Interactions among HAMP domain repeats act as an osmosensing molecular switch in group III hybrid histidine kinases from fungi.

The members of group III hybrid histidine kinases (HHK) are ubiquitous in fungi. Group III HHK have been implicated to function as osmosensors in the high osmolarity glycerol (HOG) pathway that is essential for fungal survival under high osmolarity stress. Recent literature suggests that group III HHK are also involved in conidia formation, virulence in several filamentous fungi, and are an excellent molecular target for antifungal agents. Thus, group III HHK constitute a very important group of sensor kinases. Structurally, group III HHK are distinct from Sln1p, the osmosensing HHK that regulates the HOG pathway in Saccharomyces cerevisiae. Group III HHK lack any transmembrane domain and typically contain HAMP domain repeats at the N terminus. Until now, it is not clear how group III HHK function as an osmosensor to regulate the HOG pathway. To investigate this, we undertook molecular characterization of DhNIK1, an ortholog from osmotolerant yeast Debaryomyces hansenii. We show here that DhNIK1 could complement sln1 mutation in S. cerevisiae thereby confirming its role as a bona fide osmosensor. We further investigated the role of HAMP domains by deleting them systematically. Our results clearly indicate that the HAMP4 domain is crucial for osmosensing by DhNik1p. Most importantly, we also show that the alternative interaction among the HAMP domains regulates the activity of DhNik1p like an "on-off switch" and thus provides, for the first time, an insight into the molecular mechanism of osmosensing by this group of HHKs.

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens.

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schröt, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.