Genetic Background of Variable Fumonisin Production in the Fusarium Fujikuroi Species Complex

Abstract

Fumonisin is a worldwide mycotoxin that has devastating implications for human and animal health and food security. The principal source of fumonisin contamination is the members of the Fusarium fujikuroi species complex (FFSC). A cluster (FUM gene cluster) of 16 co-expressed genes (FUM) responsible for fumonisin biosynthesis has been identified and characterized in Fusarium verticillioides. The FUM gene cluster has been detected from other members of the FFSC. In this review, the fumonisin production ability and the status of the FUM gene cluster in 3 clades (African, American, and Asian) of the FFSC have been presented. The comprehensive studies revealed that intraspecific variation is caused by several types of mutations in the FUM gene cluster. In addition, we summarized the regulatory genes involving fumonisin biosynthesis. A comparative study of fumonisin production ability and regulatory mechanism of fumonisin biosynthesis provides valuable insight for control of the mycotoxin contamination.

1. Introduction

The Fusarium fujikuroi species complex (FFSC) includes important pathogens to various crops. This species complex corresponds to mostly Fusarium moniliforme Sheild. or section Liseola defined by Wollenweber and Reinking. Fifty phylogenetically different species and 12 mating populations (MP-A to MP-L) are comprised in the FFSC at least [1, 2]. The FFSC members can be divided into three phylogenetic clades; the American, African, and Asian clades [1] and are known to produce a broad spectrum of secondary metabolites including fumonisin.

Fumonisin is a carcinogenic polyketide mycotoxin, chemically, a diester of propane-1, 2, 3-tricarboxylic acid and pentahydroxyicosane containing primary amino acid [3]. There are several types of fumonisin. Fumonisin B₁ (FB₁), B₂ (FB₂) and B₃ (FB3) are the major forms found in varieties of crops and foods. In the world, fumonisin contamination has an impact on the quality of maize products such as cornflakes, Portuguese maize bread, chips, corn starch, grits, flour, popcorn and so on [4,5] because fumonisin is heat stable. The maximum tolerable daily intake of FB₁, FB₂ and FB₃, alone or together, is 2 µg/kg body weight per day [6].

Fumonisin has adverse effects on humans and animals [7]. Fumonisin intake causes leukoencephalomalacia in horses [8], pulmonary edema, liver and pancreatic lesions in swine [9], neural tube defect in the fetus of the pregnant woman [10], skin lesions [11], liver and kidney disease in mammals [12, 13]. Consume of high concentration of FB₁ causes esophageal cancer in China and South Africa and upper gastrointestinal tract cancer in Northern Italy [7, 14]. Furthermore, fumonisin contamination in breast milk has been also reported [15]. FB₁ causes damages to the cell membranes, disruption of chlorophyll and sphingolipid biosynthesis in maize [16]. Doehlert et al. [17] suggested that high concentration fumonisin might have a deleterious effect on maize seedling emergence.

Twenty four members of the FFSC were identified as fumonisin producers. All over the world, FFSC members that produce FB₁ were detected from maize, rice, wheat, barley, oat, hops, sorghum, millet, soybean, asparagus, garlic, ginger, oil palm, mango, banana, strawberry and so on [18, 19, 20, 21]. Fusarium verticillioides, the pathogen of maize ear rot, has been known as the predominant contaminant source of fumonisin worldwide. The whole-genome sequence of F. verticillioides was revealed by Ma et al. [22]. The presence of inter and intra-specific variation in fumonisin production has been notified in the FFSC. However, fumonisin producibility in the FFSC is confused and the information of the cause of fumonisin production variability is limited so far.

2. Fumonisin biosynthetic pathway and the gene cluster

The fumonisin biosynthesis starts with the formation of linear dimethylated polyketide (20-carbon linear polyketide) as well as the condensation of the polyketide with alanine, followed by a carbonyl reduction, oxygenations and esterification with two propane-1, 2, 3- tricarboxylic acids (tricarbollytic acid-coA) [23] (Fig. 1). The 16 FUM genes involved in the fumonisin biosynthesis are clustered in the 42 kbp-stretch of the F. verticillioides genome (FUM gene cluster) (Fig. 2, Table 1). The order and orientation of FUM genes were the same in F. verticillioides and Fusarium proliferatum [24, 25]. The cluster contains a Zn(II)-2Cys6 DNA binding transcription factor (FUM21) [26], a polyketide synthase (FUM1) [24], an aminotransferase (FUM8) catalyzes the condensation of polyketide and alanine to form the 20-carbon-long fumonisin backbone [25], a cytochrome p450 monooxygenase (FUM6) catalyze hydroxylation at C-14 and C-15 [27], a C-3 carbonyl reductase (FUM13) [28], another cytochrome p450 monooxygenase (FUM12) catalyzes the hydroxylation of C-10 [29], 2-ketoglutarate deoxygenase (FUM3) catalyzes the hydroxylation of C-5 [30]. Tricarballylic acid esterification is conducted by four genes FUM7, FUM10, FUM11 and FUM14 [31, 32]. Two genes are longevity assurance factors (FUM17 and FUM18) and the terminal one is ABC transporter (FUM19) [24]. However, each gene disruption of FUM15 (cytochrome p450 monooxygenase gene), FUM16, FUM17, FUM18 and FUM19 did not affect fumonisin production in F. verticillioides [23, 31].

Figure 1: Fumonisin biosynthetic pathway in Fusarium verticillioides. The pathway shows genes and products. The final products were indicated by gray ellipses. The chemical structure of fumonisin B₁ (FB₁) was indicated at the bottom.

Figure 2: Fumonisin biosynthesis gene cluster in FFSC members. The presence of genes is indicated by black arrows whereas the absence of genes is indicated by white arrows. Gray box indicates insertional sequence.

Table 1: Genes involving fumonisin biosynthesis

Gene	Gene IDa)		Function	Reference
FUM21	FVEG_14633	Fumonisin biosynthesis gene cluster	Zn(II)2Cys6 type-transcriptional regulator	[26]
FUM1	FVEG_00316		Polyketide synthase	[24]
FUM6	FVEG_00317		Fumonisin oxygenase	[27]
FUM7	FVEG_00319		Alcohol dehydrogenase	[27]
FUM8	FVEG_00318		Aminotransferase	[25]
FUM3	FVEG_00320		Fumonisin 5-oxygenase	[30]
FUM10	FVEG_00321		Tricarballylic esterification	[31]
FUM11	FVEG_00322		Tricarballylic esterification	[31]
FUM12	FVEG_00323		Fumonisin 10-oxygenase	[29]
FUM13	FVEG_00324		C3 carbonyl reductase	[24]
FUM14	FVEG_00325		Tricarballylic esterification	[32]
FUM15	FVEG_00326		Cytochromosome P450 monooxygenase	[24]
FUM16	FVEG_00326		Fatty acyl-coenzyme A synthetase	[24]
FUM17	FVEG_00327		Similarity to tomato longevity assurance factor (ASC-1) of Alternaria alternata f. sp. lycopersici	[24]
FUM18	FVEG_00328		Similarity to tomato longevity assurance factor (ASC-1) of Alternaria alternata f. sp. lycopersici	[24]
FUM19	FVEG_00329		Similarity to ATP-binding cassette (ABC) multidrug resistant transporter	[24]
AMY1	FVEG_12951		a-Amylase	[36]
AREA	FVEG_02033		Regulator of nitrogen metabolism	[47]
ART1	FVEG_02083		Starch-responsive transcription factor	[93]
CPP1	FVEG_09543		Protein phosphatase 2A catalytic subunit	[94]
FCC1	FVEG_11306		FCK1 cyclin	[95]
FCK1	FVEG_11159		Cyclin-dependent kinase	[95]
FLBA1	FVEG_08855		Regulator of G-protein singnaling	[96]
FLBA2	FVEG_06912		Regulator of G-protein singnaling	[96]
FST1	FVEG_08441		Hoxose transporter	[97]
FUG1	FVEG_04008		Unknown, likely transcriptional regulator	[42]
FvBCK1	FVEG_05000		Mitogen-activated protein kinse (MAPK) kinase kinase	[98]
FvMK1	FVEG_05063		MAPK	[44]
FvSET1	FVEG_07811		H3K4 methyltransferase	[49]
FvSO	FVEG_08055		WW domain protein, signaling component	[45]
FvVE1	FVEG_09521		Velvet-domain-containing protein	[99]
FvVELB	FVEG_01498		Velvet-domain-containing protein	[100]
GBB1	FVEG_10291		Heterotrimeric G-protein B subunit	[101]
GBP1	FVEG_05343		Monomeric G-protein	[102]
LAE1	FVEG_05214		Global regulator of secondary metabolism	[40]
MADS1	FVEG_01965		MADS-box transcription factor	[41]
MADS2	FVEG_03759		MADS-box transcription factor	[41]
PAC1	FVEG_05393		pH regulatory component	[103]
SGE1	FVEG_09150		Transcriptional regulator	[39]
ZFR1	FVEG_09648		FCC1-dependent transcription factor	[104]
GCN5	FFUJ_00382b)		Histone 3 lysines (H3K4, H3K9) acetylase	[50]

a) Gene ID of whole genome sequence of Fusarium verticillioides strain 7600 [22]

b) Gene ID of whole genome sequence of Fusarium fujikuroi strain IMI 58289 [105]

3. Regulation of fumonisin production

Most research on the factors that affect fumonisin production has been conducted with F. verticillioides. The environmental factors impacting the fungal growth and fumonisin production are temperature, light wavelength, and humidity [33, 34, 35]. Nitrogen availability, surrounding pH, carbon nutrients also affect fumonisin production in F. verticillioides [36, 37]. Regulatory mechanisms of fumonisin production have been revealed (Fig. 3, Table 1) though these are considered as a part of complex mechanisms of fumonisin production regulation.

Figure 3: Regulatory mechanism model of expression of fumonisin biosynthesis gene cluster. The ellipses show characteristic categories. The genes with “*” are negative regulator and the genes with “†” are included in multiple characteristic categories. The histone modification is shown by dot ball. The white arrow in the ‘eu chromatin’ means expression of the FUM gene cluster.

3.1 Pathway specific activator

FUM21, a Zn(II)2Cys6 transcription factor with alternative splice forms, plays a key role in fumonisin production in F. verticillioides [26]. Sultana et al. [38] identified the stop codon causing 11 amino acids deletion in the terminal region of FUM21 resulted in fumonisin non-production in a F. fujikuroi strain. The global regulators LAE1 and SGE1 are also involved in fumonisin production [39, 40]. Disruption of MADS1 (a conserved transcription factor) exhibits a reduction of polyketide synthase gene transcription and fumonisin biosynthesis [41]. Recently, FUG1 was identified as a transcriptional regulator integral to fumonisin production in F. verticillioides [42].

3.2 Signaling components

The signaling pathway has adapted and diverged within F. verticillioides and Fusarium graminearum to regulate species-specific secondary metabolite production [43]. Disruption of MAPK (FvMk1) causes a decrease of fumonisin production in F. verticillioides [44]. Similarly, disruption of FvSo that is the homologue of Neurospora crassa SO gene involving hyphal fusion exhibited pleiotropic symptoms and reduced FB₁ production [45]. The HOG-type MAP kinase pathway was shown to be decisive for the fumonisin production in F. proliferatum [46]. Disruption of AREA that is a global regulator and involved in nitrogen utilization lost fumonisin production in F. verticillioides although its direct regulation of FUM genes remains to be unknown [47].

3.3 Epigenetic regulation

Chromatin-based regulation through histone modifications, such as acetylation or methylation affects the expression of the secondary metabolite gene cluster. The epigenetic analysis demonstrated that histone acetylation has a significant effect on FUM gene expressions and fumonisin biosynthesis [48]. Disruption of FvSET1 encoding H3K4-specific histone methyltransferase caused the loss of FB₁ production in F. verticillioides [49]. GCN5, the components of the SAGA complex, is known to be essential for acetylation of histone 3 lysines (H3K4, H3K9, H3K18, and H3K27) in F. fujikuroi. Disruption of GCN5 caused downregulation of H3K9ac and FUM1 expression [50].

4. Fumonisin production diversity

The frequency and the amount of fumonisin production in the members of FFSC have been varied in the preceding publications [51, 52]. Direct comparisons of fumonisin production among publications are difficult due to different assay methods taken in individual investigations. Consequently, the fumonisin amount indicated in this review needs to be evaluated in consideration of these difficulties.

4.1 African clade

4.1.1 F. verticillioides

Almost all F. verticillioides strains from maize have been reported to produce a high amount of fumonisin. Detected fumonisin concentration and the frequency of fumonisin producing strain were 10.6 to 4,749 µg/g and 94%, respectively, in Choi et al. [53] and 5.6 to 25, 846.4 µg/g and 97.1% in Acuna et al. [54]. The highest amount (32,756 µg/g) of fumonisin was detected in F. verticillioides from maize in Indonesia [55]. Fumonisin production has also been detected in F. verticillioides from wheat (2,050 to 14,400 µg/g) [56], rice [51], banana, and so on. F. verticillioides strains from banana in Central America and the Canary Islands were unable to produce fumonisin [57] due to the absence of the FUM gene cluster except for portions of FUM21 and FUM19 [58] (Fig. 2). Later, it was renamed Fusarium musae [59] based on the genetic difference from other F. verticillioides. Therefore, fumonisin non-producing strains are generally rear in F. verticillioides. Investigation of 245 F. verticillioides strains from maize and sorghum in North America detected several FB₂ or FB₃ producing strains and several fumonisin non-producing strains [60]. It was revealed that FB₂ or FB₃, not FB₁ production attributed to the defect of FUM12 and FUM3 [61].

4.1.2 Fusarium nygamai

Nelson et al. [62] detected fumonisin production in F. nygamai from millet and sorghum (3148 µg/g, 37% of the strains investigated). Theil et al. [63] identified one strain of F. nygamai that produced 605 µg/g fumonisin. All F. nygamai strains from poultry feeds of Argentina were found to be fumonisin producers (11.0 to 487 µg/g) [64]. The FUM gene cluster was detected from F. nygamai [65].

4.1.3 Other species

The FUM gene cluster was detected in Fusarium ramigenum and Fusarium phyllophilum (2.5 µg/g) that were reported to produce fumonisin (Fig. 2) [65, 66]. Besides, Fusarium thapsinum (30 µg/g), Fusarium napiforme (16 to 479 µg/g, 16%), Fusarium dlamni (42 to 82 µg/g, 55%), Fusarium acutatum (0.14 µg/g), Fusarium brevicatenulatum (0.15 µg/g), Fusarium pseudocircinatum (0.28 µg/g) and Fusarium pseudonygamai (trace amount) were found to produce a low amount of fumonisin [62, 66, 67, 68].

4.2 American clade

4.2.1 Fusarium subglutinans

F. subglutinans from maize produced 186 to 426 µg/g fumonisin [51, 69]. Kim et al. [70] detected fumonisin production in 7.1% in 141 strains of F. subglutinans from rice and corn grains. Reyneso et al. [71] observed 95 F. subglutinans strains from maize in Argentina produced fumonisin. Whereas, Leslie et al. [72] identified only one F. subglutinans from prairie grasses produced a trace amount of fumonisin.

4.2.2 Fusarium temperatum

F. temperatum has been recognized as a maize pathogen in Poland for three decades [73]. Waskiewicz and Stepien [74] detected 2.25 µg/g fumonisin in F. temperatum from pineapple. Wang et al. [75] reported that F. temperatum from maize in China produced fumonisin under field conditions and Fumero et al. [76] detected fumonisin production in 25% of F. temperatum strains from maize in Argentina. Recently, Fumero et al. [77] obtained the whole genome sequence of a F. subglutinans strain and a F. temperatum strain from Argentina and that suggested absence of FUM genes and fumonisin production in these strains.

4.2.3 Other species

Fusarium bulbicola and Fusarium anthophilum were found to carry the FUM gene cluster and produced fumonisin (58 to 613 µg/g, 18%) (Fig. 2) [62, 65]. These two species were fumonisin C (FC) producers. Two amino acid substitutions in FUM8 were detected between FB (F. verticillioides) and FC-producing species [65]. Darnetty and Saleh [55] found only one strain of Fusarium konzum produced 30.1 µg/g fumonisin as Leslie et al. [72] detected 120 µg/g fumonisin in this species. Fusarium begonia produced 0.07 µg/g fumonisin [66].

4.3 Asian clade

4.3.1 Fusarium proliferatum

The predominant fumonisin producing species in the Asian clade is F. proliferatum. F. proliferatum is considered the most hazardous because of the wide variety of host plants. Laday et al. [78] showed high genetic variability and host preferences in F. proliferatum. Significant intraspecific variability of growth rate was observed in F. proliferatum from garlic [79]. The highest amount (30,949 µg/g) of fumonisin production in F. proliferatum was reported as the strain from maize [80]. The frequency of fumonisin production in F. proliferatum was 38 to 100% [55, 62]. FB₁ production was detected at 156 to 21,423 µg/g of 100% [55] in F. proliferatum from maize, 6 to 7,969 µg/g of 96% from rice, maize, barley and soybean [53], 7.0 to 3,299 µg/g of 100% from pineapple [81], 5 to 922 µg/g of 100% from wheat [56], 663 to 3,503 µg/g of 99% from wine grapes [51], 155 to 2,936 µg/g of 38% from peanuts, pearl millet, sorghum, wheat and corn [62]. The FUM gene cluster was detected in F. proliferatum [65] (Fig. 2).

4.3.2 Fusarium fujikuroi

F. fujikuroi is well known as the rice pathogen. Desjardins [82] reported that 58% of F. fujikuroi strains from rice produced 5 to 1,000 µg/g fumonisin in maize culture. Subsequently, fumonisin production has been detected in F. fujikuroi from maize [53], wheat [56], strawberries [83], grapes [51] and soybeans [53]. A high amount of fumonisin production has been observed in F. fujikuroi from soybeans (5,065 µg/g) [53], rice (14,002 µg/g) [34], maize (3,149 µg/g) [53] and grapes (3,503 µg/g) [51], although different assay methods were used in these studies. The frequencies of fumonisin production in F. fujikuroi strains were 40 to 100% [51, 52, 84]. It was found that the presence of the FUM gene cluster in the genome does not certify the ability of fumonisin production as causative mutations of fumonisin non-production were detected in FUM21 and FUM7 in a F. fujikuroi strain [38]. Rosler et al. [85] detected the low expression of FUM21 in strain IMI 58289 resulted fumonisin low producibility. Recently, the causative mutations of fumonisin non-production were characterized in 44 F. fujikuroi strains: 1) pretermination of FUM21, 2) lack of a part of the FUM gene cluster, 3) the presence of insertional sequences in FUM6 [86] (Fig. 2).

4.3.3 Other species

Fusarium globosum carries the FUM cluster and produced 325 µg/g fumonisin [65, 87]. Fusarium concentricum from pineapple produced fumonisin at 10 µg/g [81]. Kim et al. [70] reported the frequency of fumonisin production in F. concentricum strains from the rice and cornfield was 6% and 3%, respectively.

4.3.4 Clade-unspecified species

Several FFSC members that have not been classified into three clades such as Fusarium polyphialidicum (3 to 19 µg/g), Fusarium ananatum (0.2 to 10 µg/g), Fusarium andiyazi (trace amount) from pineapple, maize, and other crops were reported to produce fumonisin at the low level [68, 81].

5. Overview

The level of fumonisin production depends on genetic variation, environment, and substrate [88]. In the FFSC, the ability of fumonisin production is correlated with the presence of FUM genes but not correlated with phylogenetic relationships [59, 87]. Lack of FUM genes was indicated in the species that had been reported as fumonisin producer (F. napiforme, F. dlamni, F. subglutinans, F. temperatum) [67, 77, 89, 90]. The possible reason for these irrationals is an intraspecific variation of fumonisin producibility. Strain dependent analyses should help to resolve this issue. Proctor et al. [87] suggested that FUM genes are discontinuously distributed in the FFSC. In addition, even if FUM genes are present, a mutation causing the genes non-functional results in fumonisin non-production. Genetic variations associated with fumonisin producibility have been mainly investigated in F. verticillioides, F. proliferatum and F. fujikuroi [79, 86, 91, 92] and that in other members have not been elucidated so far.

Sexual reproduction might increase the adaptation ability in an adverse environment with the creation of intraspecific genetic variations. Genetical differences responsible for secondary metabolite producibility can be identified by molecular genetic and biochemical analyses. Inter and intraspecific natural variation of fumonisin production in the FFSC has received attention because this variation has an impact on agriculture and human health. Research on genetic variation associated with fumonisin producibility in the FFSC is valuable for the welfare of mankind in the future.

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