Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity

Highlights

• Functional characterization of a Myo2 gene in F. graminearum.

• Myo2 is essential for septation and sexual development.

• Myo2 is required for virulence and mycotoxin biosynthesis.

• Time-lapse imaging of septation process in Δmyo2 mutant and wild-type.

• Lively monitoring of GFP-tagged Myo2 in hyphal and conidial development.

Abstract

Type II myosin is required for cytokinesis/septation in yeast and filamentous fungi, including Fusarium graminearum, a prevalent cause of Fusarium head blight in China. A type II myosin gene from the Chinese F. graminearum strain 5035, isolated from infected wheat spikes, was identified by screening a mutant library generated by restriction enzyme-mediated integration. Disruption of the Myo2 gene reduced mycelial growth by 50% and conidiation by 76-fold, and abolished sexual reproduction on wheat kernels. The Δmyo2 mutants also had a 97% decrease in their pathogenicity on wheat, and mycotoxin production fell to just 3.4% of the normal level. The distribution of nuclei and septa was abnormal in the mutants, and the septal ultrastructure appeared disorganized. Time-lapse imaging of septation provided direct evidence that Myo2 is required for septum initiation and formation, and revealed the dynamic behavior of GFP-tagged Myo2 during hyphal and macroconidia development, particularly in the delimiting septum of phialides and macroconidial spores. Microarray analysis identified many genes with altered expression profiles in the Δmyo2 mutant, indicating that Myo2 is required for several F. graminearum developmental processes and biological activities.

Introduction

Fusarium graminearum is an ascomycete that causes Fusarium head blight (FHB) in wheat, barley and other cereal crops world-wide. FHB pathogens produce various types of mycotoxins in the grain, posing a serious threat to human and animal health (Goswami and Kistler, 2004, Wang et al., 2011). FHB epidemics occur frequently in central China, especially along the middle and lower reaches of the Yangtze River (Qu et al., 2008, Zhang et al., 2013). Global climate change has been implicated as a potential cause for increases in the severity and geographic distribution of FHB, including northern China. Since the mid-1990s, FHB has re-emerged as a serious problem to agriculture in North America and Europe (Parry et al., 1995, Windels, 2000). Between 1998 and 2000, FHB-caused losses in the US alone were estimated to be about 3 billion US dollars (Nganje et al., 2002).

Septation/cytokinesis is one of the most important processes required for the proliferation and differentiation of fungi. Cytokinesis processes in budding yeast and fission yeast have been well studied. Type II myosins are known to play an important role in these processes in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (Schmidt et al., 2002). In S. cerevisiae, a single type II myosin encoded by the MYO1 gene is recruited to the bud neck, where it assembles with actin to form an actomyosin ring prior to septation (Bi et al., 1998, Lippincott and Li, 1998). The actomyosin ring contracts and initiates the formation of a primary septum, followed by the invagination of the plasma membrane towards the center of the neck until the disk of the septum has formed (Vallen et al., 2000). In S. pombe, myosin II and the related proteins Rng2, Cdc15 and Cdc12 are recruited sequentially to the division site in the middle of the cell (based on the position of nucleus) to form cytokinesis nodes (Pollard and Wu, 2010). When actin appears around the equator, myosin II in the cytokinesis nodes pulls the neighboring actin filaments together to assemble an actomyosin ring, which constricts simultaneously as the septum is formed (Pollard and Wu, 2010, Vavylonis et al., 2008). S. pombe contains two type II myosins encoded by the genes myo2 and myp2, the former essential for normal fission and the latter is required under stress (Bezanilla et al., 1997, May et al., 1997, Motegi et al., 2000, Mulvihill and Hyams, 2003).

The septation of filamentous fungi differs from the process in yeast because the septum contains one or more pores allowing cytoplasmic flow between adjacent compartments (Walther and Wendland, 2003). Disruption of a type II myosin (MyoB) gene in Penicillium marneffei results in aberrant chitin deposits at the division site and prevents the formation of uninucleate yeast cells at 37 °C (Cánovas et al., 2011). A distorted septation and abnormal branching were recently observed in Aspergillus nidulans with a deleted type II myosin gene (Taheri-Talesh et al., 2012). Furthermore, fungi and plants use different cell division mechanisms, and an actomyosin ring containing type II myosin is required in fungi but not in plants (Berg et al., 2001, Jürgens, 2005, Walther and Wendland, 2003). Therefore, understanding septation events unique to phytopathogenic fungi and identifying the corresponding proteins will provide novel fungal targets for the development of broad-spectrum fungicides.

Despite the significance of septation for the interactions between phytopathogenic fungi and their hosts, there has been no systematic investigation of the mechanisms of cytokinesis in phytopathogenic fungi of the F. graminearum clade, nor for a role of a type II myosin gene in mycotoxin biosynthesis in toxigenic fungi. In this study we identified a type II myosin gene, designated as Myo2, in toxigenic F. graminearum strain 5035 by screening a mutant library generated by restriction enzyme-mediated integration (REMI). Molecular and cellular analysis including time-lapse imaging of the septation process demonstrated that the type II myosin Myo2 is essential for conidiation and sexual reproduction, and plays a significant role in pathogenesis and mycotoxin production. Our data suggest that the Myo2 could be exploited as a target for the development of novel FHB control strategies.