Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 cellular integrity. Therefore, mutations affecting different elements of the pathway lead to cell lysis [4,5]. The main- tenance of cellular integrity and morphology, as well as the protection of the cell from adverse environmental con- ditions, depends on the cell wall, an essential structure that has been characterized extensively in Saccharomyces cerevisiae (reviewed in [6,7]). It has three major compo- nents: an inner layer of glucans (b-1,3 and b-1,6-glucan), chitin and an outer layer of mannoproteins. These compo- nents must be correctly assembled in order to build a fully functional structure [6,8,9]. The essentiality of the cell wall for fungal viability makes it one of the most attractive targets for therapeutic inter- vention against fungal pathogens [10]. Treatment with cell wall-perturbing agents such as the chitin-binding dyes Congo red and Calcofluor white or zymolyase, which degrades the b-1,3-glucan network, elicits a cellular survi- val response known as “compensatory mechanism” [11]. This adaptive response includes changes in the yeast tran- scriptional program that we and other groups have charac- terized, not only in yeast mutants affected at different stages of cell wall biosynthesis [12], but also in wild-type yeast cells growing under different conditions causing transient cell wall damage [13-18]. The compensatory response leads to, among other effects, an increase in the amount of b-glucan and chitin, the production of several cell wall proteins and changes in the cross-linking between cell wall polymers [19]. Although several signaling path- ways contribute to the regulation of cell wall remodeling in order to ensure cell integrity, the regulation of this com- pensatory response is controlled mainly by the MAPK Slt2p/Mpk1p (hereafter noted as Slt2p) through the cell wall integrity pathway (for a review, see [20,21]). The CWI pathway is regulated through the cell cycle, being also acti- vated in response to a variety of external stimuli and mor- phological events that cause cell wall stress, such as heat stress, hypo-osmotic shock, mating pheromones, oxidative stress, actin depolymerization, cell wall-related mutations, cell wall-stressing agents, alkaline stress and endoplasmic reticulum (ER) stress [20,22-25]. Several cell membrane proteins (Mid2, Wsc1-4 and Mtl1) [26-28] act as sensors of the CWI pathway. For further intracellular transduction of the activation signal, these sensors interact with the guanine nucleotide exchange factor (GEF) Rom2, activating the small GTPase Rho1, which then activates the yeast protein kinase C (Pkc1). The main role of activated Pkc1 is to trigger a MAPK module. Upon phosphorylation, the MAPKKK Bck1 activates a pair of redundant MAPKKs (Mkk1 and Mkk2), which phosphorylate the MAPK Slt2. Slt2 is a functional homolog of human ERK5 [29], a MAPK that is activated in response to both growth factors and physical and chemical stresses [30,31]. The dually phosphorylated (Thr190/Tyr192) form of Slt2 activates two transcription Page 2 of 17 factors: the MADS-box transcription factor Rlm1 [32] and SBF, a heterodimeric complex of two proteins, Swi4 and Swi6, which are mainly involved in the regulation of gene expression in G1/S transition [33]. Although Rlm1, acti- vated through phosphorylation by Slt2 [34], is responsible for the transcriptional activation of the majority of the genes induced in CWI adaptation responses, a non-cataly- tic mechanism of transcriptional activation mediated by SBF through Slt2 has recently been described [35]. This alternative mechanism extends the regulatory roles of the MAPK cascade. In accordance with the complexity of the cellular pro- cesses related to cell wall homeostasis in yeast, cross- talk between distinct MAPK pathways has recently been described. This complicates the simple linear “top- down” concept of signaling pathways. For example, Slt2 is activated in response to hyperosmotic shock through the HOG1 MAPK pathway [36]. Similarly, our group has shown that treating yeast cells with zymolyase also activates Slt2 in a Hog1 pathway-dependent manner [17,37]. Considering that the CWI pathway is activated under cell wall stress, our working hypothesis is that yeast strains lacking genes functionally relevant for cell wall biogenesis or pathway regulation should present a con- stitutive activation of this route. In this paper, we describe the development of a genomic-wide screening in order to identify genes whose absence produces a functional constitutive activation of the CWI pathway of Saccharomyces cerevisiae under vegetative (non-stressed) growth conditions. As a result, we have identified, for the first time at genomic scale, a map of genes that could be functionally related to the CWI pathway. Furthermore, this report gives new insights into the link between the magnitude of MAPK activation and tran- scriptional induction or cell wall remodeling events. This information can be extended to pathogenic fungi, being useful for future therapeutic purposes. Results and discussion Large-scale identification of gene deletions that activate the cell wall integrity pathway in yeast The CWI pathway of Saccharomyces cerevisiae, governed by the MAPK Slt2, is triggered under conditions that com- promise the integrity of the cell wall. Signaling through this pathway can be monitored by taking advantage of reporters driven by Rlm1-responsive promoters. We have recently developed a transcriptional reporter system, espe- cially suitable for large-scale studies, that potentially allows detecting the functional activation of the CWI pathway [38]. Essentially, this system is based on a plasmid con- struction (pJS05) that includes a transcriptional fusion of the promoter region of the MLP1 gene, one of the main effectors of the transcriptional up-regulation response