Accumulation of 4-Deoxy-7-hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-Type FgTri1 Gene

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain’s genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

F. graminearum is largely divided into two chemotypes: an NIV chemotype, which has a functional C-4 hydroxylase encoded by a cytochrome P450 monooxygenase gene, FgTri13, and a DON chemotype with a dysfunctional copy of FgTri13 [4,5]. On the basis of the name of the last precursor that accumulates in the rate-limiting deacetylation step to DON, the DON chemotype is further divided into 3-ADON and 15-ADON chemotypes. In axenic liquid culture, 3-ADON and 15-ADON chemotype strains mainly accumulate 3-ADON and 15-ADON, respectively [6,7]. Similarly, an NIV chemotype strain accumulates acetylated derivatives of NIV, namely mixtures of 4-ANIV and 4,15-diacetylnivalenol (4, [8]. The acetyl groups of these trichothecenes are slowly deacetylated to give DON and NIV when F. graminearum strains are cultured on solid medium. In 2014, the emergence of F. graminearum that synthesizes a new trichothecene unable to be detected by the validated LC-MS/MS method for known trichothecenes was reported in North America. The new trichothecene, 7-hydroxy-15-deacetylcalonectrin (7-H-15-deCAL; designated NX-2), lacked a C-8 ketone of 3-ADON [9]. Because they lack a C-8 ketone conjugated to the 9,10-double bond, NX-type trichothecenes cannot be detected by UV absorbance in high-performance liquid chromatography-based methods [10]. An NX-2 chemotype is hypothesized to have evolved from the 3-ADON chemotype because the structural organization of the trichothecene core gene cluster is the same as that of the 3-ADON chemotype [11]. Thus, the multifunctional cytochrome P450 monooxygenase FgTri1, a trichothecene noncore cluster gene responsible for hydroxylation at both C-7 and C-8, may have evolved into a monofunctional FgTri1_ NX chemotype gene by losing its C-8 hydroxylation function [10].
Fusarium strains that produce NX-type trichothecenes oxygenated at C-4 are not known to date. Outcrossing the known NX chemotype, which produces C-8 unoxygenated 4-deoxytrichothecenes, with a known NIV chemotype that has a functional copy of FgTri13 may lead to the formation of a new NX-type trichothecene oxygenated at C-4 in the natural environment. To assess the possibility of the emergence of such a new chemotype, we genetically engineered known DON and NIV chemotype strains by exchanging their FgTri1 with FgTri1_ NX chemotype .

Strong Expression of FgTri1_ NX chemotype in a FgTri1 Disruption Mutant (∆Fgtri1) of Each Chemotype
The sources of the 3-ADON, 15-ADON, and NIV chemotype strains used and the procedures used to generate the transgenic strains are described in detail in the Supplementary Materials. A FgTri1 disruption mutant (∆Fgtri1) of each DON chemotype was generated by double-crossover homologous recombination ( Figure S1). Strain NBRC 113176 generated in our previous study [12] was used as a ∆Fgtri1 strain of NIV chemotype. A synthetic FgTri1 _NX chemotype gene (1539 bp) was designed such that the 14 amino acids previously identified as being specific to the NX-2 chemotype [10] were conserved ( Figure S2). FgTri1_ NX chemotype was ectopically expressed in a ∆Fgtri1 mutant of each chemotype under the strong promoter (AnPtef) of the Aspergillus nidulans translation elongation factor gene [13] (Figure 1). As expected, the expression levels of FgTri1_ NX chemotype transcribed from the AnPtef promoter were much higher than those of the wild-type strain in each chemotype ( Figure S3). The resulting transgenic strains, ∆Fgtri1/FgTri1_ NX chemotype , proved to be useful for investigating the function of the FgTri1_ NX chemotype gene and trichothecene products. First, we sought to determine whether the transgenic NX-type trichothecene-producing strains could be further divided into NX-2 and NX-4 chemotypes by introducing the aforementioned 14 amino acid mutations into the coding region of FgTri1 in the 3-ADON and 15-ADON chemotypes, respectively. Analysis of trichothecene metabolite accumulation by thin-layer chromatography (TLC) revealed that the trichothecene profiles of the ∆Fgtri1/FgTri1_ NX chemotype strains differed from those of the wild-type and ∆Fgtri1 strains ( Figure 2a,b). In addition, the time course of accumulation of trichothecenes was quite different between the ∆Fgtri1/FgTri1_ NX chemotype strains generated from the 3-ADON and 15-ADON chemotypes. Spot 1, detected from a culture of the transgenic strain of the 3-ADON chemotype, behaved like a trichothecene intermediate at the rate-limiting step to yield spot 2 ( Figure 2a). However, the reaction was so slow that spot 1 remained even after 7 days of incubation. In contrast, no precursors were detected in a culture of the transgenic strain derived from the 15-ADON chemotype; spot 3 promptly appeared on day 3, without accumulation of any earlier intermediates (Figure 2b). The trichothecenes included in spots 1-3 were thought to have been synthesized via calonectrin (CAL), an intermediate that accumulates in the ∆Fgtri1 mutant [12,14], by the function of FgTri1_ NX chemotype ( Figure 2). The engineered biosynthetic pathways of the transgenic NX chemotypes originating from the 3-ADON and 15-ADON chemotypes are considered to proceed via 7hydroxycalonectrin (7-HCA) to each NX-type trichothecene; the metabolite is assumed to be deacetylated to yield 7-hydroxy-15-deacetylcalonectrin (7-H-15-deCAL: NX-2) and 7-hydroxy-3-deacetylcalonectrin

FgTri1_ NX chemotype Overexpressing Strain of NIV Chemotype Accumulates 4-Deoxy-7-hydroxytrichothcenes, but Not 4,7-Dihydroxytrichothecenes
Next, we focused on the possibility of the emergence of a new NX-type trichothecene oxygenated at C-4 ( Figure 3). Time-course metabolite profiles of the ∆Fgtri1/FgTri1_ NX chemotype NIV chemotype strain by TLC revealed the appearance of a trichothecene spot 4, whose retention factor (R f ) value was identical to that of spot 3, on day 5 ( Figure 3a (Figure 2c), indicating that the metabolites in the culture extract were indeed 7-HCA and NX-4 (spot 4) (Figure 3b). Thus, the final toxin product of the transgenic strain in axenic liquid culture, spot 4, was the same product included in spot 3 of the transgenic 15-ADON chemotype. Despite our efforts to identify the C-4 oxygenated NXtype trichothecenes, minor C-4 oxygenated trichothecenes, including the previously identified 7-hydroxy-4,15-diacetoxyscirpenol (7-H-4,15-DAS) [16], were barely detected, or detected in marginal quantities, in a culture of the transgenic ∆Fgtri1/FgTri1_ NX chemotype strain.

Discussion
We used the three known type B trichothecene chemotypes as hosts to investigate the effect of the expression of a recently evolved trichothecene pathway gene, FgTri1_ NX chemotype . Although the production of NX-4 by an engineered 15-ADON chemotype strain has been previously reported [10], the efficiency of 3-O-and 15-O-deacetylations of 7-HCA in an axenic culture of F. graminearum was not compared. The time-course experiment (Figure 2b) revealed that conversion of intermediate 7-HCA to NX-4 was so rapid that 7-HCA was not detected from the culture of the transgenic 15-ADON chemotype. In contrast, quite a few amounts of 7-HCA, showing a larger blue spot on TLC than that of NX-2, accumulated in the culture of the transgenic 3-ADON chemotype (Figure 2a). The very slow formation of NX-2, but not NX-4, by the transgenic 3-ADON and 15-ADON chemotypes suggested that the 15-O-acetyl group of 7-HCA is biologically more stable than the 3-O-acetyl group. This may be attributed to the function of FgTri8, a trichothecene deacetylase gene responsible for 3-ADON/15-ADON chemotype differentiation. FgTri8p_ 15-ADON chemotype C-3 deacetylase of the 15-ADON chemotype appears to accept various trichothecenes as substrates, as opposed to FgTri8p_ 3-ADON chemotype C-15 deacetylase of the 3-ADON chemotype [17]. In addition to the difference in the toxin formation kinetics between the two chemotypes, the present study unambiguously demonstrated that C-4 oxygenated trichothecenes marginally accumulated in a culture of the NIV chemotype harboring the FgTri1_ NX chemotype gene. The result suggests that CAL and 7-HCA were poor substrates of FgTri13p, supporting our previous finding of the specificity of the FgTri13p enzyme towards trichothecene intermediates [12]. These findings shed light on the biosynthetic pathway of NIV after CAL in F. graminearum. Thus, outcrossing of the currently occurring NX-2 chemotype [3,9,10,[18][19][20] and NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene. The Tri13 genes are widely distributed among trichothecene-producing FIESC (F. incarnatum-equiseti species complex) and Sambucinum species complex [21]. A previous study reported that the amino acid sequence similarity of Tri13p ranges from 76% to 100% [22]. The sequence diversity indicates that there is a possibility for Tri13p to hydroxylate the C-4 unoxygenated NX-type trichothecene in other Fusarium species. In fact, an isolate of Fusarium equiseti, a member of FIESC, was reported to accumulate a small amount of 7-H-4,15-DAS in axenic culture [16]. The mechanisms underlying the diversity of trichothecene side-chain variations need to be elucidated by analyzing the functions of Tri1 and Tri13 of various trichothecene-producing Fusarium species in terms of the function and substrate specificity of the biosynthetic monooxygenases they encode.

Construction of Transformation Vectors
FgTri1 gene disruption vectors were constructed by replacing the coding region of FgTri1 with a hygromycin B phosphotransferase gene (hph) cassette by using different methods. For the construction of pH3∆FgTri1-hph ( Figure S1a), an hph cassette without trpC terminator from pCSN43 [25] and the flanking regions of FgTri1 concatenated with 16-bp overlapping sequences were PCR-amplified using the primers listed in Table S1 and assembled directionally into a SmaI-linearized pUC19 vector using a NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) according to the instruction manual. pJCM∆FgTri1-hph was constructed by inverse PCR as described previously ( Figure S1b) [12,26]. A trichothecene C-7 hydroxylase-encoding FgTri1_ NX chemotype gene was synthetized by Eurofins Genomics (K.K., Tokyo, Japan) and supplied in a cloning plasmid, pEX-A2J1. The FgTri1_ NX chemotype expression vector was constructed as follows: the plasmid was digested with PacI and AscI restriction endonucleases, and the synthetic gene, cloned between the PacI and AscI recognition sites, was excised from the vector backbone. An overexpression vector, pAnTef-neo [17], which contains the neomycin phosphotransferase gene (neo) cassette, was digested with the same enzymes and ligated with the synthetic FgTri1_ NX chemotype gene using a Ligation-Convenience Kit (Nippon Gene, CO., LTD., Tokyo, Japan) to construct the destination vector, pAnTef-FgTri1_ NX chemotype -neo ( Figure S2).

Nucleic Acid Manipulations
Genomic DNA was extracted from fungal tissues cultured in YG medium with or without selection markers for 3 days, using a Nucleon PhytoPure kit (GE Healthcare Bioscience, Tokyo, Japan) according to the instruction manual. RNA extraction and cDNA synthesis (from 1 µg of total RNA) were performed as previously reported [28]. The cDNA was used as a template for RT-PCR. In the RT-PCR analysis, FgTri1 and the ubiquitinconjugating enzyme gene (Ubc; as a positive control) were amplified using the primer sets listed in Table S1 and detected under UV ( Figure S3).

Trichothecene Analysis
Trichothecene cultures were collected at appropriate time points during cultivation for up to 7 days. Trichothecene was extracted from the culture supernatant by adding an equal volume of ethyl acetate. Dried extract was applied onto a TLC plate, developed (ethyl acetate: toluene = 3:1), and detected as blue-purple spots based on the NBP/TEPA color reaction [12]. LC-MS/MS analysis for trichothecene confirmation was carried out using an Eksigent ekspert ultraLC 100-XL system (Dublin, CA, USA) equipped with a C18 reverse-phase column (PEGASIL ODS SP100-3; 2ϕ × 100 mm, Senshu Scientific Co., Ltd., Tokyo, Japan), as previously described [12,17].