Summary: Histidine kinases (HK) sense and transduce via phosphorylation events many intra‐ and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N‐terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape‐function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs.

PPZ1 orthologs, novel members of a phosphoprotein phosphatase family of phosphatases, are found only in fungi. They regulate diverse physiological processes in fungi e.g. ion homeostasis, cell size, cell integrity, etc. Although they are an important determinant of salt tolerance in fungi, their physiological role remained unexplored in any halotolerant species. In this context we report here molecular and functional characterization of DhPPZ1 from Debaryomyces hansenii, which is one of the most halotolerant and osmotolerant species of yeast. Our results showed that DhPPZ1 knock-out strain displayed higher tolerance to toxic cations, and unlike in Saccharomyces cerevisiae, Na(+)/H(+) antiporter appeared to have an important role in this process. Besides salt tolerance, DhPPZ1 also had role in cell wall integrity and growth in D. hansenii. We have also identified a short, serine-arginine-rich sequence motif in DhPpz1p that is essential for its role in salt tolerance but not in other physiological processes. Taken together, these results underscore a distinct role of DhPpz1p in D. hansenii and illustrate an example of how organisms utilize the same molecular tool box differently to garner adaptive fitness for their respective ecological niches.

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1–5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1–5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1–5 protein inside experimental data-based models showed how two chains of HAMP1–5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1–5 structure mediate its function.

The hybrid histidine kinase 3 (HHK3) is a highly conserved sensor kinase in fungi that regulates the downstream HOG/p38 mitogenactivated protein kinase (MAPK). In addition to its role in osmoadaptation, HHK3 is involved in hyphal morphogenesis, conidiation, virulence and cellular adaptation to oxidative stress. However, the molecular mechanisms by which it controls these processes remain obscure. Moreover, HHK3 is a molecular target for antifungal agents such as fludioxonil which thereby interferes with the HOG/p38 pathway, leading to the abnormal accumulation of glycerol and subsequent cell lysis. Here, we used a chemical genomics approach with the yeast Saccharomyces cerevisiae to better understand the fungicidal action of fludioxonil and also the role of HHK3 in fungal growth and physiology. Our results indicated that the abnormal accumulation of glycerol is not the primary cause of fludioxonil toxicity. Fludioxonil appears to impair endosomal trafficking in the fungal cells. We found that the components of class C core vacuole-endosome tethering (CORVET) complex are essential for yeast viability in the presence of a sub-threshold dose of fludioxonil and that their over expression alleviates fludioxonil toxicity. We also noted that by impeding secretory vesicles trafficking, fludioxonil inhibits hyphal growth in the opportunistic fungal pathogen Candida albicans. Our results suggest that HHK3 regulates fungal hyphal growth by affecting vesicle trafficking. Together, our results reveal an important role of CORVET complex in the fungicidal action of fludioxonil downstream of HHK3.

Clathrin is a major coat protein involved in vesicle formation during endocytosis and transport in the endosomal/trans Golgi system. Clathrin is required for normal growth of yeast (Saccharomyces cerevisiae) and in some genetic backgrounds deletion of the clathrin heavy chain gene (CHC1) is lethal. Our lab defined a locus referred to as "suppressor of clathrin deficiency" (SCD1). In the presence of the scd1-v allele ("v" - viable), yeast cells lacking clathrin heavy chain survive but grow slowly, are morphologically abnormal and have many membrane trafficking defects. In the presence of scd1-i ("i"- inviable), chc1∆ causes lethality. As a strategy to identify SCD1, we used pooled linkage analysis and whole genome sequencing. Here, we report that PAL2 (YHR097C) is the SCD1 locus. pal2∆ is synthetic lethal with chc1∆; whereas a deletion of its paralog, PAL1, is not synthetic lethal with clathrin deficiency. Like Pal1, Pal2 has two NPF motifs that are potential binding sites for EH domain proteins such as the early endocytic factor Ede1, and Pal2 associates with Ede1. Also, GFP-tagged Pal2p localizes to cortical patches containing other immobile phase endocytic coat factors. Overall, our data show that PAL2 is the SCD1 locus and the Pal2 protein has characteristics of an early factor involved in clathrin-mediated endocytosis.

Fludioxonil belongs to the phenylpyrrole group of fungicides with a broad antifungal spectrum that has been widely used in agricultural practices for the past thirty years. Although fludioxonil is known to exert its fungicidal action through group III hybrid histidine kinases, the downstream effector of its cytotoxicity is poorly understood. In this study, we utilized a S. cerevisiae model to decipher the cytotoxic effect of fludioxonil. Through genome wide transposon mutagenesis, we have identified Bem2, a Rho GTPase activating protein, which is involved in this process. The deletion of BEM2 resulted in fludioxonil resistance. Our results showed that both the GAP and morphogenesis checkpoint activities of Bem2 were important for this. We also provided the genetic evidence that the role of Bem2 in the cell wall integrity (CWI) pathway and cell cycle regulation could contribute to the fludioxonil resistance phenotype.