wild-type cells and the rtg2 mutant grown in 1 or 50 mM (NH 4 ) 2 SO 4 with or without 2% ethanol (v/v). Experiments were performed in triplicate from independent inductions. Error bars represent the standard deviation between experiments. Asterisk denotes a p < 0.01 for samples relative to wild type in media lacking ethanol (Ctl). growth. A mutant lacking peroxisomes was not required for ethanol-dependent filamentous growth, indicating that this is not the case (Figures 5A-C, pex3 ). However, the pex3 mutant did show some defect (Figure 5B), and Cit2p, which is a target of RTG, was induced by ethanol ( Figure 4D). These proteins regulate the glyoxylate cycle (Jazwinski, 2013) and it is possible that that metabolic pathway plays a role in regulating ethanol-inducible filamentous growth. Therefore, the mitochondrial retrograde pathway regulates ethanol-inducible filamentous growth in a manner that is separate from TOR, fMAPK, and HOG, and partly independent of peroxisome function.

Regulation of the TCA Cycle Underlies the Role of the Mitochondrial Retrograde Pathway in Controlling Ethanol-Inducible Filamentous Growth
The tricarboxylic acid (TCA or citric acid/Krebs) cycle functions through a series of reactions to generate ATP and produce reducing agents necessary for mitochondrial electron transport and energy generation. The TCA cycle is compromised in cells experiencing mitochondrial defects, but flux through the pathway can be maintained by the action of the RTG pathway (Liu and Butow, 1999;Lin et al., 2011), which is a major function of the RTG pathway (Butow and Avadhani, 2004). Glutamate can suppress the requirement for the retrograde pathway in the TCA cycle by increasing metabolic flux (Liu and Butow, 1999). Glutamate suppressed the defect in ethanol-inducible filamentous growth of the rtg1 , rtg2 , and rtg3 mutants (Figures 6A-C). The role of the RTG pathway in regulating ethanol-inducible invasion suggests that mitochondrial respiration is important for ethanoldependent invasive growth. Consistent with this possibility, rho 0 cells, which lack a functional mitochondria, were defective for ethanol-inducible invasive growth (Figures 6A-C). Thus, one function of the RTG pathway in ethanol-dependent filamentous growth is to stimulate flux through the TCA cycle. Glutamate did not suppress the invasive growth defect of the flo11 mutant (Figures 6A-C). Given that Flo11p is the main cell adhesion molecule that regulates filamentous growth, these results suggest that glutamate-dependent invasive growth in rtg mutants is mediated (in some manner) through Flo11p.
The mitochondrial retrograde pathway has also been shown to regulate deoxyribonucleotide pools by impacting the rate of threonine metabolism (Hartman, 2007). Hydroxyurea induces a cell-cycle delay (Adams and Lindsay, 1967) and reduces the rate of DNA synthesis (Niu et al., 2008), and accordingly triggers a filamentation-like response (Jiang and Kang, 2003). Ethanol may impact threonine levels and DNA synthesis rates and induce retrograde-dependent filamentation. However, hydroxyurea, unlike ethanol, did not cause invasive growth in SLAD medium ( Figure S4B). Moreover, the elongated cell morphology induced by hydroxyurea was retrograde-independent ( Figure  S4C). Therefore, the mitochondrial retrograde pathway probably does not regulate ethanol-dependent filamentous growth by influencing the rate of threonine metabolism.
Several other mutants that are defective in pathways surrounding the TCA cycle, ethanol uptake and metabolism, signaling, and the cell cycle were examined for a role in ethanolinducible filamentous growth ( Figure S5). Most of the mutants examined showed a detectable reduction in ethanol-inducible invasive growth ( Figure S5). Two mutants stood out. One lacked Adh2p, which might be expected as that protein catalyzes the conversion of ethanol to acetaldehyde (Bennetzen and Hall, 1982;Young and Pilgrim, 1985;Dickinson et al., 2003). The other lacked Csf1p ( Figure S5), a protein that is required for fermentation at low temperatures (Tokai et al., 2000). Notably, the wine yeast P24, which does not invade the agar in SLAD medium, is defective for growth at low temperatures (García-Ríos et al., 2014). Thus, the regulators of ethanol-inducible filamentous growth may encompass a more diverse collection of proteins than has been defined here.

Ethanol-Inducible Filamentous Growth Requires the Polarisome and Occurs through Induction of FLO11 Expression
Filamentous growth involves at least three major regulatory changes. One is an increase in cell length, which is mediated by a delay in the cell cycle (Kron et al., 1994) and by an increase in polarized growth by a Cdc42p-dependent mechanism that involves the polarisome (Cullen and Sprague, 2002). The formin Bni1p (Evangelista et al., 1997) and accessory proteins Bud6p, Pea2p, and Spa2p comprise the polarisome (Amberg et al., 1997;Sagot et al., 2002;Graziano et al., 2011;Tu et al., 2012). Another change is a switch in polarity to distal-unipolar budding that requires the distal-pole landmark Bud8p (Gimeno et al., 1992;Cullen and Sprague, 2002). Bud8p is a distal-pole marker that localizes to the distal pole of the cell (Harkins et al., 2001). The third change, as discussed above, is an increase in adhesion mediated by the cell adhesion molecule Flo11p (Lambrechts et al., 1996;Lo and Dranginis, 1996;Guo et al., 2000). The different aspects of filamentous growth are genetically separable and can be examined by mutants that specifically compromise each aspect of the response (Cullen and Sprague, 2002). Mutants were examined that were specifically defective for polarized growth (bud6 ), polarity reorganization (bud8 ), or cell adhesion (flo11 ). Ethanol-inducible filamentous growth occurred in cells lacking Bud8 (Figures 7A-C, bud8 ), which indicates that ethanol does not function mainly through the switch in polarity. Ethanol-inducible filamentous growth was reduced in cells lacking the polarisome component Bud6p (Figures 7A-C, bud6 ). Thus, ethanol induces filamentous growth by a mechanism that is partly dependent on the increase in polarized growth driven by the polarisome. This is consistent with studies of fusel alcohols, which induce dramatic changes in cell length (Dickinson, 1996;Lorenz et al., 2000). Ethanol-inducible filamentous growth was also dependent on Flo11p (Figures 7A-C, flo11 ). Consistent with this result, ethanol stimulated the expression of the FLO11 gene ( Figure 7D). Therefore, ethanol-inducible filamentous growth, which is controlled by the RTG pathway, requires polarisome function and occurs by a mechanism that involves Flo11pdependent transcriptional induction.

DISCUSSION
Filamentous growth in yeast has been mainly studied in one strain background ( 1278b; Gimeno et al., 1992), in part because most laboratory strains have lost filamentation properties due to genetic manipulation in the laboratory (Liu et al., 1996;Dowell et al., 2010;Chin et al., 2012). Although filamentous growth is common among "wild" S. cerevisiae strains (Carstens et al., 1998;Sidari et al., 2014), the triggers of filamentous growth have not been extensively characterized in other backgrounds. By   Figure 1B. Asterisk denotes a p < 0.01 for samples relative to wild type invasion in SLAD. Double asterisk denotes a p < 0.01 for samples relative to wild type invasion in SLAD with ethanol. (C) Colony peripheries from the plates in panel (A) were examined by microscopy 20X magnification. Bar, 50 µm. (D) Ethanol stimulates FLO11 expression in SLAD medium. Cells were incubated in SLAD (orange bar) or SLAD with ethanol (blue bar). Gene expression was examined by qPCR at time 45 min and normalized to a control transcript (ACT1). Error bar represents standard difference between samples. Asterisk denotes a p < 0.01. examining a collection of wine yeast, we show that most wine strains undergo filamentous growth. The strains also showed a high degree of phenotypic variation. Phenotypic variation is common among individual strains (Dowell et al., 2010) and may not be surprising given that these strains have undergone selection based on flavor, cold-sensitivity, alcohol tolerance, and flocculation (Suzzi et al., 1984;Fleet, 2003;Borneman et al., 2011).
We show here that nitrogen limitation and carbon limitation induce filamentous growth in most strains. This is consistent with previous claims that nitrogen limitation (Gimeno and Fink, 1994) and carbon limitation (Cullen and Sprague, 2000) trigger the filamentation response. We also show that ethanol and fusel alcohols induce filamentous growth. Ethanol (Dickinson, 1994;Lorenz et al., 2000) and fusel alcohols (Dickinson, 1996;Chen and Fink, 2006) are known to stimulate filamentous growth. Fusel alcohols induced filamentous growth under nutrient-replete conditions, and ethanol stimulated filamentous growth under nitrogen-limiting conditions. Ethanol is a byproduct of glycolysis, whereas fusel alcohols are by-products of Ehrlich reactions. Thus, the two types of alcohols may provide information about different nutritional states. During alcoholic fermentation, S. cerevisiae produces ethanol when it has reached a maximum population density that corresponds with consumption of nitrogen (Beltran et al., 2005). Because nitrogen limitation is itself a trigger for filamentous growth, ethanol may be a coincidence detector of nitrogen levels and TCA compromise. Alternatively, glucose uptake correlates with the rate of the TCA cycle (Heyland et al., 2009). We also identify a potential role for the glyoxylate cycle in regulating ethanol-dependent filamentous growth. Thus, ethanol production may be a readout of nitrogen or glucose availability.
The cellular response to mitochondrial stress is important for biological responses in many systems. Generally speaking, cellular responses to mitochondrial disfunction have been implicated in cancer (Guha and Avadhani, 2013), aging (Friis et al., 2014;da Cunha et al., 2015;Jazwinski, 2015), development (Berkowitz et al., 2016), and inter-organellar homeostasis (Liu and Butow, 2006). Here, we show that the fungal-specific RTG pathway controls ethanol-inducible invasive growth in yeast. Lorenz and Heitman argued that the fMAPK pathway mediates the response to alcohols (Lorenz et al., 2000), and we show that it may play a minor role. Here we establish the RTG pathway as a key pathway in the response. How does the RTG pathway control ethanol-dependent filamentous growth without involving other major filamentation regulatory pathways? One possibility is that the RTG pathway is part of the sensing/signaling mechanism that controls the rate of flux through the TCA cycle (Liu and Butow, 1999;Lin et al., 2011). TCA cycle rate is dependent on carbon and nitrogen levels, which are key inducers of filamentous growth in yeast and other fungal species. Canonical metabolic regulatory pathways that control filamentous growth also control TCA cycle flux including Snf1 (Hedbacker and Carlson, 2008) and TOR (Komeili et al., 2000); thus, TCA cycle activity may be a nexus for monitoring nutritional health.
The connection between TCA cycle flux and filamentous growth may be relevant from the perspective of pathogenecity. TCA cycle flux has been connected to the evolution of pathogenicity in filamentous fungi (Hogan et al., 2015) and apicomplexan parasites (Oppenheim et al., 2014). TCA cycle reprogramming is becoming increasingly tied to developmental transitions in pathogens ranging from C. albicans (Askew et al., 2009;Guedouari et al., 2014;Grahl et al., 2015), to Plasmodium falciparum (Ke et al., 2015) to Yersinia pseudotuberculosis (Bucker et al., 2014). The boost in TCA cycle flux is critical for phagososomal escape of the bacterial pathogen Francisella (Ramond et al., 2014). Moreover, the fungal RTG pathway is responsible for evasion of programmed cell death in yeast cells growing on non-repressing carbon sources (Guaragnella et al., 2013). Both the RTG pathway and relief of carbon catabolite repression are required for programmed cell death resistance. Evasion of programmed cell death and filamentous growth may be two hallmarks that fungi must acquire to become pathogenic. Our study therefore connects TCA cycle flux, as regulated by the RTG pathway, to an aspect of filamentous growth. Perhaps TCA flux controls developmental and morphogenetic responses in other eukaryotic systems.

AUTHOR CONTRIBUTIONS
BG designed and performed experiments. GB designed experiments. AM designed experiments. MJT designed experiments. PC helped with experimental design and writing the paper.

FUNDING
PC is supported from grants from the NIH (GM098629 and DE022720). The work was supported by the Ministry of Economy and Competitiveness, Spain (Grant no. AGL2013-47300-C3). BG is grateful to the pre-doctoral fellowship from the University Rovira i Virgili and the Oenological Biotechnology research group for a mobility grant.

ACKNOWLEDGMENTS
Thanks to Charlie Boone for generously providing the MATa 1278b deletion collection, and Zhengchang Liu for providing the pCIT2-lacZ plasmid. Thanks to Javier Rodriguez for technical assistance.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphys. 2017.00148/full#supplementary-material