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

Abstract

The Fusarium fujikuroi species complex (FFSC) is a cosmopolitan fungal lineage with production of a broad spectrum of secondary metabolites including mycotoxins, pigments and plant hormones. The FFSC includes many important plant pathogens. Fusarium fujikuroi, a member of the FFSC, causes rice bakane disease and has been recognized as the exclusive gibberellin (GA) producer for a long time. However, other species such as Fusarium proliferatum, Fusarium sacchari and Fusarium konzum in the FFSC were also identified to produce GA in recent 20 years. GA biosynthesis is conferred by a gene cluster including 7 adjacent genes (GA gene cluster). Expression of the GA genes is activated under limited nitrogen conditions. GA low- or non-production in most FFSC species was revealed to attribute to a partial deletion of the GA gene cluster, malfunction or low expression of the GA genes although the cause has not been fully elucidated. It has been reported that transcriptional factors, signaling components, global regulators and histone modification are involved in regulation of the GA gene expression.

Introduction

The Fusarium fujikuroi species complex (FFSC) includes important plant pathogens. This species complex comprises more than 50 phylogenetically different species [1], including 12 mating populations (MP-A to MP-L) [2]. According to the phylogenetic analyses, the FFSC was divided into the American, African, and Asian clades [1]. Recently, three clades were proved to be evolved from a common ancestor and the Asian clade was branched in the earliest time [3]. The FFSC members have been intensively studied for producibility of various secondary metabolites, including mycotoxins: fusarin [4], fusaric acid [5], beauvericin [6], moniliformin [7], fumonisin (FUM) [8], pigments: neurosporaxanthin [9], bikaverin [10] and fusarubin [11], and plant hormones: gibberellins (GAs) [12], auxins [13], and cytokinin [13]. The mycotoxins and plant hormones (especially GAs) that the FFSC produces are involved in plant diseases. Fusarium fujikuroi is a member of the Asian clade, being intensively studied for its pathogenicity to induce rice bakanae disease which causes 30% to 95% yield loss in the world [14].

F. fujikuroi causes rice bakanae disease by production of GAs. GA is a family of diterpenoids containing 136 chemical forms whereas the bioactive forms as plant hormone are confined to GA₁, GA₃, GA₄ and GA₇ [15]. In most plants, the final GA products are GA₁ and GA₄, while the main product is GA₃ in F. fujikuroi [16]. GA₃ promotes seed germination [17] and responses to abiotic stress [18], and enhances fruit growth [19] and flowering [20], while, GA₃ production of F. fujikuroi in rice plant induces bakanae disease. The diseased seedlings have yellowish to pale leaves and are taller than healthy seedlings. During growth, diseased rice may die because F. fujikuroi attacks all organs [14]. GA₃ production is not necessary for F. fujikuroi to survive in plants or in vitro but confers F. fujikuroi an advantage to invade into the rice root cells [21].

2. GA biosynthesis pathway and the gene cluster

In F. fujikuroi, GA biosynthesis is conferred by a gene cluster that consists of 7 adjacent genes in chromosome Ⅴ, including the P450 mono oxygenase genes (P450-1, P450-2, P450-3 and P450-4), the ent-copalyl diphosphate/ent-kaurene synthase gene (CPS/KS), the geranylgeranyl diphosphate synthase gene (GGS2) and the desaturase (DES) [21, 22] (Fig. 1, Table 1). GA biosynthesis starts with farnesyl diphosphate (FDP) produced from acetyl-CoA by mevalonic acid pathway (Fig. 2). Geranylgeranyl diphosphate (GGDP) is generated from FDP by GGS2 [22]. GGDP is transferred into the GA-specific intermediate ent-kaurene by CPS/KS [22]. Ent-kaurene is converted into ent-kaurenoic acid by P450-4 then into ent-7α-hydroxy-kaurenoic acid by P450-1 [22]. GA₁₂ and GA₁₄ are synthesized from ent-7α-hydroxy-kaurenoic acid by multifunctional P450-1 [23]. P450-2 catalyzes GA₁₄ to GA₄ [24]. GA₁ and GA₇ are generated from GA₄ by P450-3 and DES, respectively [24, 25]. The main product GA₃ is converted from GA₇ by P450-3 [25] (Fig. 2). GA biosynthesis pathway in F. fujikuroi is different from that in high order plants, suggesting independent evolution of GA production between Fusarium and plants [26].

Figure 1: Gibberellin biosynthesis gene cluster in the FFSC members. The presence of genes is indicated by black arrows whereas the absence of genes is indicated by white arrows.

Figure 2: Gibberellin biosynthesis pathway in Fusarium fujikuroi. The pathway shows genes, enzymes and products. The main pathway is shown by bold arrows. FDP, GGDP and CPP means farnesyl diphosphate, geranylgeranyl diphosphate, and ent-copalyl diphosphate, respectively. The final products are indicated by gray ellipses. The chemical structure of gibberellin A₃ (GA₃) was indicated at the bottom.

Table 1: Genes involving gibberellin biosynthesis

Gene	Gene ID a)		Function b)	Reference
DES	FFUJ_14331	Gibberellin biosynthesis gene cluster	Desaturase	[21]
P450-4	FFUJ_14332		P450 monooxygenase
P450-1	FFUJ_14333		P450 monooxygenase
P450-2	FFUJ_14334		P450 monooxygenase
GGS2	FFUJ_14335		Geranylgeranyl diphosphate synthase
CPS/KS	FFUJ_14336		Ent-copalyl diphosphate synthase/ent-kaurene synthase
P450-3	FFUJ_14337		P450 monooxygenase
FfSge1	FFUJ_07864		SM gene expression regulator Sge1	[32]
FfHda1	FFUJ_09787		Histone deacetylases	[50]
FfHda2	FFUJ_01551		Histone deacetylases
AreA	FFUJ_06143*		GATA transcription factor AreA	[27]
AreB	FFUJ_05048*		GATA transcription factor AreB
meaB	FFUJ_03322*		bZIP transcription factor MeaB	[36]
Ffvel1	FFUJ_01649*		Velvet complex VeA	[46]
Ffvel2	FFUJ_01561*		Velvet complex VelB
FfLae1	FFUJ_00592*		Velvet complex
FfAc	FFUJ_01428*		Adenylyl cyclase	[40]
FfG1	FFUJ_08667*		G protein alpha subunit	[38]
FfG3	FFUJ_04487*		G protein alpha subunit
Ffpka2	FFUJ_06244*		Protein kinase A2 catalytic subunit 2
wcoA	FFUJ_13691*		White collar 1-like protein	[42]
tor	FFUJ_06537*		Target of rapamycin protein	[43]
GLN1	FFUJ_07224*		Glutamine synthetase	[33]
cpr-Gf	FFUJ_04716*		Cytochrome P450 oxidoreductase	[35]

a) Gene ID of whole genome sequence of strain IMI 58289 [21]

b) Functions were referred to the “References” column.

*: FFUJ number was not indicated in the reference and it was determined by high score with BLAST (homology) search.

3. Regulation of gibberellin production

3.1 Transcriptional factors

Regulatory mechanism model of the GA gene cluster revealed so far was summarized in Fig. 3. The number of transcriptional factor (TF) genes were compared among several FFSC members by Wiemann et al. [21]. In F. fujikuroi, 950 TF genes were present, that was more than F. verticillioides (640) and F. circinatum (841), however, it was close to F. mangiferae (945). These TFs were classified into families according to the putative binding domains [21]. F. fujikuroi had 235 TF families, which was more than other members. In addition, 53 TFs with low homology (less than 60% identity) to TFs in F. verticillioides, F. circinatum and F. mangiferae, were detected in F. fujikuroi [21]. GA production in F. fujikuroi depends on the nitrogen conditions. GA production in F. fujikuoi increases under limited nitrogen conditions and vice versa [21]. Expression of GA genes is repressed under high nitrogen concentration [27]. A GATA-type TF AreA which directly binds to the GATA motifs in promoters in the GA gene cluster, positively regulates GA gene expressions except P450-3 [28] (Fig. 3). The positive regulation is conducted by utilization of alternative nitrogen sources (nitrate, arginine, urea and allantoin) if preferred nitrogen sources (glutamine and ammonium) are absent [29, 30]. Another GATA-type TF AreB is essential for expression of the GA genes [31]. Unlike AreA, AreB can affect gene expression positively and negatively [31] (Fig. 3). The TF FfSge1 is an orthologue of Sge1 in Fusarium oxysporum though FfSge1 does not regulate conidiogenesis in F. fujikuroi as Sge1 [32]. FfSge1 upregulates expression of the GA genes under limited nitrogen conditions [32] (Fig. 3). The glutamine synthetase (GS) encoded gene GLN1 has positive effect on GA gene expressions [33] (Table 1) although glutamine itself is known to repress synthesis of nitrogen metabolites [34] (Fig. 3). The cytochrome P450 oxidoreductase gene cpr-Gf was proved to be essential for GA gene expressions, especially to P450-1, P450-2 and P450-4 [35] (Table 1, Fig. 3). The negative TF of GA production is the basic leucine zipper (bZIP) TF MeaB [36] (Fig. 3). In F. fujikuroi, MeaB generates the large transcript (MeaB^L) in sufficient nitrogen and the small transcript (MeaB^S) in limited nitrogen conditions. AreA binds to MeaB^L and represses its expression [36].

Figure 3: Regulatory mechanism model of expression of gibberellin biosynthesis gene cluster. The transcription factor is indicated by ellipses whereas the velvet complex is indicated by white rectangle. The synthetase is indicated by circle, and the signaling components were indicated by rhombus. And the gene is indicated by black rectangle. The positive effect is emphasized by bold arrow whereas the negative effect is indicated by imaginary arrow. The histone modification is shown by dot ball. The white arrow in the ‘euchromatin’ means expression of gibberellin biosynthesis genes.

3.2 Signaling components

In fungi, secondary metabolites biosynthesis is significantly influenced by external conditions. Heterotrimeric G proteins that are known as the major signaling components are essential for various secondary metabolites biosynthesis to sense and transduce the external signals [37]. However, gene disruption of the heterotrimeric G subunit FfG1 or FfG3 had no effect on GA biosynthesis [38] (Table 1). The cyclic adenosine monophosphate (cAMP) dependent protein kinase A (Pka) pathway is one of the best characterized signaling pathways implicating various cellular activities [39]. Disruption of the adenylyl cyclase gene FfAc that generate cAMP reduced GA production in F. fujikuroi with dramatic decrease of CPS/KS transcription [40] (Table 1). It has been demonstrated that FfAc and FfPka2 have positive effect on GA biosynthesis [38, 40] (Table 1, Fig. 3). As for response to light, the White Collar complex (WC complex) which comprises the WC protein wc-1 and wc-2 was found in fungi [41]. Disruption of the wcoA that is an ortholog of wc-1 in F. fujikuroi decreased GA production either in the dark or under continuous illumination [42] (Table 1, Fig. 3). In addition, tor that encodes the target of rapamycin kinase, involving in nutrient signal, is known to be essential for GA gene expression in F. fujikuroi [43] (Table 1, Fig. 3).

3.3 Global regulator

Global regulators are also associated with secondary metabolites biosynthesis. The most characterized global regulator is velvet complex which was revealed in Aspergillus spp. [44, 45]. FfVel1 and FfVel2 were identified as component genes of velvet complex in F. fujikuroi [46] (Table 1). These were proved to positively regulate expression of GA genes in F. fujikuroi [46] (Fig. 3). In addition, another velvet-like regulator FfLae1 (homolog of Lae1 in Aspergillus spp.) is also revealed to be an activator for GA biosynthesis [46] (Table 1, Fig. 3).

3.4 Epigenetic regulation

It has been known that chromatin modification is involved in gene expressions [47]. In fungi, expression of secondary metabolites biosynthesis genes was proved to be regulated by chromatin-modifying enzymes [47]. Histone-modifying enzymes can be markers to facilitate or repress gene transcription by changing chromatin structure, such as acetylation of lysine 9 at histone H3 (H3K9ac) and dimethylation of lysine 4 at histone H3 (H3K4me2), which positively regulate gene transcription. On the contrary, trimethylation of histone H3 lysine 9 (H3K9me3) represses gene transcription [47, 48]. In F. fujikuroi, H3K9ac was enriched in nuclei whereas H3K9me3 was enriched mainly in periphery of nuclei [21]. H3K9ac and H3K4me2 occurred in several chromatin regions in F. fujikuroi under limited nitrogen conditions, whereas H3K9me3 was not observed [21]. H3K9ac occurred not only in the region of GA gene cluster but also in bikaverin and FUM biosynthesis gene clusters. While, H3K4me2 occurred only in the region of GA gene cluster, especially in P450-4 and P450-2 [21] (Fig. 3). Recently, association of TFs and histone-modification in F. fujikuroi was proposed as “deletion AreA and AreB results in reduced H3K9ac levels at GA gene cluster which is in line with an abolished biosynthesis of GA” [49]. Histone acetylation is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs) [48]. F. fujikuroi carries two HDAC genes, FfHda1 and FfHda2 (Table 1), and disruption of either gene resulted in decrease of GA gene expression and GA production [50] (Fig. 3).

4. Gibberellin production diversity

4.1 Asian clade

4.1.1 F. fujikuroi

Intriguingly, GA producibility has been proved to be diverse in F. fujikuroi in recent 30 years although F. fujikuroi is well known as GA producer. In addition, negative tendency of GA producibility and FUM producibility has been observed in F. fujikuroi [51]. GA production was detected in FUM non-producing F. fujikuroi strains [51]. Fifty-eight F. fujikuroi strains isolated from diseased rice produced 1 to 74 μg/g GA, but FUM production were detected only in 8 strains of them; mean concentration of fumonisin B₁ and fumonisin B₂ was 380 μg/g and 23 μg/g, respectively [52]. Ten F. fujikuroi strains that induced rice seedlings yellowish and slender leaves were proved to produce GA₃ while trace level of FUM [53]. According to the phylogenetic and pathogenicity analyses, Niehaus et al. [54] found that two pathotypes with phylogenetically different are present in F. fujikuroi: bakanae type producing GA but a little or no FUM and stunting type producing FUM but not GA. Suga et al. [51] designated G- and F-group in F. fujikuroi based on the results of phylogenetic analyses and FUM and GA production assays. G-group produces a significant amount of GA but no FUM, whereas F-group produces a significant amount of FUM but GA at very low or undetectable level. G-group strains induced abnormal elongation on rice seedlings while seedlings infected with F-group strains did not show the symptom [51]. Bao et al. [55] revealed that GA low producibility in F-group strains attributed to low expression of P450-1, P450-4 and P450-2 in the GA gene cluster at least. According to Piombo et al. [56], GA production difference were detected among three F. fujikuroi strains. The strain with the highest GA₃ production induced abnormal elongation on rice seedlings whereas the strain with the lowest production induced stunting. GA production of these strains were affected by temperature in vivo. In addition, FUM production was detected in the GA low-producer in vitro. Expression of CPS/KS was in accordance with GA production among these strains.

4.1.2 F. proliferatum

GA production was absent in most F. proliferatum strains [57]. The cause of GA non-production in F. proliferatum has been investigated with D02945 and D00502 strains. Mutations in P450-4 and deficient expression of P450-1 might be the cause of GA non-production in these strains [58, 59]. In addition, inactivation of GGS2 and CPS/KS by mutations in the coding and 5’-noncoding region, and nonfunctional DES were also involved in GA non-production in D00502 [59]. GA production of these strains were recovered by complementation of functional GA genes from F. fujikuroi [58, 59]. Therefore, regulation of GA gene cluster in these F. proliferatum strains are functional. TFs binding to the GA gene cluster have also been studied in F. proliferatum. The mutations were detected in GATA motifs in the promoter region between P450-4 and P450-1 in D02945 although sequence of the GA gene cluster in F. proliferatum has high homology to that in F. fujikuroi. The mutations negatively affect binding of the GATA TFs and the transcription of P450-4 and P450-1 [58]. Later, GA production was exceptionally detected in several stains of F. proliferatum. The GA production of KGL0401 was even higher than a F. fujikuroi strain [60]. The orchid-associated F. proliferatum strain ET1 produced significant amount of GA₄ and GA₇ [61]. GA production in the F. proliferatum strains might be utilized as endophytes rather than pathogens in plant [62].

4.1.3 F. sacchari

GA₃ production has been detected in F. sacchari (MP-B) strains B-7610, B-12756 and B-1732 although the production level was lower than F. fujikuroi [62, 63]. Most of F. sacchari strains are GA non-producers. Inactive GGS2 and CPS/KS and low expression of DES might be the causes of GA low or non-production in F. sacchari [62].

4.2 American clade

4.2.1 F. konzum

It was reported that F. konzum (MP-I) did not produce GA [57] despite the presence of the whole GA gene cluster in the genome (Fig. 1). GA non-production is due to no GA oxidase activities [62]. However, several F. konzum strains were revealed to produce GA. I-10653 strain produced GA₁ [64] and I-11616 and I-11893 produced a lot of GA₁ and a little GA₃ [62]. DES and CPS/KS expression were detected in the GA producer but not in the non-producer of F. konzum [57].

4.2.2 Other species

Five F. subglutinans strains did not produce GA although they retained the whole GA gene cluster [57] (Fig. 1). GA non-production in two F. circinatum strains and one F. anthophilum strain might be associated to deletion of the GA gene cluster [57]. The Only DES remains in most of F. circinatum strains [65], however, in the GA non-producer Fsp34, the whole GA gene cluster are present [66]. Only DES and P450-4 remain in F. anthophilum [1] (Fig. 1).

4.3 African clade

GA production has not been detected in the African clade although the number of the strains in each species for investigation are limited. For example, 10 [67] and 5 strains were investigated for the GA non-production in F. verticillioides [57]. Five strains of each F. thapsinum and F. nygamai, one strain of each F. udum and F. napiforme did not produce GA [57]. Some species retain partial GA gene cluster. Only DES and P450-4 remain in F. verticillioides [67]. Transferring FvDES into F. fujikuroi DES deletion mutant recovered GA production in the mutant [67]. Only P450-3 remains in F. napiforme [1] (Fig. 1).

5. Overview

Among the FFSC, F. fujikuroi has been recognized as the unique GA producer for longtime, for which it induces rice bakanae disease. GA production of F. fujikuroi depends on expression of the GA genes (P450-1 to P450-4, DES, GGS2 and CPS/KS) which are closely correlated to nitrogen conditions [21] and are positively or negatively regulated by TFs, signaling components, global regulators, and histones. Intriguingly, GA producibility was diverse among different F. fujikuroi strains. GA non-production was detected in some strains, following non-occasion of typical bakanae symptom [51, 54]. According to phylogenetic analyses, pathogenicity analyses and GA production assay, F. fujikuroi was designated as G-group (GA producer) and F-group (GA non-producer) [51]. Extraordinary low- or non-expression of the GA genes was identified in the representative F-group strains [55, 68]. Single GA gene transferring from the G-group strain increased GA production and expression of other GA genes simultaneously of the F-group strains [55].

GA production is not detected in most FFSC members. One of causes of GA non-production in most FFSC members is deletion of the GA gene cluster. For example, only DES is present in most of F. circinatum strains [65] and only P450-3 is present in F. napiform [1]. Only DES and P450-4 remain in F. anthophilum [1] and in F. verticillioides [67]. In addition, malfunction or low- to non-expression of the GA genes also result in GA non-production. Deficient expression of P450-1 and mutations in P450-4, GGS2 and CPS/KS were identified to associate to GA non-production in F. proliferatum [58, 59]. In active GGS2 and CPS/KS, following low expression of DES was detected in F. sacchari [62]. In GA non-producers of F. kozum, DES and CPS/KS did not express [57].

Recently, GA producibility was detected in several strains of other FFSC members, such as F. proliferatum [60, 61, 62], F. sacchari [62, 63], and F. konzum [62, 64]. Researches of the GA genes are beneficial to elucidate mechanism of GA production of the FFSC, furthermore, to explore control methods of fungal disease at genetic level in the future.

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