*Article* **MrHex1 is Required for Woronin Body Formation, Fungal Development and Virulence in** *Metarhizium robertsii*

#### **Guirong Tang 1, Yanfang Shang 1, Shiqing Li 1,2 and Chengshu Wang 1,2,3,\***


Received: 2 August 2020; Accepted: 11 September 2020; Published: 14 September 2020

**Abstract:** The Woronin body (WB) is a peroxisome-derived dense-core vesicle, a self-assembling hexagonal crystal of a single protein Hex1. This organelle is specific to the ascomycete fungi belonging to the Pezizomycotina subphylum by functioning in sealing septal pores in response to mycelium damage and the control of cell heterogeneity. We retrieved all available Hex1-domain containing proteins of different fungi from the GenBank database and found considerable length variations among 460 obtained Hex1 proteins. However, a highly conserved Hex1 domain containing 75 amino acid residues with a specific S/A-R/S-L consensus motif for targeting peroxisome is present at the carboxy-terminus of each protein. A homologous *Hex1* gene, named *MrHex1*, was deleted in the entomopathogenic fungus *Metarhizium robertsii.* It was found that MrHex1 was responsible for WB formation in *M. robertsii* and involved in sealing septal pores to maintain cell integrity and heterogeneity. Different assays indicated that, relative to the wild-type (WT) strain, Δ*Mrhex1* demonstrated a growth defect on a solid medium and substantial reductions of conidiation, appressorium formation and topical infectivity against insect hosts. However, there was no obvious virulence difference between WT and mutants during injection of insects. We also found that Δ*MrHex1* could tolerate different stress conditions like the WT and the gene-rescued mutant of *M. robertsii*, which is in contrast to the reports of the stress-response defects of the *Hex1* null mutants of other fungal species. In addition to revealing the phenotypic/functional alterations of the *Hex1* deletion mutants between different pathotype fungi, the results of this study may benefit the understanding of the evolution and WB-control of fungal entomopathogenicity.

**Keywords:** Woronin body; conidiation; stress response; appressorium formation; virulence; *Metarhizium robertsii*
