Functional analysis of the mitochondrial gene mitofilin in the filamentous entomopathogenic fungus Beauveria bassiana

https://doi.org/10.1016/j.fgb.2019.103250Get rights and content

Highlights

Abstract

Mitofilin acts as an essential organizer that maintains the complex architecture of the mitochondrial inner membrane (IM). In the present study, a yeast ortholog of mitofilin was characterized in the filamentous entomopathogenic fungus Beauveria bassiana; hence, it was named BbMtf. Mitochondrial localization was observed for B. bassiana mitofilin, and loss of this protein altered both the overall morphology and crista junction of the mitochondrial IM. Disruption of BbMtf resulted in reduced ATP synthesis and germination on the oligotrophic surface compared to the control. The ΔBbMtf mutant did not display significant variation in mycelial growth and stress tolerance. However, the BbMtf gene was required for conidiation and blastospore formation, and its absence led to a significant reduction in conidiation (40%) and blastospore yield (70%) in the mutant strain compared to the control. In addition, the development of the ΔBbMtf mutant in the host hemocoel was also significantly impaired, with a reduction of approximately 80% in spore concentration. Finally, disruption of BbMtf significantly attenuated fungal pathogenicity against insect hosts. Mitofilin, therefore, maintains the function of the mitochondrial IM, which contributes to the development and virulence of B. bassiana as a biocontrol fungus.

Introduction

Mitochondria are double membrane-bound organelles that are ubiquitous in eukaryotes, and they function as powerhouses to generate energy in the form of adenosine triphosphate (ATP) for cellular activities (Roger et al., 2017). The inner membrane (IM) comprises two domains, i.e., the boundary membrane and the crista membrane, which protrudes into the mitochondrial matrix to form cristae. These membranous structures provide an enlarged surface area for many processes, such as oxidative phosphorylation (van der Bliek et al., 2017). This complex architecture is maintained with the help of the mitochondrial inner membrane organization system (MINOS), in which mitofilin acts as an essential organizer (Zerbes et al., 2012). Mitofilin, a mitochondrial inner membrane protein, was initially identified as a heart muscle protein in humans (Odgren et al., 1996) and was subsequently recognized in yeast (Hoppins et al., 2011) and protists (Mun et al., 2010). Mitofilin/Fcj1 contributes to the architecture of the mitochondrial inner membrane. In Saccharomyces cerevisiae, Fcj1 (a mitofilin ortholog) is specifically enriched within crista junctions (CJs) (Rabl et al., 2009). Disruption of the mitofilin gene results in an absence of CJs and accumulation of inner membrane stacks within the mitochondrial matrix (Rabl et al., 2009, Harner et al., 2011). Mitofilin's role in regulation of crista morphology is not only conserved among mammalian, insect and nematode cells but is also critical for mitochondrial function (John et al., 2005, Mun et al., 2010, Tsai et al., 2018). Additionally, mitofilin is involved in other processes independent of crista organization. In human cells, mitofilin mediates the mitochondrial localization of poly(ADP-ribose)polymerase-1 (PARP-1), which plays a role in maintaining the integrity of the mitochondrial genome (Rossi et al., 2009). Mitofilin interacts with a translocase from the outer membrane (OM) and promotes mitochondrial protein biogenesis by mediating protein import into the intermembrane space (von der Malsburg et al., 2011). Currently, the physiological role of mitofilin in filamentous fungi remains unknown.

The filamentous fungus Beauveria bassiana was first recognized as an insect pathogen and has been used to control arthropod pests in many countries (Xiao et al., 2012, Ying et al., 2013). In B. bassiana, mitochondria contribute during the entire lifecycle, including the saprophytic and pathogenic stages (Ding et al., 2018). B. bassiana could be used as an ideal model fungus to explore the biological roles of mitofilin in a filamentous mycopathogen, due to its established genome sequence and tractable genetic manipulation system (Xiao et al., 2012, Ying et al., 2013).

In this study, we described the role of a protein identified in B. bassiana that is orthologous to S. cerevisiae mitofilin. B. bassiana mitofilin is ubiquitously localized to the mitochondria, and loss of this protein results in abnormal morphogenesis of the mitochondrial IM. Furthermore, mitofilin contributes to the potential use of B. bassiana for biological pest control due to its important roles in fungal sporulation and virulence.

Section snippets

Fungal strains and growth conditions

The wild-type strain B. bassiana ARSEF2860 (referred as Bb2860) was obtained from the US Plant, Soil and Nutrition Laboratory (Tower Road, Ithaca, NY, USA) and used as the parent strain for construction of the gene disruption mutant. The wild-type and mutant strains were maintained using the standard method (Ding et al., 2018) on Sabouraud dextrose agar (SDAY: 4% glucose, 1% peptone and 1.5% agar plus 1% yeast extract). For conidiation, fungal strains were cultured at 25 °C on SDAY plates for

Bioinformatic analysis of mitofilin and construction of the gene disruption and complementation strains

Mitofilin (Locus tag: BBA_08545) (E-value: 4e-24) was identified by searching the B. bassiana genome using the S. cerevisiae ortholog as a query; the resulting gene was defined as BbMtf. The ORF sequence of BbMtf was 2196 bp long and two introns are present within the genomic sequence. Domain analysis of the deduced protein sequence revealed that BbMtf contains a mitofilin domain, conserved among mitofilin homologs from other eukaryotes (Supporting Information Table S2). Phylogenetic analysis

Discussion

Mitofilin functions as a conservative organizer in maintaining the architecture of the mitochondrial inner membrane (Odgren et al., 1996, Mun et al., 2010, Hoppins et al., 2011). The present study has provided initial clues toward understanding the role of mitofilin in filamentous fungi. In this study, we report that mitofilin dominates mitochondrial structure and function, and as a result, loss of mitofilin influences the development and virulence of the filamentous fungus B. bassiana.

The

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

We greatly thank Dr. She-Long Zhang (Equipment and Technology Service Platform, College of Life Sciences, Zhejiang University) and Dr. Jun-Ying Li (Analytical Center of Agrobiology and Environmental Sciences, Zhejiang University) for technical assistance with fluorescence and electron microscopy, respectively.

Funding

This study was jointly supported by National Key R & D Program of China (2017YFD0200400) and the Fundamental Research Funds for the Central Universities (2019FZA6002).

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