Calcineurin-Responsive Zinc Finger Transcription Factor CRZ1 of Botrytis cinerea Is Required for Growth, Development, and Full Virulence on Bean Plants

Abstract

Recently, we showed that the α subunit BCG1 of a heterotrimeric G protein is an upstream activator of the Ca ²⁺ /calmodulin-dependent phosphatase calcineurin in the gray mold fungus Botrytis cinerea . To identify the transcription factor acting downstream of BCG1 and calcineurin, we cloned the gene encoding the B. cinerea homologue of CRZ1 (“CRaZy,” c alcineurin- r esponsive z inc finger transcription factor), the mediator of calcineurin function in yeast. BcCRZ1 is able to partially complement the corresponding Saccharomyces cerevisiae mutant, and the subcellular localization of the green fluorescent protein-BcCRZ1 fusion product in yeast cells depends on the calcium level and calcineurin activity. Bc crz1 deletion mutants are not able to grow on minimal media and grow slowly on media containing plant extracts. Hyphal morphology, conidiation, and sclerotium formation are impaired. The cell wall and membrane integrity, stress response (extreme pH, H ₂ O ₂ , Ca ²⁺ , Li ⁺ ), and ability of the hyphae to penetrate the intact plant surface are affected in the mutants. However, BcCRZ1 is almost dispensable for the conidium-derived infection of bean plants. The addition of Mg ²⁺ restores the growth rate, conidiation, and penetration and improves the cell wall integrity but has no impact on sclerotium formation or hypersensitivity to Ca ²⁺ and H ₂ O ₂ . The expression of a set of recently identified BCG1- and calcineurin-dependent genes is also affected in ΔBc crz1 mutants, confirming that this transcription factor acts downstream of calcineurin in B. cinerea . Since the Bc crz1 mutants still respond to calcineurin inhibitors, we conclude that BcCRZ1 is not the only target of calcineurin.