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. 2023 Apr 17;14:1148771. doi: 10.3389/fmicb.2023.1148771

Figure 2.

Figure 2

Genetic manipulations of the Tri4–Tri6–Tri5–Tri10 locus in Fusarium. Deregulation of Tri6 and Tri10 occurs in cases when the targeted gene disruptions based on homologous recombination change the length of the core region of the trichothecene gene cluster. Vector integrations via single or double crossover homologous recombination for each subfigure are described in detail in Supplementary Figure 3. (A) Genetically engineered cluster core region of 15-ADON chemotype F. graminearum GZ3639 (Chen et al., 2000). The structure of the cluster core region is illustrated. Transformants, G11-1, D6-2, and B4-1, were obtained by homologous integration of vectors pProm5 (containing PTri5 alone), pProm5GUS-11 (containing PTri5 fused to GUS; PTri5::GUS), and pGUSTRI6P5 (containing both PTri5::GUS and FsTri6), respectively. Integration of pProm5, pProm5GUS-11, and pGUSTRI6P5 through the Tri5 promoter region extends the Tri5–Tri6 intergenic region by 6.2 kb (G11-1), 8.5 kb (D6-2), and 11.8 kb (B4-1), respectively. While the wild-type GZ3639 accumulates less than 5 mg/L of trichothecenes by shake culture on liquid medium with glucose, elongation of the cluster core region resulted in increased production of 15-ADON (up to 75 mg/L by G11-1, up to 225 mg/L by D6-2, and up to 1,200 mg/L by B4-1), irrespective of the presence or absence of FsTri6 on the vector. Ectopic integration of pGUSTRI6P5 (e.g., up to 54 mg/l by strain B9-1; not shown in the subfigure) did not yield 15-ADON above the amount obtained by strain G11-1 lacking FsTri6. Notably, compared with strain B9-1, much stronger GUS activity was detected from strain B4-1 (50- to 100-fold increase), in which PTri5::GUS is located at the cluster core region. (B) Genetically engineered cluster core region of F. sporotrichioides strain NRRL 3299 (Tag et al., 2001). The structure of the cluster core region of each derived mutant strain is illustrated. The resulting ΔTri6 strain contains a 3′-truncated Tri6 gene followed by an hph cassette and then a 5′-truncated Tri6 gene, and is expected to have a 5.7 kb extension at the Tri6 region. The ↑Tri10 and ΔTri10 strains were constructed from the same plasmid pTri10-1 which could undergo single or double crossover (Continued)FIGURE 2 (Continued)homologous recombination events at the upstream and/or downstream regions, respectively. The ↑Tri10 strain contains a disrupted copy of Tri10 followed by a functional copy of Tri10 downstream of Tri5 with a 6.6 kb extension, while the ΔTri10 strain contains only a disrupted copy of Tri10 with a 1.6 kb extension in the same region (a ΔTri10 strain described in Supplementary Figure 3B only). (C) Disrupted Tri6 in F. graminearum strain NRRL 31084 (PH-1) (Seong et al., 2009). The PH1Δtri6 strain was generated by replacing the Tri6 region with an hph cassette, resulting in a slight perturbation within the cluster core region. The region is extended by 0.3 kb in PH1Δtri6. (D) Wild-type and mutated Tri6 promoters without structural perturbation of the cluster core region in F. graminearum strain JCM 9873 (Nakajima et al., 2020). PTri6/m0 (complemented) and PTri6/m15 (mutated) strains were designed by applying a two-step transformation process involving double crossover homologous recombination events. All fifteen 5′-GATA-3′ AreA-binding consensus sequences were mutated to 5′-GGTG-3′ in the wild-type Tri4–Tri6 bidirectional promoter in PTri6/m15. Both PTri6/m0 and PTri6/m15 strains are marker free and introduce no perturbation within the core cluster region. (E) Two types of Tri6 disrupted mutants of F. graminearum strain JCM 9873. Both ΔTri6 tk (Nakajima et al., 2014) and ΔTri6-nsm strains were generated to study the effect of Tri6 deletion. The ΔTri6 tk strain was generated using a one-step transformation process and contains a selection marker cassette replacing the Tri6 coding region, which results in a 7.0 kb extension in the cluster. The ΔTri6-nsm strain was generated using a two-step transformation process and contains a dysfunctional copy of the Tri6 gene containing a nonsense mutation, which does not result in perturbation within the cluster region. The relative expression level of Tri10 (represented as Tri10/GPD) was determined using the expression of GPD as an endogenous reference in the same RNA samples. The Tri10/GPD ratio only increased in ΔTri6 tk, while that in ΔTri6-nsm was similar to the expression level in the wild-type.