ABSTRACT
Calmodulin is a ubiquitous highly conserved calcium binding protein involved in cell signalling. Previous studies in our laboratory suggested a role for calmodulin in prepenetration morphogenesis in Colletotrichum trifolii, the causal agent of alfalfa anthracnose. In this report, we describe the cloning, sequencing and partial characterization of the calmodulin gene from C. trifolii. The gene is present as a single copy in the genome of C. trifolii and its predicted amino acid sequence shows considerable homology to other fungal calmodulins. The gene is most highly expressed during conidial germination and appressorial development. Using a Neurospora crassa inducible promoter driving the calmodulin gene in antisense orientation, transformants were obtained with constitutive levels of antisense calmodulin expression. Upon induction, transformants did not develop appressoria and were not pathogenic on alfalfa plants.
Subject(s)
Calmodulin/genetics , Calmodulin/metabolism , Colletotrichum/growth & development , Amino Acid Sequence , Calmodulin/chemistry , Cloning, Molecular , Colletotrichum/genetics , Colletotrichum/metabolism , Gene Expression Regulation, Fungal , Molecular Sequence Data , Plasmids/genetics , Promoter Regions, Genetic , RNA, Antisense/metabolism , RNA, Fungal/metabolism , Sequence Analysis, DNAABSTRACT
Spore germination and appressorium formation are important steps in the process of fungal development and pathogenesis. These prepenetration events, which begin with spore attachment and culminate with appressorium maturation, a common scheme for many pathogenic fungi, are prerequisites for penetration of host external barriers and subsequent colonization. Conditions for in vitro spore germination and appressorium development in Colletotrichum trifolii are described. In addition, effects of Ca(sup2+) and calmodulin on these processes have been examined. Results indicate that, as for other pathogenic fungi, appressorium development is induced on a hard surface. The data suggest that disturbance of calcium homeostasis, by ethylene-bis(oxy-ethylenenitrolo)tetraacetic acid (EGTA) or calcium channel blockers, impairs appressorium development. Moreover, calmodulin inhibitors affect both germination and differentiation, implying that the Ca(sup2+)/calmodulin signal transduction pathway is important in the early development of C. trifolii on the plant host surface.
