Factors of the Fusarium verticillioides-maize environment modulating fumonisin production

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1 Critical Reviews in Microbiology, 2010, 1 11, Early Online RESEARCH ARTICLE Factors of the Fusarium verticillioides-maize environment modulating fumonisin production Adeline Picot 1,2,3, Christian Barreau 2, Laëtitia Pinson-Gadais 2, Daniel Caron 1, Christian Lannou 3, and Florence Richard-Forget 2 1 ARVALIS - Institut du végétal, Baziège, France, 2 Institut National de la Recherche Agronomique, UR1264, MycSA, Villenave d Ornon, France, and 3 Institut National de la Recherche Agronomique, UMR1290, BIOGER-CPP, Versailles-Grignon, France Abstract Fumonisins are mycotoxins mainly produced by two Fusarium species: F. verticillioides and F. proliferatum. These toxins are of great concern due to their widespread contamination in maize and their adverse effects on animal and human health. In the past decade, progress was made in identifying the genes required for fumonisin biosynthesis. Additionally, molecular mechanisms involved in the regulation of fumonisin production have been very recently elucidated. By covering the latest advances concerning the factors modulating fumonisin production, this review aims at presenting an integrated approach of the overall mechanisms involved in the regulation of fumonisin biosynthesis during maize kernel colonization. Keywords: Maize-pathogen interaction; toxin regulation; ecophysiological factors; ph; nutrient sources Introduction Fumonisins are polyketide-derived mycotoxins produced by several species of the Fusarium section Liseola, including F. verticillioides (Sacc.), Nirenberg (teleomorph Gibberella moniliformis Wineland), F. proliferatum (Matsush.), Nirenberg ex Gerlach & Nirenberg (teleomorph Gibberella intermedia Kuhlman). These fungal pathogens are the major causal agents of fusarium ear rot, one of the most important diseases affecting maize production worldwide. Fumonisin-contaminated maize products are a major food safety concern since their ingestion has been associated with increased incidence of human esophageal cancer in China (Li et al., 1980; Yang 1980) and in South Africa (Jaskiewicz et al., 1987; Makaula et al., 1996). Based on human epidemiological surveys and on recent studies reporting the involvement of fumonisins in developmental abnormalities in mice, it has been suggested that fumonisin consumption may be related to human neural tube defects and associated birth defects such as craniofacial abnormalities (Marasas et al., 2004). Fumonisins are also suspected to be responsible for animal toxicoses including leukoencephalomalacia in horses (Kellerman et al., 1990) and pulmonary edema and hydrothorax in swine (Harrison et al., 1990). According to the European Union regulation, fumonisin content in unprocessed maize cannot exceed 4 mg kg 1 for human consumption. The chemical structure of fumonisins consists of an aminopentol backbone with one tricarballylic acid on each side chain and one or more hydroxyl groups (Bezuidenhout et al., 1988). Due to their structural similarity with sphinganine, they may act as specific inhibitors of sphingolipid biosynthesis, which are major constituents of cell membranes and important components of many signalling pathways (Wang et al., 1991; Riley et al., 1994; Merrill et al., 2001). While approximately 60 distinct fumonisin molecules have been identified, fumonisin B1 (FB1) is the predominant fumonisin encountered on maize kernels (Bartók et al., 2006). In the past decade, tremendous progress has been made in identifying the fungal genes required for fumonisin biosynthesis. As it commonly occurs in toxigenic fungi, these genes are organized in a cluster designated as the FUM gene cluster (Proctor et al., 1999; Proctor et al., Address for Correspondence: Florence Richard-Forget; Tel: ; fforget@bordeaux.inra.fr (Received 18 December 2009; revised 28 January 2010; accepted 22 February 2010) ISSN X print/issn online 2010 Informa UK Ltd DOI: /

2 2 A. Picot et al. 2003). However, the mechanism by which this cluster is regulated is far from being fully understood and has become an important topic of current research. Multiple environmental factors including ph, water availability or nutrient sources are often pointed out as key factors regulating fumonisin production. Additionally, various fungal genes have been very recently shown to be involved in fumonisin regulation. However, up to now, published data devoted to fumonisin regulation have often considered each regulatory mechanism separately, resulting in a scattered literature, whereas each fungal gene involved in fumonisin regulation is part of a general regulatory pattern, governed by the interaction with maize during kernel colonization. So far, strategies for reducing fumonisin contamination have mainly focused on reducing fungal attacks. For instance, plant breeders often select maize varieties based on disease visual symptoms. Yet, symptomless maize ears may also be toxin-contaminated, as it has often been reported for fusarium ear rot disease (Munkvold and Desjardins 1997; Desjardins and Plattner 1998). Consequently, limiting visual fungal contamination may not be the most effective method for reducing mycotoxin contamination in maize kernels. Developing approaches leading to a down-regulation of the fumonisin biosynthetic pathway is therefore a complementary strategy to consider (Duvick, 2001). These approaches are also of particular relevance when mycotoxins are part of fungal pathogenicity (Duvick, 2001). For example, it has been demonstrated that deoxynivalenol, a mycotoxin produced by F. graminearum, may help the fungus to colonize maize ears (Harris et al., 1999). Although there is no compelling evidence that fumonisins participate in the disease development, strategies aiming at fumonisin reduction remain of great interest (Duvick, 2001). In this context, understanding the conditions that favor or restrain fumonisin production during maize kernel colonization is crucial to develop approaches that allow fumonisin reduction from pre- to post-harvest. The aim of this review is to present the different mechanisms that are known to be involved in the regulation of fumonisin biosynthesis during maize kernel colonization. After briefly describing the genes involved in the fumonisin biosynthesis and its regulation, this paper reviews the factors of the F. verticillioides-maize environment that are known to affect fumonisin production, including 1) temperature and water availability, 2) ph and nutrients sources, and 3) plant natural defenses. Genes associated with fumonisin biosynthesis and its regulation In the past ten years, sixteen co-regulated genes have been identified to form the FUM gene cluster in F. verticillioides, located within an approximate 46 kb region on chromosome 1 (Proctor et al., 2003; Brown et al., 2007). The first gene isolated was a polyketide synthase gene (FUM1) which encodes an enzyme catalyzing the first step of the fumonisin biosynthetic pathway (Proctor et al., 1999). Downstream of FUM1, ten other genes that encode most of the enzymatic activities required for fumonisin biosynthesis have been identified (Proctor et al., 2003). The FUM gene cluster also includes genes that, when disrupted, show little or no effect on fumonisin production, suggesting that their function may be either redundant or not required for fumonisin biosynthesis (Desjardins and Proctor, 2007). While some of the steps leading to fumonisin biosynthesis have been elucidated, there is still much to learn about the regulatory mechanisms involved in fumonisin production. As for other fungal secondary metabolite gene clusters, a regulatory gene, named FUM21, has been recently discovered adjacent to FUM1 in the FUM cluster (Brown et al., 2007). Additionally, ten other genes, found outside this cluster, are known to be involved in fumonisin regulation thanks to mutational analysis (FCC1, PAC1, ZFR1, FCK1, GBP1, GBB1, FST1, CPP1, AREA, FvVE1) (Shim and Woloshuk, 2001; Flaherty et al., 2003; Flaherty and Woloshuk, 2004; Bluhm and Woloshuk, 2006; Sagaram et al., 2006a; Sagaram and Shim, 2007; Bluhm et al., 2008; Choi and Shim, 2008; Kim and Woloshuk, 2008; Myung et al., 2009). Data on these genes are summarized in Table 1, which is an updated version of a similar table published by Sagaram et al. (2006b). They are global regulators or broad-domain transcription factors regulating diverse cellular functions in response to external signals (Brown et al., 2007). However, their exact role and importance in fumonisin production during host colonization is far from complete. Identification and functional characterization of downstream derivatives are therefore necessary to better understand how they could regulate FUM gene expression over the course of host colonization. Nevertheless, the role and function of four of these genes (PAC1, AREA, ZFR1, and FST1) involved in ph homeostasis or nutrient catabolite regulation have been more clearly defined and provide new insights into the molecular mechanisms that regulate aspects of fumonisin production during maize kernel colonization by F. verticillioides. They will be thoroughly described in the third paragraph. Effect of temperature and water availability on fumonisin production Water activity (a w ) and temperature are considered to be the main abiotic factors influencing fumonisin production (Sanchis et al., 2006). The effects of these factors on fumonisin biosynthesis by F. verticillioides and

3 Modulation of fumonisin production in maize 3 Table 1. Updated version of the genes associated with FB1 regulation in F. verticillioides, published by Sagaram et al. (2006). *Genes reported in Sagaram et al. (2006). FB1 production in mutants bearing the disrupted gene FUM gene expression (on mature maize kernels, if in mutants bearing the Other phenotype traits of the Gene Name Encoding gene not mentioned) disrupted gene mutant Regulatory Mode Reference FCC1* C-type cyclin No FB1 production on No expression of FUM5 Severe reduction in Postive Shim et al. (2001) mature maize kernels. gene on maize kernels. conidiation Decreased FB1 production in Similar levels of FUM5 acidic defined media gene in acidic defined media PAC1* PACC group of ph regulatory Increased FB1 production Higher expression of Severly impaired growth at Negative Flaherty et al. (2003) genes under acidic ph and on FUM1 gene under acidic alkaline ph mature maize kernels, blocks and alkaline conditions in FB1 production under alkaline ph suppressed ZFR1* Zn(II)2Cys6 binuclear cluster Decreased FB1 production No reports No effect on growth and Positive Flaherty et al. (2004) development FCK1* Cyclin-dependent kinase Decreased FB1 production Reduced expression of Severe reduction in growth Positive, physically Bluhm et al. (2006) FUM1 gene and conidiation and enhanced interacts with Fcc1 pigmentation GBP1* Developmentally regulated Increased FB1 production Increased expression of No effect on growth and Negative Sagaram et al. monomeric G protein FUM1 and FUM8 genes development (2006) GBB1 Heterotrimeric G protein β Decreased FB1 production Reduced expression of Reduced hyphal development Postive Sagaram et al. subunit FUM1 and FUM8 genes but no effect on fungal (2007) virulence CPP1 Putative protein phosphatase Increased FB1 production Increased expression of Reduced radial growth, Negative Choi et al. (2007) 2A catalytic subunit FUM1 gene reduced conidia germination rates, increased macroconidia formation and hyphal swelling AREA Regulator of nitrogen No production of FB1 on Reduced expression No effect on growth on blister Positive Kim et al. (2008) metabolism blister and mature maize of FUM1, FUM8 and kernels, severe reduced kernels FUM12 genes growth on mature maize kernels FST1 Putative sugar transporter Decreased FB1 production No reports No effect on growth, kernel Positive Bluhm et al. (2008) colonization or kernel acidification FvVE1 Regulator of sexual No FB1 production on No expression of FUM1, Effect on cell wall integrity, Positive Myung et al. (2009) development and secondary mature maize and rice FUM8 and FUM21 genes cell surface hydrophobicity, metabolism kernels hyphal polarity and conidiation pattern (Li et al. 2006) No reports Positive Brown et al. (2007) FUM21 Predicted Zn(II)-2Cys6 DNA-binding transcription factor Little to no FB1 production Low expression of FUM1 and FUM8 genes in the early stages of growth

4 4 A. Picot et al. F. proliferatum have been largely studied in vitro on maize grains. Higher water availability generally results in higher fumonisin production and higher fungal growth (Marín et al., 1999a; Marín et al., 1999b; Samapundo et al., 2005). Optimal conditions for fumonisin production were described at a w and at temperatures between 20 C and 30 C for F. verticillioides and between 15 C and 25 C for F. proliferatum (Marín et al., 1999b; Samapundo et al., 2005). However, at temperatures that are nonoptimal for fungal biomass accumulation, fumonisin production relative to fungal growth was greater at lower a w values, indicating that a w stress may enhance fumonisin production (Marín et al., 1999a; Samapundo et al., 2005). Similarly, a recent in vitro study showed that low a w values (at 7 and 10 MPa, equivalent to and a w ) significantly increased FUM1 gene expression whereas F. verticillioides growth was severely affected (Jurado et al., 2008). Consequently, during maize kernel ripening, while water availability progressively decreases because of starch formation, the FUM gene expression may be activated (Jurado et al., 2008; Figure 1). Under field conditions, fumonisin contents in kernels was inversely related to June rainfall in maize hybrids located in different regions of the United States, indicating that dry weather, just before or at pollination, is probably an important environmental factor affecting fumonisin production (Shelby et al., 1994). Similar results were obtained in field experiments in Canada (Miller et al., 1995). Another field report, in Poland, recorded the highest fumonisin contamination in three seasons over seven, that were characterized by the highest temperatures at pollination time, but not necessarily by the lowest precipitations (Pascale et al., 2002). Yet, we do not know whether this increase in fumonisin production during dry and/or hot summers results from an enhanced fungal development in the maize ear or from an up-regulation of the FUM gene expression. Effect of ph and nutritional factors on fumonisin production In addition to ecophysiological factors, physicochemical, and nutritional factors are of paramount importance in modulating fumonisin production. Among these factors, ph or C:N ratio variations during the course of maize ripening are thought to be key factors modulating fumonisin production. Figure 1 attempts to schematically describe these maize physiological changes that could favor fumonisin production during maize kernel ripening. In liquid cultures, fumonisin production by F. proliferatum occurred at final ph ranging from 2 to 4.5 (Keller et al., 1997). Optimal ph for fumonisin production ranged from 3.0 to 3.5 whereas ph above 3.5 enhanced fungal growth (Keller et al., 1997). Flaherty et al. (2003) demonstrated that repression of fumonisin production occurred at alkaline ph. The authors identified the gene PAC1 in F. verticillioides, an ortholog of the PacC gene described in Aspergillus nidulans (Tilburn et al., 1995), as a repressor of fumonisin biosynthesis under alkaline conditions: the PAC1-disrupted mutant, but not the wild type, was able to produce fumonisins under alkaline conditions. To our knowledge, no studies have monitored the evolution of kernel ph in planta during the colonization of maize ears Maize kernel physiological changes Fungal host interaction Water availability C : N ratio Acidification (due to the fungal metabolism?) Oxidative stress Maize kernel substrate C sources ( starch and amylopectin) N availability 9-LOX oxylipin pathway H 2 and ROS production Fungal genes ZFRI FSTI AREA PACI psi factors FUM cluster Figure 1. Schematic representation of the factors enhancing fumonisin production in association with identified fungal regulatory genes, during maize kernel ripening. Dotted arrows indicate putative pathways that affect the expression of the FUM gene cluster. The dotted line ended with bar means alleviation of Pac1 repression of FUM genes under acidic conditions. The question mark stands for hypothetical effects on FUM gene expression, demonstrated in other pathosystems (A. parasiticus, F. graminearum). ( ) indicates an increase whereas ( ) indicates a decrease.

5 Modulation of fumonisin production in maize 5 by F. verticillioides and the potential in vivo effects of ph variations on fumonisin production are still unknown. The substrate from which F. verticillioides draws its carbon and nitrogen sources also plays a critical role in fumonisin regulation. The influence of nutrient sources on fumonisin production by F. verticillioides and F. proliferatum in liquid cultures has been investigated by Jiménez et al. (2003). A positive relationship was obtained between fumonisin production and sugar concentration, independently of the carbohydrates used (glucose, fructose, rhamnose, sucrose, maltose, and trehalose). On the contrary, decreasing amino-acid concentration from 10 g l 1 to 1 g l 1 (nine amino acids were tested: serine, threonine, glutamic acid, aspartic acid, valine, isoleucine, methionine, glycine, alanine, and cystine) led to a significant increase in fumonisin production and to a decrease in mycelial mass. The authors concluded that fumonisin production is positively influenced by an increase in the C:N ratio whereas fungal growth is reduced (Jiménez et al., 2003). In a recent in vitro study, the expression of F. proliferatum FUM1 and FUM8 genes significantly increased when nitrogen sources decreased in the culture medium, supporting the idea that N-starvation induces FUM gene expression and fumonisin production (Kohut et al., 2009). Inversely, when ammonium phosphate, as a source of nitrogen, was supplemented to maize kernels, repression of FB1 production occurred (Shim and Woloshuk, 1999). All these studies suggest that the nitrogen metabolism contributes to regulate fumonisin production. Recently, Kim and Woloshuk (2008) have demonstrated that expression of AREA, a known regulator of nitrogen metabolism, induces a positive regulation of fumonisin production. On the one hand, the AREAdisrupted F. verticillioides strain did not produce FB1 on mature maize kernels. On the other hand, the mutant that constitutively expresses AREA (AREA-CE) was able to produce FB1 on mature kernels even with the addition of ammonium phosphate, which was repressive in the wild type. These results indicate that expression of AREA is a required condition for FB1 production. However, on blister kernels, neither the wild type, nor AREA-CE could produce FB1, suggesting that additional molecular mechanisms are able to induce repression of FUM gene expression. By adding amylopectin, a starch component, to the blister kernels, FB1 was produced by both the wild type and AREA-CE, which suggests that amylopectin may also be a key component in inducing fumonisin production. However, this supplementation also led to a decrease in kernel ph, which may also account for the induction in fumonisin production (Kim and Woloshuk, 2008). According to these authors, fumonisin repression in blister kernels was due to high ph conditions which resulted from an overabundance of nitrogen compared to carbon sources. The use of nitrogen sources by the fungus could actually lead to an alkalization of the maize kernels (Kim and Woloshuk, 2008) since it has been shown that blister kernels (characterized by a low C:N ratio) became alkalized whereas dent kernels (characterized by a high C:N ratio) became acidified 10 days after inoculation with F. verticillioides strain (Bluhm and Woloshuk, 2005). However, it still remains difficult to determine whether the addition of amylopectin was an indirect cause of fumonisin production via kernel acidification or had a direct effect on fumonisin production. Yet, multiple pieces of evidence strongly suggest that amylopectin metabolism is not only an indirect cause of fumonisin production due to kernel acidification but seems to be an important cue inducing fumonisin production during colonization of maize kernels. A mutant of F. verticillioides impaired in its ability to hydrolyze starch produced significantly lower levels of FB1 on mature kernels, even though it was able to colonize them (Bluhm and Woloshuk, 2005). In the same study, four maize mutants with a reduced content of starch (38 45% relative to dry weight) compared to the conventional hybrid (72% relative to dry weight) were inoculated with a F. verticillioides strain. FB1 production was lower in the maize mutants than in the conventional hybrid (Bluhm and Woloshuk, 2005). Field experiments conducted in 2000 and 2001 in Italy by Blandino and Reyneri (2007) also showed that waxy maize hybrids, which contained more amylopectin, were more contaminated with fumonisin than normal hybrids. These results are consistent with field observations on the kinetics of fumonisin production. Maize kernel development is characterized by a rapid accumulation of starch during kernel ripening and amylopectin begins to accumulate in maize kernels from 15 to 40 days after pollination (Zhang et al., 2008). Field studies monitoring fumonisin production according to kernel stages show that fumonisin production begins to be detected three to five weeks after silking (Reid et al., 1999; Bush et al., 2004), which is more or less concomitant with amylopectin accumulation in maize kernels. During maize kernel colonization by F. verticillioides, the sugar availability appears therefore to be of critical importance for fumonisin production. At a molecular level, recent advances have been made in clarifying the regulatory mechanisms that link the sugar uptake by the fungus and fumonisin production. Bluhm et al. (2008) have characterized the role of ZFR1, a gene encoding a putative zinc-finger transcription factor, previously identified for its possible role in regulating fumonisin biosynthesis (Flaherty and Woloshuk, 2004). Growth of the zfr1-disrupted mutant was significantly lower than that of the wild type on minimal media containing either glucose, maltose, amylopectin, or dextrin. Growth was also significantly reduced on endosperm fractions of the kernels, characterized by higher amounts of starch. These observations led the authors to hypothesize

6 6 A. Picot et al. that the mutant was impaired in the perception or utilization of sugars. Surprisingly, this impairment was not due to an inability to hydrolyze starch (the expression of amylolytic genes was even higher than in the wild type strain on endosperm tissues) nor to an inability to perceive carbohydrates (hexokinase and glucokinase activity was similar in the wild-type and the mutant) but it was most probably due to an inability to perceive or fully utilize the products of sugar hydrolysis. Using microarrays, the authors identified three putative sugar transporter genes (FST1, FST2 and FST4) for which expression was reduced in the Δzfr1 strain, indicating that ZFR1 controls the expression of important genes involved in perceiving sugars. Moreover, when disrupting FST1 in another mutant, mycelial growth and kernel colonization was not affected but FB1 production was reduced by 82% compared to the wild type (Bluhm et al., 2008). Based on these results, Bluhm et al. (2008) hypothesized that FST1 detects specific starch products during maize kernel colonization and activates signal transduction pathways that in turn induce the fumonisin biosynthetic pathway. It has also to be underlined that FST1 disruption did not impede kernel acidification during fungal colonization but did reduce FB1 production. This result strongly supports the idea that amylopectin metabolism is not simply an indirect cause of fumonisin induction via kernel acidification, as firstly suggested by Kim and Woloshuk (2008). These recent studies give a better understanding of the complex regulation of fumonisin biosynthesis by nitrogen and carbon sources available to the fungus. During kernel colonization, the combined effects of sugar and nitrogen metabolism along with the ph variations play an important role in inducing the fumonisin biosynthetic pathway (Figure 1). FST1, AREA, and PAC1 would here act as a key linkage between primary and secondary metabolism. However, further studies are still needed to clarify the different steps linking the sugar and nitrogen uptake with the ph and their impact on the induction of the fumonisin biosynthetic pathway. Effect of plant natural defenses on fumonisin production Physiological changes, in terms of nutrient composition and kernel ph evolution, occur during maize ripening that influence fumonisin production. The maize-fungus interaction also leads to physiological and molecular perturbations in the plant cell, triggering plant defense mechanisms, which may influence positively or negatively fungal growth and toxin production. One of the first biochemical events following the plant infection by a pathogen is the production of reactive oxygen species (ROS), including H 2, involved in many signalling transduction pathways. The accumulation of these highly phytotoxic molecules induces a programmed cell death, leading to the hypersensitive response. This mechanism induces perturbation of the oxidative state of the plant cell which may interfere with the fungus metabolism. To our knowledge, no published data are available on the effects of H 2 or other ROS on fumonisin biosynthesis. Nevertheless, some data are available concerning the role of H 2 in deoxynivalenol (DON) production by F. graminearum. An increase in DON production and an up-regulation of various genes involved in the DON biosynthetic pathway were observed when exogenous H 2 was added to liquid cultures of F. graminearum (Ponts et al., 2006; Ponts et al., 2007). These effects were inversed when catalase, which depletes H 2 from the medium, was supplemented in the liquid cultures (Ponts et al., 2007). These results suggest that an increase in the oxidative stress enhances DON accumulation. In Aspergillus parasiticus, it has also been shown that higher levels of oxidative stress result in enhanced aflatoxin production (Jayashree and Subramanyam, 2000; Narasaiah et al., 2006; Reverberi et al., 2008). Because the fumonisin biosynthetic pathway contains many oxidation steps, it is tempting to speculate that fumonisin biosynthesis may also be enhanced by increasing the levels of ROS, but it still has to be demonstrated. The plant oxylipin pathway is a major defense signalling pathway. The oxylipins function as signals that trigger the plant defense response (Howe and Schilmiller, 2002). They also contribute to increase the oxidative stress of the plant cell. The oxylipin biosynthesis begins with the oxidation of free polyunsaturated fatty acids, chiefly linoleic (C18:2) and linolenic acid (C18:3), through the action of lipoxygenases. The main lipoxygenases are referred to as 9-LOXs and 13-LOXs as oxidation occurs either at the position 9 or 13 of the carbon chains, respectively. The resulting two fatty hydroperoxides induce two distinct biosynthetic pathways. 13-LOXs products lead to the formation of jasmonic acid and its derivatives, well-known key signalling molecules that regulate the expression of certain defense-related genes (Turner et al., 2002). 9-LOXs products lead to less known metabolites but numerous studies on different fungal/host interaction led Gao et al. (2007) to suggest their implication as defense factors in response to fungal attack. However, other data suggested that 9-LOXs products could increase sporulation as well as mycotoxin biosynthesis in certain fungal pathogens (Brodhagen and Keller, 2006; Sagaram et al., 2006b). According to these authors, by mimicking the fungal oxylipins, called psi factors for precocious sexual inducers, plant 9-LOXs pathways could be sensed by the fungus itself to regulate mycotoxin biosynthesis and sporulation. This hypothesis was strongly supported by Gao et al. (2007). They constructed

7 Modulation of fumonisin production in maize 7 a mutant maize line in which a 9-LOX gene (ZmLOX3) was knocked out, resulting in reduced levels of several derivatives of 9-LOXs. Fumonisin production and fungal spore production significantly decreased when inoculating F. verticillioides on kernels of this maize mutant, compared to the wild type. In contrast, the fungus grew similarly on both maize lines. They concluded that ZmLOX3-derived oxylipins are required to facilitate mycotoxin and conidia production by F. verticillioides but have no effect on fungal growth. In addition, the maize mutant line showed decreased susceptibility to the two other pathogens Colletotrichum graminicola and Cochliobolus heterostrophus, which suggests similar effects of ZmLOX3-derived oxylipins on other pathogens. These data were further supported by another study conducted on A. nidulans (Brodhagen et al., 2008). These authors inserted the plant ZmLOX3 9-LOX gene into a wild-type strain and a mutant Δppo strain of A. nidulans. This Δppo is impaired in its ability to produce oxylipin, conidia and sterigmatocystin, a precursor of aflatoxin. Introduction of ZmLOX3 enhanced production of conidia and sterigmatocystin in both strains, suggesting that the maize 9-LOX gene mimics the effect of the endogenous fungal oxylipins involved in spore production and toxin production. Taken together, these two studies support the idea of an oxylipin cross-talk between the fungus and its host plant. The host oxylipin pathway would be perceived by some fungal pathogens and would not only stimulate their spore production but also their toxin production. However, the outcomes of the maize oxylipin pathway on fungal metabolism and toxin production are far more complex and less predictable. Though 9-LOX activity seems to be a susceptibility factor in the maize-aspergillus interaction, as suggested by the study of Brodhagen et al. (2008), Gao et al. (2009) obtained unexpected increased levels of aflatoxin on the mutant maize line with the ZmLOX3 gene disrupted. More presumably, disruption of ZmLOX3 in the maize line must activate a wide range of other unknown downstream derivatives inducing different regulatory effects. It seems then that disruption of ZmLOX3 in maize can result in both an activation of the aflatoxin biosynthetic genes and a repression of the fumonisin biosynthetic genes (Gao et al., 2009). Previously, we have illustrated that metabolites with pro-oxidant properties (H 2, lipid peroxides) may act as stimulator of toxin biosynthesis. In contrast, fumonisin production, as well as aflatoxin and trichothecenes B production, can be inhibited by adding plant natural antioxidants to in vitro fungal cultures. This inhibitory effect of antioxidants is consistent with the idea developed above that fumonisin biosynthesis may be stimulated by oxidative stress. Reviews of in vitro antioxidants inhibitors of trichothecenes B and aflatoxin biosynthesis have been recently published (Boutigny et al., 2008; Holmes et al., 2008). Plant natural antioxidant compounds and more particularly phenolics have often been reported to have antifungal properties (Beekrum et al., 2003; Kim et al., 2004; Kim et al., 2006; Nesci et al., 2007). Phenolics are major components of plant cell walls (Wallace and Fry 1994) and are most likely encountered by the fungus during plant infection. Their inhibitory effects on disease spread might be explained by a rapid accumulation of these compounds within the infection site (Nicholson and Hammerschidt, 1992), triggering inactivation of fungal enzymes and/or reinforcement of plant cell walls (Bell, 1981) which would act as a mechanical barrier against the pathogen. In F. verticillioides and F. proliferatum, there is evidence that antioxidant compounds are able to affect the activity of hydrolytic enzymes (N-acetyl-β-D-glucosaminidase, β-d-glucosidase, and Table 2. Plant natural antioxidant compounds at concentrations showing specific inhibitory effect to fumonisin production with little or no inhibition on fungal growth. Class phenolic compounds cyclic terpenes Antioxidant compounds Concentration tested Reduction of fumonisin production* Media Time** Species*** References liquid medium 21 d 1 strain Fv Beekrum et al., 2003 chlorophorin 0.8; 1; 1.45 µg ml 1 iroko 1 µg ml 1 89 liquid medium 21 d 1 strain Fv Beekrum et al., 2003 maakianin 1 µg ml 1 91 liquid medium 21 d 1 strain Fv Beekrum et al., 2003 caffeic acid 1 µg ml 1 90 liquid medium 21 d 1 strain Fv Beekrum et al., µg g maize kernels d 1 strain Fv, Samapundo et al., strain Fp ferulic acid 1 µg ml 1 90 liquid medium 21 d 1 strain Fv Beekrum et al., 2003 vanillic acid 1 µg ml 1 90 liquid medium 21 d 1 strain Fv Beekrum et al., µg g maize kernels d 1 strain Fv, 1 strain Fp Samapundo et al., 2007 limonene 75 µg g 1 45 maize kernels 28 d 1 strain Fv Dambolena et al., 2008 thymol 75 µg g 1 49 maize kernels 28 d 1 strain Fv Dambolena et al., 2008 *Percentage of fumonisin reduction compared to untreated control **Number of days after inoculation before fumonisin production was measured ***Species tested, F. verticillioides (Fv) and F. proliferatum (Fp)

8 8 A. Picot et al. α-d-galactosidase) (Reynoso et al., 2002), required for the degradation of maize cell walls. The consequence is that these compounds disturb the colonization of maize kernels (Marín et al., 1998b). However, it is noteworthy that at lower concentrations, some antioxidant compounds can inhibit toxin biosynthesis without affecting fungal growth (Holmes et al., 2008). Table 2 presents a list of studies reporting the inhibitory effect of plant natural antioxidant compounds on fumonisin biosynthesis at concentrations showing little to no inhibition on fungal growth. Nevertheless, this specific effect of antioxidant compounds on the toxin biosynthetic pathway remains to be elucidated. One hypothesis is that antioxidant compounds may directly block some of the steps of the fumonisin biosynthetic pathway. This pathway includes many oxidation steps that require high oxygen levels. In A. parasiticus, it has been demonstrated that oxygen requirements progressively increase during the different steps of aflatoxin biosynthesis, and are lowest in the non-toxigenic strain and highest in the aflatoxin producing strain (Narasaiah et al., 2006). Consequently, it can be hypothesized that antioxidant compounds, by trapping oxygen molecules, may limit the availability of oxygen molecules required for some fumonisin biosynthetic reactions. Another hypothesis is that antioxidant compounds may modify the redox equilibrium of the fungal cell, therefore inducing a regulation cascade upstream of the fumonisin biosynthetic pathway that will eventually inhibit or reduce fumonisin production. Alternatively, antioxidant compounds may reduce or suppress upstream signals, such as oxidative stress, that induce toxin biosynthetic pathway (Kim et al., 2007). For instance, it has been shown that caffeic acid induces an increased expression of certain fungal antioxidant enzymes, such as alkyl hydroperoxide reductase, leading to an attenuation of the fungal oxidative stress and to reduced levels of aflatoxin production without affecting fungal growth (Kim et al., 2007). In summary, compelling evidence shows that changes in the oxidative state of the plant cell, through the production of ROS and/or lipids peroxidation, interfere with the fungal metabolism. As described in Figure 1, the fungus seems therefore to benefit from the plant 9-LOX genes products to activate its own oxylipin pathway which will positively regulate sporogenesis and fumonisin production (Gao et al., 2007). Though not demonstrated in the F. verticillioides-maize pathosystem, the oxidative burst may also activate toxin production. Preventing oxidative stress might therefore help to impede toxin accumulation. Nevertheless, as stated by Magbanua et al. (2007), the plant cell must strike a balance in the oxidative state that is sufficient to trigger defense signalling pathways but low enough to prevent oxidative stress that would lead to a toxin accumulation. Fumonisin production as a response to stressful environmental conditions? Depending on its environmental growth conditions, the fungus will sense various external signals and will respond by regulating its production of secondary metabolites (Yu and Keller, 2005). Throughout this review, we mentioned various examples suggesting that stressful conditions for fungal growth (acidic conditions, a w stress, nitrogen-starving conditions, or oxidative stress) may enhance fumonisin production (Keller et al., 1997; Shim and Woloshuk, 1999; Jiménez et al., 2003; Jurado et al., 2008; Kohut et al., 2009). In the course of maize kernel colonization, F. verticillioides may be subject to a certain number of these stressful conditions leading to a stimulation of fumonisin production. This could result in situations in which fumonisin accumulation occurs in maize kernels with a limited fungal development (Jurado et al., 2008) and could account for fumonisin-contaminated ears with little or no visible symptoms. Mycotoxin production has been viewed as an adaptation of the fungus to stressful environmental conditions for growth (Schmidt-Heydt et al., 2008). However, the benefit for the fungus to produce higher levels of fumonisins in such conditions remains an open issue. Indeed, fumonisin production represents a metabolic cost for the fungus. Moreover, the fact that deletion of fumonisin production has no effect on aggressiveness in maize kernels does not support the idea that fumonisin production facilitates maize ear colonization (Desjardins et al., 2002). However opposite results were recently obtained on maize seedling blight by Glenn et al. (2008), who conferred both fumonisin production and pathogenicity on maize seedlings to a Fusarium verticillioides banana strain, by complementing this non-toxigenic strain with the FUM cluster. Therefore, to which extent fumonisins are involved in the fungus pathogenicity still remains to be clarified. An alternative hypothesis is that fumonisin would help the fungus to compete in its natural environment with other microorganisms. However, although several field and in vitro studies showed that F. verticillioides is dominant against F. graminearum, A. flavus and Penicillium spp., none of these studies could directly link this competitive advantage to fumonisin production (Wicklow et al., 1988; Marín et al., 1998a; Reid et al., 1999; Zorzete et al., 2008). Conclusion Fumonisin regulation is a complex process governed by environmental conditions and by the interaction with the host plant. Although some steps of the molecular regulation of fumonisin production have been recently elucidated, further studies are needed to piece together

9 Modulation of fumonisin production in maize 9 the signalling and regulatory networks, involving both fungal primary and secondary metabolism. The use of recent multiple microarrays allowing high-throughput screening will provide, into the next few years, additional evidence on differential expression of fungal genes under a wide variety of conditions and will therefore help characterizing the fungal genes involved in fumonisin regulation. A more complete understanding of the factors regulating fumonisin biosynthesis during maize kernel colonization is required to the development of novel methods aiming at reducing fumonisin contamination in field. Among the different strategies currently underway, genetic engineering offers great expectations but implies years of experimentations from lab to field to be really effective and raises the issue of public acceptance. Though promising genes encoding fumonisin degrading enzymes have been identified such as the fumonisin esterase and amine oxidase genes of Exophiala spinifera (Duvick, 2001), none of these genes have yet been successfully introduced in maize. Consequently, to our opinion, plant breeding currently offers the best and safer perspective to reduce fumonisin contamination in maize crops. Promising candidates may be phenolic acids since their inhibitory effects towards fumonisin biosynthesis are well documented in vitro. However, more studies, and especially in vivo studies, are needed to clarify their potential role in inhibiting the fumonisin biosynthetic pathway. Acknowledgements The authors would like to thank Sophia Ahmed for her proof reading. Declaration of interest The authors are grateful to ARVALIS-Institut du végétal and the ANRT (National Agency for Research and Technology) for their financial support as part of a PhD grant. 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