Developmental roles of the F. oxysporum velvet proteins
An inventory of the velvet protein family in F. oxysporum detected three members, VeA, VelB and VelC. Deletion of veA and velB had profound effects on hyphal growth and development. Major phenotypes of the mutants include a flat colony morphology, premature asexual development and increased conidiation in submerged culture, which are reminiscent of veA mutants in other fungal species (Calvo, 2008), such as A. nidulans, A. fumigatus, N. crassa, Penicillium chrysogenum and Fusarium graminearum (Kim et al., 2002; Kato et al., 2003; Bayram et al., 2008b; Hoff et al., 2010; Jiang et al., 2011; Park et al., 2012). In contrast, the ΔvelC mutant showed only minor developmental phenotypes. This suggests that VelC has an auxiliary role in F. oxysporum, as previously reported in A. nidulans and A. fumigatus (Sarikaya Bayram et al., 2010; Park et al., 2012).
Most of the velvet phenotypes are more severe in ΔveA than in ΔvelB mutants. This suggests that VeA has additional functions that are independent of VelB, as previously reported in Aspergillus (Bayram et al., 2008a; Bayram and Braus, 2012). We speculate that some of these additional VeA functions could be carried out in association with VelC. This idea is supported by genetic evidence showing a significant contribution of VelC in light-dependent functions such as repression of submerged conidiation or chromatin remodelling at secondary metabolite gene clusters (Figs 2C, 3B and 4B). Our yeast two-hybrid results suggest that VeA can interact both with VelB and with VelC, consistent with a hypothetical model in which VeA forms at least two distinct complexes (Fig. 7). In vivo protein interaction experiments are required to corroborate this hypothesis.
Figure 7. Roles of the F. oxysporum velvet complex in regulation of hyphal growth and development. Proposed model for the role of different velvet complex proteins in hyphal growth and conidiation. VeA forms complexes with VelB or VelC that have both overlapping and contrasting functions in different developmental processes. Interactions depicted in the model are based on yeast two-hybrid analysis and on genetic evidence. For simplicity, putative interactions VelB–VelB and VelB–VelC are not shown. Model partially based on that proposed by Bayram and Braus (2012).
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The increase in length and size of microconidia was more severe in ΔvelB than in ΔveA, which is in contrast to most other mutant phenotypes. Unexpectedly, deletion of veA in the ΔvelB background recapitulated the less severe ΔveA phenotype. Together with the finding that ΔvelC displays the opposite phenotype, namely smaller and shorter conidia, this result suggests a hypothetical model in which VeA–VelB and VeA–VelC complexes have opposite roles in determination of conidia size: positive for VeA–VelC and negative for the VeA–VelB (Fig. 7). Deletion of velB would result in loss of the negatively regulating complex and increased conidia size, whereas deletion of velC creates the opposite effect. Knockout of veA abolishes both complexes, leading to a moderate increase in conidia size. Such exquisite fine-tuning of conidial development is consistent with the ability of Fusarium to differentiate different types of asexual spores termed microconidia, macroconidia and chlamydospores. Even though F. oxysporum generally does not produce macroconidia in submerged culture, macroconidia-like spores were frequently observed in cultures of the ΔvelB mutant. Likewise, knockout of veA in Fusarium verticillioides led to a marked increase in the ratio of macroconidia to microconidia (Li et al., 2006).
Yeast two-hybrid experiments also detected a self-interaction VelB–VelB, which was recently described in A. nidulans (Sarikaya Bayram et al., 2010), as well as a previously unreported interaction VelB–VelC whose biological role is currently unknown. Unexpectedly, we failed to detect a F. oxysporum orthologue of VosA, the fourth member of the velvet protein family in Aspergillus (Ni and Yu, 2007). Lack of VosA appears to be specific for the genus Fusarium, since F. graminearum, F. verticillioides and F. solani also lack VosA orthologues while other ascomycetes such as N. crassa, Chaetomium globosum or M. oryzae all have predicted VosA proteins (Fig. 1). VosA is primarily involved in regulation of sporogenesis and trehalose biogenesis of A. nidulans (Ni and Yu, 2007) and forms a light-regulated complex with VelB (Sarikaya Bayram et al., 2010). It is currently unknown whether the VosA-specific functions in Fusarium are taken over by the other velvet complex proteins.
The conidiation phenotype of the F. oxysporum ΔlaeA mutants was opposite to that of ΔveA, decreased versus increased respectively. P. chrysogenum ΔlaeA mutants also displayed reduced conidiation, in contrast to ΔveA mutants (Hoff et al., 2010). This indicates that, as previously suggested in A. nidulans (Sarikaya Bayram et al., 2010), F. oxysporum LaeA inhibits certain developmental functions of velvet.
In summary, our data suggest that F. oxysporum velvet proteins form different complexes, some of which could have competing functions. VeA has a key role in complex formation, since it is the only velvet-like protein to interact with LaeA in the yeast two-hybrid assay. This finding explains the generally more severe phenotype of the ΔveA mutant relative to ΔvelB and ΔvelC mutants. However, our interaction data are solely based on yeast two-hybrid experiments and thus should be viewed with caution. A recent study confirmed in vivo interaction of VeA and LaeA in the nucleus of Fusarium fujikuroi, using an alternative approach based on bimolecular fluorescence complementation (Wiemann et al., 2010).
Velvet participates in chromatin remodelling and transcriptional activation of secondary metabolite gene clusters
In this study, we establish a light-dependent role for the velvet complex in biosynthesis of three secondary metabolites, ferricrocin, triacetylfusarinine C and BEA. In A. nidulans, VeA and LaeA govern biosynthesis of the secondary metabolites sterigmatocystin, penicillin and lovastatin (Bok and Keller, 2004; Calvo, 2008; Bayram et al., 2008a). VeA was also shown to activate expression of genes involved in biosynthesis of cephalosporin C in Acremonium chrysogenum (Dreyer et al., 2007), or production of the deleterious mycotoxins fumonisin, fusarin C, trichotecene or deoxynivalenol in the plant pathogens F. verticillioides, F. fujikuroi and F. graminearum (Myung et al., 2009; Wiemann et al., 2010; Jiang et al., 2011; Merhej et al., 2012). Transcriptional profiling in the human pathogen A. fumigatus revealed that LaeA controls expression of 13 of 22 secondary metabolite gene clusters, including those involved in biosynthesis of siderophores and mycotoxins (Perrin et al., 2007). Likewise, LaeA regulates expression of multiple secondary metabolism gene clusters in the plant parasite Aspergillus flavus (Georgianna et al., 2010). LaeA deletion caused downregulation of gene clusters encoding biosynthesis of bikaverin, fumonisin, fusaric acid, fusarin and two unknown secondary metabolite clusters (Butchko et al., 2012).
Importantly, our work correlates reduced transcript levels in the F. oxysporum velvet complex mutants with a significant decrease in chromatin accessibility at the ferricrocin and BEA gene clusters (Figs 3 and 4). This result fits the generally accepted view that tightly positioned nucleosomes repress gene activity whereas loss of nucleosome positioning leads to transcriptional activation (Felsenfeld and Groudine, 2003). Moreover, these findings strongly suggest that the main regulatory function of the velvet complex in F. oxysporum resides in the modification of chromatin structure at the target loci. The exact mechanism of chromatin remodelling by velvet remains to be elucidated but is likely to depend on LaeA function. Like other fungal orthologues, F. oxysporum LaeA contains a conserved domain of methyltransferase SAM binding residues (Bayram and Braus, 2012). Site-directed mutation of the s-adenosyl methionine binding site in A. nidulans LaeA resulted in a loss-of-function phenotype, suggesting that LaeA acts as a methyltransferase (Bok et al., 2006). Chromatin modification by LaeA was recently shown to involve reversal of histone H3 lysine 9 trimethylation and the concomitant removal of heterochromatic marks (Reyes-Dominguez et al., 2010).
Velvet controls fungal virulence factors such as the mycotoxin BEA
Our study establishes a conserved role of the velvet complex during infection of F. oxysporum on both plant and mammalian hosts. LaeA had a more severe effect on virulence than VeA, indicating that regulation of secondary metabolism is a key contribution of the velvet complex during infection. In support of this idea, production of BEA and fusaric acid, two known mycotoxins, was impaired in the velvet complex mutants during growth in human blood and on tomato roots. BEA and its close structural relative enniatin are cyclic hexadepsipeptides consisting of alternating d-α-hydroxy-isovaleryl-(2-hydroxy-3-methylbutanoic acid) and amino acid-units (Jestoi, 2008). BEA was first isolated from the culture of the entomopathogenic fungus B. bassiana (Hamill et al., 1969), and its production has been reported in several species of the genus Fusarium including different formae speciales of F. oxysporum (Logrieco et al., 1998; Moretti et al., 2002; Song et al., 2008).
We confirmed that BEA is a virulence factor of F. oxysporum by deleting the NRPS responsible for BEA biosynthesis. The Δbeas mutants were attenuated in virulence on mice and on tomato plants. Targeted knockout of the beas gene in the entomopathogen B. bassiana revealed a significant role of this mycotoxin in virulence on insect hosts (Xu et al., 2008). Mutants of the cereal pathogen F. avenaceum lacking enniatin synthetase exhibited significantly reduced virulence in an infection assay on potato tubers (Herrmann et al., 1996a). These compounds display a wide array of biological activities including antiviral, antibacterial, nematicidal, insecticidal and cytotoxic (Jestoi, 2008). So far, however, their exact function during infection of plant and animal hosts remains unknown. BEA and enniatins act as potent and specific inhibitors of ABC-type multidrug efflux pumps such as Saccharomyces cerevisiae Pdr5p (Hiraga et al., 2005) and human ABCB1 and ABCG2 (Dornetshuber et al., 2009). Moreover, BEAS induces membrane scrambling and apoptosis in human erythrocytes (Qadri et al., 2011), while enniatin causes necrosis of potato tuber tissue, suggesting that cyclic hexadepsipeptides can trigger cell death both in mammalian and in plant cells (Herrmann et al., 1996b).
Components of the velvet complex have previously been associated with virulence in fungal human and plant pathogens. A role of LaeA in virulence of A. fumigatus was partly attributed to regulation of biosynthesis of gliotoxin (Sugui et al., 2007), an epidithiodioxopiperazine that induces apoptosis in different human cell types (Scharf et al., 2012). Interestingly, reduced virulence of ΔlaeA mutants of A. fumigatus was accompanied by decreased levels of pulmonary gliotoxin and changes in susceptibility to host phagocytes ex vivo (Bok and Keller, 2004; Bok et al., 2005). In the dimorphic human pathogen Histoplasma capsulatum, the orthologues of VosA, VelB and VeA are required for correct switching from hyphal to yeast stage and for full virulence (Webster and Sil, 2008; Laskowski-Peak et al., 2012). Null mutants in veA or laeA of the mycotoxigenic fungus A. flavus failed to metabolize host cell lipid reserves, resulting in inhibition by oleic acid and reduced seed colonization (Amaike and Keller, 2009). Strains of F. graminearum lacking VeA or VelB showed reduced in vitro expression of trichothecene and deoxynivalenol biosynthesis genes and attenuated virulence on wheat heads (Jiang et al., 2011; Lee et al., 2012; Merhej et al., 2012), while F. fujikuroi ΔveA, ΔvelB and ΔlaeA mutants caused less bakanae etiolation symptoms on rice seedlings, likely due to impaired biosynthesis of gibberelic acid (Wiemann et al., 2010), and F. verticillioides ΔveA strains displayed reduced fumonisin and fusarin production and infection on maize seedlings (Myung et al., 2009; 2012). In a recent study, production of the host selective polyketide T-toxin in the corn pathogen Cochliobolus heterostrophus was reduced in ΔveA or ΔlaeA mutants and increased in veA or laeA overexpression strains (Wu et al., 2012). In contrast, veA was dispensable for virulence in two other plant pathogens, Mycosphaerella graminicola (Choi and Goodwin, 2011) and Dothistroma septosporum (Chettri et al., 2012).
The reduction in virulence of the Δbeas mutants was less severe than that of the ΔveA and ΔlaeA mutants, suggesting that the velvet complex controls additional pathogenicity functions. We found that velvet is also required for production of fusaric acid (5-n-butyl-2-pyridine carboxylic acid) during growth of F. oxysporum in human blood. Fusaric acid was identified more than 50 years ago as a phytotoxin produced by F. oxysporum on tomato plants, and has been implicated in the development of vascular wilt symptoms by altering water permeability of the plant plasma membrane and causing solute leakage (Gäumann, 1957; 1958). Although we were unable to detect fusaric acid in infected tomato plants, possibly due to a low level of production or to instability of the compound in the plant tissue, it cannot be excluded that F. oxysporum produces fusaric acid during specific stages of infection. The recent identification of the fusaric acid biosynthetic gene cluster in F. verticillioides (Butchko et al., 2012) opens the way for future studies on the role of this mycotoxin in fungal virulence. Moreover, mutants in a third target gene of the velvet complex encoding an ABC3 multidrug transporter were also attenuated in virulence on tomato plants. Interestingly, the orthologous abc3 gene in M. oryzae functions as a virulence factor on rice plants (Sun et al., 2006). Additional putative virulence genes controlled by velvet include those encoding two NRPSs that participate in biosynthesis of the siderophores ferricrocin and triacetylfusarinine C. While the role of siderophores during infection of F. oxysporum has not been determined, a recent study found that the regulator of iron homeostasis HapX is required for virulence on mice and on tomato plants (López-Berges et al., 2012). Finally, important developmental functions of LaeA may account for the reduced virulence of the ΔlaeA mutant during late stages of infection. Reduced conidiation of the mutant could lead to a delay in colonization of the vascular bundles and attenuated wilt symptoms. Collectively, these results demonstrate that the velvet complex controls expression of multiple virulence-related genes in F. oxysporum, some of which are specific for either plant or mammalian infection while others, such as beas, contribute to virulence on both types of hosts.