Disruption of CRZ1 in Bipolaris maydis Causes Hypersecretion of Ophiobolin A, Resulting in Mycelial Growth Inhibition

Calcineurin responsive zinc finger 1 (CRZ1) is a transcription factor related to the calcineurin signaling pathway. In several filamentous fungi, disruption of CRZ1 gene has been reported to have suppressive effects on mycelial growth, suggesting that CRZ1 regulates genes involved in functions such as cell wall integrity, vesicular transport, and ion homeostasis. To investigate the functions of CRZ1 in Bipolaris maydis, we generated CRZ1-disrupted strains (Δcrz1). Morphological observations revealed that mycelial growth of Δcrz1 was inhibited, compared to that of the wild-type strain. Interestingly, high amounts of the secondary metabolite ophiobolin A (OA) were detected in Δcrz1 by LC/MS analysis. In this study, we genetically and chemically demonstrated that the growth suppression in Δcrz1 was caused by the hypersecretion of OA. We identified two OA biosynthesis-related genes, the terpene synthase OPH1 and the cytochrome P450 OPH2. The expression analysis in Δcrz1 found that both OPH1 and OPH2 were significantly up-regulated, indicating that disruption of CRZ1 increases expression of OPH1 and OPH2. We generated disrupted strains of OPH1 and OPH2 in Δcrz1 (Δcrz1Δoph1 and Δcrz1Δoph2, respectively). OA was not detected in Δcrz1Δoph1 nor Δcrz1Δoph2 by LC/MS analysis, suggesting that these genes are essential for OA biosynthesis. In addition, morphological observations showed that wild-type level mycelial growth was restored in both Δcrz1Δoph1 and Δcrz1Δoph2, confirming that the growth suppression of Δcrz1 was caused by the hypersecreted OA. This demonstrates that the disruption of CRZ1 increases the expression of OA biosynthesis-related genes, resulting in the hypersecretion of OA, which inhibits mycelial growth.