Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity
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.
Section snippets
Strains, plasmids and culture conditions
F. graminearum wild-type strain 5035 and its derivatives were cultured at 28 °C on PDA and PDB for mycelium growth, and in CMC medium for conidiation (Xu et al., 2010). YTS (1 g L−1 yeast extract, 1 g L−1 tryptone; 1 M sucrose; 1% agarose) was used for transformation. Escherichia coli strain DH5α was used for general cloning and was cultured in Luria–Bertani broth at 37 °C.
The Myo2 gene replacement vector was constructed by amplifying a 1-kb upstream flanking sequence and a 1-kb downstream flanking
Identification of a F. graminearum insertion mutant lacking type II myosin activity
We generated a library of REMI mutants from the toxigenic F. graminearum strain 5035, isolated from an infected wheat spike harvested in Wuhan, China, where FHB epidemics occurs frequently (Zhang et al., 2007). We screened ∼6000 transformants and identified 48 mutants with aberrant phenotypes when grown in potato dextrose agar (PDA). We selected mutant M594 for further analysis because of its aberrant growth and strikingly low frequency of conidiation. Southern blot analysis and TAIL-PCR
Discussion
We have carried out the first functional characterization of a type II myosin gene from a phytopathogenic fungal species, F. graminearum responsible for FHB in cereal crops. We used a combination of molecular, cellular, chemical and biological assays to confirm that Myo2 plays a key role in septation, and is required for the development, pathogenicity and metabolic potential of this fungal pathogen.
Disruption of the Myo2 gene interfered with both asexual growth and sexual reproduction in F.
Acknowledgments
This work was supported by the National Basic Research Program of China (973 program 2009CB118806, 2013CB127801), the National Natural Science Foundation of China (31272004, 31271718, 31201475) and the Ministry of Agriculture of China (2011ZX08002-001).
References (53)
- et al.
Membrane traffic: a driving force in cytokinesis
Trends Cell Biol.
(2005) - et al.
Cytokinesis in higher plants
Cell
(1996) - et al.
Septation and cytokinesis in fungi
Fungal Genet. Biol.
(2003) - et al.
A model for integration of DNA into the genome during transformation of Fusarium graminearum
Fungal Genet. Biol.
(2008) - et al.
Disruption of the chitin synthase gene CHS1 from Fusarium asiaticum results in an altered structure of cell walls and reduced virulence
Fungal Genet. Biol.
(2010) - et al.
Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China
Mycol. Res.
(2007) - et al.
A millennial myosin census
Mol. Biol. Cell
(2001) - et al.
Identification of a second Myosin-II in Schizosaccharomyces pombe, Myp2p is conditionally required for cytokinesis
Mol. Biol. Cell
(1997) - et al.
Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis
J. Cell Biol.
(1998) - et al.
The structure and synthesis of the fungal cell wall
BioEssays
(2006)
The sporulation-specific enzymes encoded by the DIT1 and DIT2 genes catalyze a two-step reaction leading to a soluble ll-dityrosine-containing precursor of the yeast spore wall
Proc. Natl. Acad. Sci. USA
The fungal type II myosin in Penicillium marneffei, MyoB is essential for chitin deposition at nascent septation sites but not actin localization
Eukaryote Cell
Combined metabonomic and quantitative real-time PCR reveal systems metabolic changes of Fusarium graminearum induced by Tri5 gene deletion
J. Proteome Res.
Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission
J. Cell Biol.
Reduced virulence of trichothecene-nonproducing mutants of Gibberella zeae in wheat field tests
Mol. Plant–Microbe lnteract.
Magnaporthe grisea pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues
Plant Cell.
Analysis of ergosterol in single kernel and ground grain by gas chromatography–mass spectrometry
J. Agric. Food Chem.
Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II
J. Cell Biol.
The biosynthetic pathway for aurofusarin in Fusarium graminearum reveals a close link between the naphthoquinones and naphthopyrones
Mol. Microbiol.
Heading for disaster, Fusarium graminearum on cereal crops
Mol. Plant Pathol.
Pathogenicity and in planta mycotoxin accumulation among members of the Fusarium graminearum species complex on wheat and rice
Phytopathology
GIP2, a putative transcription factor that regulates the aurofusarin biosynthetic gene cluster in Gibberella zeae
Appl. Environ. Microbiol.
Cytokinesis in higher plants
Annu. Rev. Plant Biol.
Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe
J. Cell Biol.
Mass flow and pressure-driven hyphal extension in Neurospora crassa
Microbiology
Cited by (24)
MoRts1, a regulatory subunit of PP2A, is required for fungal development and pathogenicity of Magnaporthe oryzae
2023, Microbiological ResearchPhenamacril is a reversible and noncompetitive inhibitor of Fusarium class I myosin
2019, Journal of Biological ChemistryCitation Excerpt :The Mg2+-dependent ATPase activity of the motor domain utilizes the energy stored in ATP to produce unidirectional movement along polar actin filaments. Thereby, myosin isoforms facilitate directional cargo-transport processes, local constriction, and other specialized energy-requiring tasks within the cell (8, 13–17). Since the establishment of baseline sensitivity of Fusarium graminearum to phenamacril in 2008 (18), both laboratory (3, 4, 18–20) and field-resistant strains (5) have been characterized in China, where the compound is widely used to control Fusarium-induced infections of cereals (5, 6).
Resistance risk assessment of Fusarium oxysporum f. sp. melonis against phenamacril, a myosin inhibitor
2018, Pesticide Biochemistry and PhysiologyReal-time imaging of the growth-inhibitory effect of JS399-19 on Fusarium
2016, Pesticide Biochemistry and PhysiologyCitation Excerpt :They are involved in essential cellular events in the cytoskeletal apparatus. Type II has been associated with septation and sexual development in F. graminearum [55]. Similarly, in Aspergillus nidulans, type II and V myosin have been shown to be important for septation and hyphal tip extension rate [56].
A mutation in the converter subdomain of Aspergillus nidulans MyoB blocks constriction of the actomyosin ring in cytokinesis
2015, Fungal Genetics and BiologyCitation Excerpt :Despite this lack of diversity, fungal myosins play essential roles in a wide range of functions, ranging from cytokinesis to endocytosis and vesicle motility (Steinberg, 2000). Deletion of myosin II genes in A. nidulans (Taheri-Talesh et al., 2012), Fusarium solani (Song et al., 2013), and Penicillium marneffei (Cánovas et al., 2011) effectively blocks both septation and conidiation. Type II myosin, like actin, goes through a stepwise process of coalescence at septation sites (Delgado-Álvarez et al., 2014; Taheri-Talesh et al., 2012), appearing first as cytoplasmic strings before condensing into a compact cortical ring.