title: Cch1 and Mid1 are functionally required for vegetative growth under low-calcium conditions in the phytopathogenic ascomycete Botrytis cinerea.
abstract: In the filamentous phytopathogen Botrytis cinerea, the Ca(2+)/calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The cch1 and mid1 genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca(2+)-chelating agents EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The impact of external Ca(2+) was investigated by supplementing with CaCl(2) and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1-green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in B. cinerea.
authors.affiliation: Westfälische Wilhelms-Universität Münster, Institute of Biology and Biotechnology of Plants, Münster, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Harren
authors.forename: Bettina
authors.initials: B
authors.lastname: Tudzynski
mesh_terms: D061245:Axenic Culture; D020171:Botrytis / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002467:Cell Nucleus; D003470:Culture Media; D005656:Fungal Proteins / Q000502:physiology*; D055786:Gene Knockout Techniques; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013172:Spores, Fungal / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D003470:Culture Media; D005656:Fungal Proteins; D002118:Calcium