Opposite Nuclear Dynamics of Two FRH-Dominated Frequency Proteins Orchestrate Non-Rhythmic Conidiation in Beauveria bassiana

Abstract

Non-rhythmic conidiation favors large-scale production of conidia serving as active ingredients of fungal insecticides, but its regulatory mechanism is unknown. Here, we report that two FREQUENCY (FRQ) proteins (Frq1/2) governed by a unique FRQ-interacting RNA helicase (FRH) orchestrate this valuable trait in Beauveria bassiana, an asexual insect-pathogenic fungus. Frq1 (964 aa) and Frq2 (583 aa) exhibited opposite expression dynamics (rhythms) in nucleus and steadily high expression levels in cytoplasm under light or in darkness no matter whether one of them was present or absent. Such opposite nuclear dynamics presented a total FRQ (pooled Frq1/2) level sufficient to persistently activate central developmental pathway in daytime and nighttime and supports continuous (non-rhythmic) conidiation for rapid maximization of conidial production in a fashion independent of photoperiod change. Importantly, both nuclear dynamics and cytoplasmic stability of Frq1 and Frq2 were abolished in the absence of the FRH-coding gene nonessential for the fungal viability, highlighting an indispensability of FRH for the behaviors of Frq1 and Frq2 in both nucleus and cytoplasm. These findings uncover a novel circadian system more complicated than the well-known Neurospora model that controls rhythmic conidiation, and provide a novel insight into molecular control of non-rhythmic conidiation in B. bassiana.

DIM5/KMT1 controls fungal insect pathogenicity and genome stability by methylation of histone H3K4, H3K9 and H3K36

“…In B. bassiana, the key CDP activator genes brlA and abaA have been shown to act as master regulators of hyphal invasion into insect body, dimorphic transition and aerial conidiation [58]. Expression of both CDP genes was sharply repressed in the absence of vvd [61] or each of two genes encoding frequency proteins (Frq1 and Frq2), which have opposite rhythms in nucleus to persistently activate the CDP genes in daytime and nighttime and hence support nonrhythmic conidiation independent of photoperiod change [62]. In our Δdim5 mutant, abolished transcription of brlA and abaA at early developmental stage and marked repression of vvd and frq1 in the transcriptome could be associated with the severe defects in aerial conidiation and submerged dimorphic transition in vivo and in vitro.…”

“…Interestingly, none of the CDP genes required for conidiation appeared in the list of DEGs. Instead, three other developmentmediated genes (Figure 6(d)) were significantly repressed, including both the clock gene frq1 (BBA_01528) and the blue-light receptor gene vvd (BBA_02876) essential for the CDP activation and the asexual cycle in vivo and in vitro of B. bassiana [61][62][63] and another sporulation-specific gene (BBA_04401) functionally unknown yet. Moreover, 100 dysregulated genes (up/down ratio: 50:50) were putatively involved in transmembrane transport, cellular homeostasis and drug resistance, implicating their comprehensive effects on a wide array of cellular processes and biological aspects.…”

“…Samples taken from the 3-day-old cultures were stained with the cell wall-specific dye calcofluor white and observed under a microscope to reveal conidiation status of each strain at the early stage of development. From day 5 onwards, conidial yield in three samples taken every 2 days from each plate culture with a cork borer (5 mm diameter) was measured as the number of conidia per unit area (cm 2 ) of culture as described previously [61,62]. Meanwhile, biomass levels of each strain were measured from cellophane-overlaid SDAY cultures initiated with the same method.…”

The secondary metabolite regulator, BbSmr1, is a central regulator of conidiation via the BrlA‐AbaA‐WetA pathway in Beauveria bassiana

“…We thought other regulatory factors still existed upstream of BbBrlA to control conidial development and BbSmr1 acted as a major influential factor of the central pathway. Recently, two FRH‐dominated frequency proteins from B. bassiana were found to orchestrate non‐rhythmic conidiation (Tong et al ., 2020). Presently, while we still know few UDA members in entomopathogenic fungi and do not know their relationship in conidiation, our study for BbSmr1 does provide a basis to further explore more conidiation‐related regulatory proteins and to clarify their roles.…”

Essential Role of COP9 Signalosome Subunit 5 (Csn5) in Insect Pathogenicity and Asexual Development of Beauveria bassiana

“…The conidiation status of each strain was microscopically examined using the samples taken from 3- or 6-day-old cultures and stained with calcofluor white (cell wall-specific dye). From day 5 onward, conidial yield in three samples taken every 2 days from each plate culture with a cork borer (5 mm diameter) was quantified as the number of conidia per unit area (cm 2 ), as described previously [ 48 , 49 ]. Meanwhile, biomass levels were measured from cellophane-overlaid SDAY cultures every 2 days from day 4 onward.…”