ORF/ Gene YCR090C YDL173W YDR290W YGR022C YLR250W YLR338W YMR119W- A YNL058C YNL105W YNR014W YPL158C ERC1 FES1 FPK1 GAT2 GLG2 KSP1 LEA1 MOG1 MSP1 PHM8 RMD11 ROX1 SEC72 SUV3 YPR115W — — SSSS — — SSSS — — — — — — SSSSS — SS — — SS — — — — — — — — — — SSS SSS — R — — — — SSS — — — — — — — SS — — — — — — — — S S S — SS — — — — — — SS — — — SSSSS — — — R SS — — SSSSS SSSSS — R S — — — — — — SSSS SS SS — — — — S — — — — — — — — — — — SS — — SSSSS — — — — — — — — SSSSS — — SSS SSSSS — SSS — SSSS — SSS — Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 Table 2 Sensitivity test on yeast deletant strains Sensitivity Congo red Caspofungin SDS Caffeine Hygromycin B Sensitivity to Congo red (100 μg/ml), Caspofungin (40 ng/ml), SDS (200 μg/ml), Caffeine (12 mM) and Hygromycin B (50 μg/ml) using a spot dilution assay (see Methods for details) is shown. Hypersensitivity levels are shown as the number of spot dilutions ("S”) in which no cell growth is scored. “R” refers to a resistant phenotype and “—” indicates no difference in growth relative to the wild-type strain. Genes of unknown function are grouped in the upper part of the table. from yeast cells stained with CW measured by flow cytometry and the biochemical determination of chitin [74]. Remarkably, 19 (~30%) strains contained more than twice the amount of chitin than the wild-type strain (Figure 4), denoting that a cell wall compensatory mechanism is triggered in these cells. As described above for Slt2 activation, a wide range of chitin content was observed, suggesting that increased deposition of this polymer is adapted to specific cellular requirements. In some of these mutants (nbp2Δ, sla1Δ, vrp1Δ, ylr338wΔ, gas1Δ and arc18Δ) elevated chitin levels and genetic interactions with mutations involved in chitin synthesis have previously been described [75]. In con- trast, the remaining 45 mutants did not record an evi- dent increase in CW binding. Interestingly, about half of those mutants with increased chitin levels have been related functionally to cell wall and/or morphogenesis, Page 8 of 17 whereas within the group of mutants lacking signifi- cantly increased chitin deposition only 18% were assigned to this functional group. These data suggest a functional link between the chitin-related mechanism and activation of the CWI pathway by cell wall and morphogenesis alterations. Eight out of ten mutants with maximum Slt2 phosphorylation, most of them cell wall related (see Table 1), recorded higher chitin levels, reinforcing the idea that this polymer plays a key role in yeast for salvaging the cell under conditions that jeopar- dize cell integrity. However, the identification of mutants with no signifi- cant variations in the amount of chitin indicates that CWI pathway activation could be due to alternative stimuli or internal inputs on this signaling route. In this regard, gat2Δ and lea1Δ mutants are two examples of special interest because they have strong Slt2 activation without affecting their chitin content (Table 1 and Figure 5a), despite they showed cell wall alterations. GAT2 encodes for a poorly characterized putative zinc finger transcription factor, while LEA1 gene product is involved in RNA spli- cing, although its mutation shows synthetic sick inter- action with several cell wall related genes, such as CHS1 or CHS5 [40]. In order to gain further insights into the origin of pathway activation in these mutants, we decided to con- struct double mutants deleting ROM2 or BCK1 in the gat2Δ and lea1Δ backgrounds. Rom2 is the major GEF for Rho1 that is responsible for relaying signals from cell sur- face to Rho1 for its activation [76,77], while Bck1 is the first element of the CWI pathway MAPK module. These mutants allowed us to distinguish whether the phosphory- lation of Slt2 was the result of cell wall stress sensing or otherwise took place directly through the MAPK module of the route independently of upstream elements. After investigating Slt2 phosphorylation in single (gat2Δ and lea1Δ) and double (gat2Δrom2Δ; gat2Δbck1Δ; lea1Δrom2Δ; lea1Δbck1Δ) mutants, it was evident that MAPK activation was fully dependent on Bck1, whereas the lack of Rom2 did not affect the Slt2 activation in gat2Δ and lea1Δ strains (Figure 5b). Nevertheless, participation of other Rho1 GEFs like Rom1 or Tus1 can not be ruled out. This is in contrast to the activation of the CWI path- way by the cell wall stress caused by Congo red, in which Rom2 is demanded for Slt2 activation [37]. These results support the notion that particular cell wall alterations could trigger specific adaptive responses through the CWI pathway. MAPK phosphorylation vs. transcriptional activation An important aspect we wanted to address about the functioning of the CWI pathway was the association between the magnitude of Slt2 phosphorylation and the concomitant effect on gene expression. To achieve this goal, a selected group of mutants, representing different