Abstract
Simple SummaryNutrient metabolism is closely related to the growth, development, and pathogenicity of pathogenic fungi. The nitrogen catabolite repression (NCR) pathway is a well-known fungal nitrogen source regulation path, in which NmrA plays an important regulatory role. Here, we reported a negative regulatory protein MaNmrA, the NmrA homologous protein, in the entomopathogenic fungus Metarhizium acridum, and found that it played important roles in carbon and nitrogen metabolism, growth, stress tolerance, and virulence of M. acridum. Our work will provide a theoretical basis for further exploring the functions of NCR pathway related genes in entomopathogenic fungi.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.
Highlights
Entomopathogenic fungi are important insect pathogenic microbes and play important roles in the control of agricultural pests [1]
Based on the important regulatory role of NmrA in the nitrogen catabolite repression (NCR) pathway, we firstly focus on the role of MaNmrA in nitrogen utilization
NmrA binds to the highly conserved C-terminal of GATA transcription factor AreA to negatively regulate the activity of AreA in the presence of a preferential nitrogen source, while NmrA is separated from the NmrA-AreA dimer to activate the expression of AreA and other genes involved in nitrogen catabolism and release the inhibition of nitrogen metabolism under nitrogen starvation condition [23,36,37]
Summary
Entomopathogenic fungi are important insect pathogenic microbes and play important roles in the control of agricultural pests [1]. Conidia of entomopathogenic fungi firstly adhere to the host cuticle, germinate to form infection structure appressoria, followed by penetrating the host cuticle under the action of turgor pressure and cuticle degrading enzymes, colonizing in the host hemolymph, and killing them [3,4]. For pathogenic bacteria, such as Bacillus thuringiensis (Bt), once Bt enters the host, it will produce different types of toxins or toxic proteins, which can destroy the host’s immune systems and lead to the death of the host [5,6]. Conidia are the effective infection unit of pathogenic fungi, the activity, infection, and pathogenicity of the conidia are disturbed by the external environment, such as nutritional conditions, temperature, humidity and UV-B, etc. [7]
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