The Zinc Finger Transcription Factor BbCmr1 Regulates Conidium Maturation in Beauveria bassiana

ABSTRACT The entomopathogenic fungus Beauveria bassiana is a typical filamentous fungus and has been used for pest biocontrol. Conidia are the main active agents of fungal pesticides; however, we know little about conidial developmental mechanisms and less about maturation mechanisms. We found that a Zn2Cys6 transcription factor of B. bassiana (named BbCmr1) was mainly expressed in late-stage conidia and was involved in conidium maturation regulation. Deletion of Bbcmr1 impaired the conidial cell wall and resulted in a lower conidial germination rate under UV (UV), heat shock, H2O2, Congo red (CR) and SDS stresses compared to the wild type. Transcription levels of the genes associated with conidial wall components and trehalose synthase were significantly reduced in the ΔBbcmr1 mutant. Further analysis found that BbCmr1 functions by upregulating BbWetA, a well-known transcription factor in the central development of BrlA-AbaA-WetA. The expression of Bbcmr1 was positively regulated by BbBrlA. These results indicated that BbCmr1 played important roles in conidium maturation by interacting with the central development pathway, which provided insight into the conidial development networks in B. bassiana. IMPORTANCE Conidium maturation is a pivotal event in conidial development and affects fungal survival ability under various biotic/abiotic stresses. Although many transcription factors have been reported to regulate conidial development, we know little about the molecular mechanism of conidium maturation. Here, we demonstrated that the transcription factor BbCmr1 of B. bassiana was involved in conidium maturation, regulating cell wall structure, the expression of cell wall-related proteins, and trehalose synthesis. BbCmr1 orchestrated conidium maturation by interplaying with the central development pathway BrlA-AbaA-WetA. BbBrlA positively regulated the expression of Bbcmr1, and the latter positively regulated BbwetA expression, which forms a regulatory network mediating conidial development. This finding was critical to understand the molecular regulatory networks of conidial development in B. bassiana and provided avenues to engineer insect fungal pathogens with high-quality conidia.

The authors report on a Zn2Cys6 transcription factor of B. bassiana, BbCmr1, that is part of the transcription factors in the central development pathway BrlA-AbaA-WetA in conidium maturation. Targeted gene knockout of Bbcmr1 resulted in conidial cell wall impaired, lower conidial germination rate under abiotic stresses. Further analysis found that Bbcmr1 positively regulated BbwetA expression, and BbBrlA positively regulated the expression of Bbcmr1, which forms a regulatory network mediating conidial development. This finding was critical to understand the molecular regulatory networks of conidial development in B. bassiana and provided avenues to engineer insect fungal pathogens with high-quality conidia. Overall, the data are presented in a logical manner, and the manuscript is well-written.
A few minor points are listed below. 1) Fig. 2, panel D, Fig. 3 panel C-E, Fig. 8, there are markings on the images to show significant difference between or among the test samples, either better images could be provided or the markings can be replaced. 2) Lin 175-198, author described that Bbcmr1 regulates the expression of genes involved in conidium maturation. Would recommend adding some comments on that Smr1, BrlA and WetA genes were also differentially expressed in ΔBbcmr1 strain.
Reviewer #2 (Comments for the Author): The study by Chen et al. focuses on the role of the zinc finger transcription factor BbCmr1 in conidium maturation of the entomopathogenic fungus Beauveria bassiana. The entire work is scientifically sound. The authors have done a nice job combining diverse cellular and molecular biology techniques to satisfactorily answer the research questions. As a general recommendation, try to avoid using excessive jargon. See below some minor comments. line 52: "a fungal invertebrate pathogen", "an invertebrate pathogenic fungi" or "a fungal pathogen of invertebrates" reads better than "an invertebrate fungal pathogen" line 58: cuticle-degrading hydrolases Line 139 and Fig

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Thank you for submitting your paper to Microbiology Spectrum. Overall, the data are presented in a logical manner, and the manuscript is well-written.
A few minor points are listed below. 1) Fig. 2, panel D, Fig. 3 panel C-E, Fig. 8, there are markings on the images to show significant difference between or among the test samples, either better images could be provided or the markings can be replaced.
2) Lin 175-198, author described that Bbcmr1 regulates the expression of genes involved in conidium maturation. Would recommend adding some comments on that Smr1, BrlA and WetA genes were also differentially expressed in ΔBbcmr1 strain.

Christina Cuomo
Editor, Microbiology Spectrum Dear Dr. Christina Cuomo We are grateful to you and the two reviewers for the constructive comments on our manuscript entitled "The zinc finger transcription factor BbCmr1 regulates conidium maturation in Beauveria bassiana". The manuscript has been revised based on the Reviewers' suggestions. We reorganized multipanel figures to be assembled into one file. Introduction, Results, Discussion, and Materials and Methods have been modified in response to specific reviewers' comments. We feel that the quality of our paper has been substantially improved after revision and appreciate your consideration of this work. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed. To ensure there are no delays in publication of your article, please ensure that the sequence data (PRJNA777036) is promptly released.
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