Antisense expression of the calmodulin gene from Colletotrichum trifolii impairs prepenetration development

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. ß : S 0 3 7 8 -1 0 9 7 ( 0 0 ) 0 0 3 9 2 -X

Mitogen-Activated Protein Kinase Signaling in Plant-Interacting Fungi: Distinct Messages from Conserved Messengers

“…These protein complexes then bind to calcineurin A, a highly conserved protein phosphatase (Luan, 2009). In phytopathogenic fungi, CaMs and CNBs have been involved in virulence (Lee and Lee, 1998;Warwar et al, 2000;Uhm et al, 2003;Harel et al, 2006;Ahn and Suh, 2007;Egan et al, 2009;Cervantes-Chávez et al, 2011), and comparative genomics of the Ca 2+ /CaM/CNB pathway suggest that it is highly conserved in fungi (Rispail et al, 2009).…”

Cdc42 Is Required for Proper Growth and Development in the Fungal Pathogen Colletotrichum trifolii

“…Spore germination is a key step in the colonization process of a new environment by filamentous fungi. Recent studies of C. trifolii have revealed that various signaling molecules, including calcium, cyclic AMP (cAMP), calmodulin, and protein kinases, are important players in regulating spore germination (14,46,50). Here we described the involvement of the small GTPase Cdc42 in this process; it will be of interest to examine the relationship between Cdc42 and these other signaling components.…”

“…Previous studies of C. trifolii have shown that an increase in endogenous cAMP levels by using cAMP-elevating agents, such as 8-Br-cAMP, sodium fluoride, and 3-isobutyl-1-methylxanthine, also results in induction of appressorial differentiation on a noninductive surface, suggesting a possible linkage between CtCdc42 and the cAMP signaling pathway (50). Moreover, several signaling molecules in C. trifolii that are important for appressorium development have been isolated and characterized, including the catalytic subunit of PKA, calmodulin, and lipid-induced protein kinase (14,46,49,50). Molecular analysis of the connection of CtCdc42 with these molecules will improve the understanding of signaling pathways involved in appressorium development in C. trifolii.…”

“…The mechanism(s) of appressorium development has been intensively studied in phytopathogenic fungi, including Magnaporthe grisea and other Colletotrichum spp. The results of these studies indicated that cAMP/PKA-and MAPK-mediated signaling pathways are operative during appressorium development (10,46,(48)(49)(50). Interestingly, disruption of MAGB, encoding an inhibitory group I subunit of a heterotrimeric G protein (G␣i), affects the appressorium formation and pathogenicity of M. grisea (26), suggesting the possibility that G protein may participate in the process of appressorium formation.…”

Restriction enzyme improves the efficiency of genetic transformations in Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao

“…In this sense, genetic transformation is a powerful instrument to study the pathogenicity mechanisms of this fungus at the molecular level. Transformation is widely employed in genetic studies in fungi and has been used in gene disruption (Cummings et al, 1999;Fujimoto et al, 2002;Jin et al, 2005;Seong et al, 2005), gene replacement (Oeser et al, 2002) and gene silencing (Warwar et al, 2000). Mutants have been demonstrated to be a valuable resource for genetic and molecular analysis.…”