MaNCP1, a C2H2 Zinc Finger Protein, Governs the Conidiation Pattern Shift through Regulating the Reductive Pathway for Nitric Oxide Synthesis in the Filamentous Fungus Metarhizium acridum.

Asexual sporulation is the most common reproduction mode of fungi. Most filamentous fungi have two conidiation patterns, normal conidiation and microcycle conidiation, which may be regulated by nutritional conditions. Nitrogen source can affect the fungal conidiation pattern, but the regulatory mechanism is not fully understood. In this study, we report a C2H2 zinc finger protein, MaNCP1, which has typical transcription factor characteristics and is screened from the subtractive library regulated by nitrate in the entomopathogenic fungus Metarhizium acridum. MaNCP1 and its N-terminal play critical roles in the conidiation pattern shift. Further study shows that MaNCP1 interacts with MaNmrA, which also contributes to the conidiation pattern shift and is involved in the reductive pathway of nitric oxide (NO) synthesis. Intriguingly, the conidiation pattern of the MaNCP1-disruption strain (ΔMaNCP1) can be restored to microcycle conidiation when grown on the microcycle conidiation medium, SYA, supplemented with NO donor or overexpressing MaNmrA in ΔMaNCP1. Here, we reveal that MaNCP1 governs the conidiation pattern shift through regulating the reductive synthesis of NO by physically targeting MaNmrA in M. acridum. This work provides new mechanistic insights into how changes in nitrogen utilization are linked to the regulation of fungal morphological changes. IMPORTANCE Fungal conidia play important roles in the response to environmental stimuli and evasion of the host immune system. The nitrogen source is one of the main factors affecting shifts in fungal conidiation patterns, but the regulatory mechanism involved is not fully understood. In this work, we report that the C2H2 zinc finger protein, MaNCP1, governs the conidiation pattern shift in M. acridum by targeting the MaNmrA gene, thereby altering the regulation of the reductive pathway for NO synthesis. This work provides further insights into how the nutritional environment can regulate the morphogenesis of filamentous fungi.

MaNmrA, a Negative Transcription Regulator in Nitrogen Catabolite Repression Pathway, Contributes to Nutrient Utilization, Stress Resistance, and Virulence in Entomopathogenic Fungus Metarhizium acridum.

The NCR pathway plays an important regulatory role in the nitrogen metabolism of filamentous fungi. NmrA, a central negative regulatory protein in the NCR pathway and a key factor in sensing to the carbon metabolism, plays important roles in pathogenic fungal nutrition metabolism. In this study, we characterized the functions of MaNmrA in the insect pathogenic fungus M. acridum. Multiple sequence alignments found that the conserved domain (NAD/NADP binding domain) of MaNmrA was highly conservative with its homologues proteins. Deletion of MaNmrA improved the utilization of multiple carbon sources (such as glucose, mannose, sucrose, and trehalose) and non-preferred nitrogen sources (such as NaNO3 and urea), significantly delayed the conidial germination rate and reduced the conidial yield. The MaNmrA-disruption strain (ΔMaNmrA) significantly decreased tolerances to UV-B and heat-shock, and it also increased the sensitivity to the hypertonic substance sorbitol, oxygen stress substance H2O2, and cell wall destroyer calcofluor white, indicating that loss of MaNmrA affected cell wall integrity, tolerances to hypertonic and oxidative stress. Bioassays demonstrated that disruption of MaNmrA decreased the virulence in both topical inoculation and intrahemocoel injection tests. Further studies revealed that the appressorium formation, turgor pressure, and colonization in hemolymph were significantly reduced in the absence of MaNmrA. Our work will deepen the functional cognition of MaNmrA and make a contribution to the study of its homologous proteins.

MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum.

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and ß-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.