The Essential and the Nonessential Roles of Four Clock Elements in the Circadian Rhythm of Metarhizium robertsii

FRQ (frequency protein), FRH (FRQ-interacting RNA helicase), and WC1 and WC2 (white collar proteins) are major clock elements that govern the circadian rhythm in Neurospora crassa. However, deletion of frh is lethal for the viability of N. crassa, making it elusive whether FRH is essential or nonessential for the circadian rhythm. This needs clarification in a fungus where frh deletion is not lethal. Here, the nuclear FRH ortholog proved nonessential for the circadian rhythm of Metarhizium robertsii. The nucleocytoplasmic shuttling of M. robertsii FRQ, WC1, and WC2 orthologs was light-dependent. Yeast two-hybrid assay validated interactions of FRQ with FRH and WC1 instead of FRH with WC1 and WC2 or FRQ with WC2. The circadian rhythm well, shown as conidiation rings of tint and dark in 15 d-old plate cultures grown at 25 °C in a light/dark cycle of 12:12, was abolished in the absence of frq or wc1, partially disturbed in the absence of wc2, but unaffected in the absence of frh. These results indicate a requirement of either FRQ or WC1 instead of FRH for the fungal circadian rhythm. Further analyses of frq and frh mutants revealed the dispensable and the limited roles of FRQ and FRH in the insect-pathogenic lifecycle of M. robertsii, respectively.