Trichodiene is a sesquiterpene hydrocarbon and the precursor of trichothecene mycotoxins produced by Fusarium and other fungi. Interestingly, utilizing trichodiene as a volatile treatment has been shown to reduce mycotoxin production in Fusarium graminearum-infected wheat heads. This research developed a pilot-scale fungal fermentation method to produce trichodiene as a biofumigant to mitigate mycotoxin contamination. A TRI4 mutant strain of Fusarium sporotrichioides was used to mass-produce trichodiene. Xanthotoxin, a furanocoumarin produced by parsnips, was used to enhance trichodiene production in this mutant. Xanthotoxin treatments significantly increased trichodiene yield and were found to promote lipid droplet release from the fungal germlings. Benchtop scale evaluations were conducted to determine the impacts of xanthotoxin concentration, fermentation time, extraction solvent, and filtration on overall yield. After optimal conditions were identified, the fungal cultures treated with xanthotoxin were fermented for 1 week in a 30 L bioreactor. After an organic extraction of the fungal culture and concentration of the extract, trichodiene was isolated using silica gel column chromatography. Purified trichodiene reduced mycotoxin production in F. graminearum in a dose-dependent manner. This research will allow the production of trichodiene in sufficient quantities to further evaluate its efficacy as a biofumigant to suppress mycotoxin production in F. graminearum.IMPORTANCEFood contamination from microbial toxins is a threat to human and animal health. Globally, the pathogen Fusarium graminearum causes annual losses in billions of dollars for cereal farmers and producers. Previous studies have shown that the fungal terpene trichodiene can suppress the production of vomitoxin (deoxynivalenol) by F. graminearum. We developed a way to scale production of trichodiene and use it to inhibit F. graminearum toxin contamination. A mutant strain of Fusarium sporotrichioides that produces trichodiene was grown in large 30 L fermenters and treated with xanthotoxin, a natural compound made by parsnips. Xanthotoxin caused lipid droplet release from the fungus and increased trichodiene yield. The purified trichodiene effectively reduced toxin production by F. graminearum by direct contact or as a volatile. Based on these research findings, trichodiene can be produced using common large-scale fermentation methods. Field formulations can now be developed to suppress mycotoxin contamination in food and feed.

RNA interference (RNAi) technology has been widely used to protect plants from pests and diseases. Fusarium graminearum causes Fusarium head blight (FHB) in wheat and barley, which not only results in grain yield losses but also threatens food safety due to mycotoxin contamination of grains. F. graminearum trichothecene (TRI) genes are required for trichothecene biosynthesis. Our recent study showed that TRI5 is critical for NX trichothecene production during F. graminearum infection of barley heads. To reduce mycotoxin contamination and disease using RNAi, we generated a hairpin construct targeting the TRI5 gene and introduced it into the NX-producing F. graminearum strain NRRL44211. Compared with 44211, TRI5 RNAi mutants had significantly reduced TRI5 expression and NX toxin production in vitro. Barley heads inoculated with TRI5 RNAi mutants or 44211 showed similar disease levels; however, barley heads inoculated with TRI5 RNAi mutants had significantly reduced NX toxin compared with 44211-inoculated heads. Therefore, a plant RNAi vector targeting the same TRI5 RNAi region was generated and introduced into the barley cultivar Golden Promise. FHB virulence assays showed that both FHB severity and NX-3 were significantly reduced in TRI5 RNAi transgenic lines compared with controls. Small RNA (sRNA) sequencing of both F. graminearum TRI5 RNAi mutants and barley TRI5 RNAi lines revealed sRNA reads that mapped to the TRI5 sequence, and the sRNA mapping patterns were remarkably similar in Fusarium and barley. Our results indicate that TRI5 can serve as an effective RNAi target to reduce mycotoxin contamination and improve food safety. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Trichothecenes are a family of toxic metabolites produced by multiple fungal species. All trichothecene analogs include an epoxide-containing tricyclic structure known as 12,13-epoxytrichothec-9-ene (EPT) but differ by the presence, absence and types of substituents attached to EPT. Among the 21 known genes associated with trichothecene biosynthesis, tri14 is one of only three that are universally found in all trichothecene-producing fungi. Recent studies have revealed that the tri14-encoded protein, Tri14, enhances the biosynthetic reaction that forms EPT, a reaction previously thought to occur spontaneously. In our study, we assessed the impact of tri14 deletion on the biology of Trichoderma arundinaceum, a producer of the trichothecene harzianum A (HA). The results revealed that tri14 deletion reduced HA production by 69%, an outcome that was associated with diminished antifungal activity. To our knowledge, this is the first study showing that tri14 is required for wild-type production of a trichothecene analog by a fungal organism. tri14 deletion also had moderate effects on the expression of some other trichothecene biosynthetic genes, as well as in the production of metabolites beyond HA. These results suggest that Tri14 plays a crucial role in EPT formation, leading to diverse downstream effects on the biology of T. arundinaceum.

Soybean (Glycine max) is one of the most economically important crops in the world. Production of soybean can be severely impacted by many diseases, including soybean rust. Elicitor treatments have been utilized to enhance plant resistance against multiple diseases. To investigate whether elicitor treatment can induce soybean resistance, pilot experiments were conducted to test the effects of elicitors (chitin, laminarin, and co-treated with both) on the reactive oxygen species (ROS) burst in five soybean genotypes. We discovered that all elicitor treatments induced an ROS burst with different levels. The expression of several plant defense genes was upregulated in soybean Williams 82 following elicitor treatments. GmCERK1, GmRbohD, GmPR1, GmPR2, GmPAL, and GmCHS exhibited the highest expression at 3 h post-elicitor treatments. Interestingly, co-treatment with chitin and laminarian significantly enhanced the expression of GmPAL and GmCHS. Soybean rust severity was evaluated on plants with elicitor treatment prior to Phakopsora pachyrhizi inoculation. A 5-point scale, with 5 as the highest, was used. With chitin treatment, the severities were reduced to 2.0 and 1.9 in Williams 82 and PI 200526, respectively. Controls without elicitor treatments had severities of 4.2 and 3.8, which were significantly (P < 0.001) higher than the severities in the genotypes with elicitor treatments. To the best of our knowledge, this is the first demonstration of the effects of elicitors chitin and laminarin on inducing resistance in soybean against P. pachyrhizi infection. The information from this research will be useful for development of an alternative method to control soybean rust or other diseases in crops.

Fusarium graminearum is the primary causal agent of Fusarium head blight (FHB), a devastating fungal disease on wheat, barley, and other grains. During infection, F. graminearum produces trichothecene mycotoxins, predominately deoxynivalenol (DON), that contaminate grain and reduce grain yield and quality. Although DON functions as a virulence factor to promote F. graminearum spread in the wheat head, it is not essential for establishing initial infection in wheat or barley. When fungal pathogens, such as F. graminearum, infect a host plant, they secrete hundreds of protein effectors that interfere with plant immunity to promote disease. A recent study identified hundreds of putative effector-encoding genes that are conserved across six Fusarium species. In the current study, we selected a subset of 50 conserved effectors from F. graminearum PH-1 and determined their expression on wheat heads over a 7-day infection period. Gene expression analysis revealed that several genes were highly induced in wheat heads during fungal infection. One of them was a putative rhamnogalacturonan acetylesterase homolog (FgRGAE), which was also highly induced in barley heads. FHB virulence assays showed that deletion mutants of FgRGAE significantly reduced initial infection and DON accumulation in wheat and barley heads compared with wild-type controls. Replacing the FgRGAE::Hyg deletion construct with an FgRGAEORF+::Gen construct at the native locus restored FHB disease to wild-type levels in both wheat and barley heads. FgRGAE may serve as an ideal target to reduce FHB and mycotoxin contamination in wheat and barley. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The fungal pathogen Fusarium graminearum causes Fusarium head blight (FHB) on wheat and produces trichothecene mycotoxins that contaminate grains. Deoxynivalenol (DON) and its acetylated derivatives, including 3-acetyl-DON (3-ADON) and 15-acetyl-DON (15-ADON), are the most common trichothecenes found in contaminated grains, which causes food and feed safety issues. Approaches that detoxify DON can reduce FHB and mycotoxin contamination. Our previous study showed that transgenic Arabidopsis thaliana expressing a F. graminearum 3-O-acetyltransferase self-protection enzyme (FgTri101), converted DON to 3-ADON and excreted 3-ADON out of plant cells to protect plant growth and development. The goal of the current study was to identify the transporter involved in 3-ADON excretion and utilize it to reduce toxicity and FHB. To identify trichothecene transporter candidates, transcriptomic studies were conducted on FgTri101 transgenic A. thaliana seedlings treated with DON (50 mg/L, 24 h) versus untreated controls. Transcriptomic analyses revealed that three transporter genes, including two A. thaliana detoxification genes (AtDTX1 and AtDTX3) and one ABC transporter (ABCB4), were upregulated by DON treatment. Atdtx1 mutant transported 3-ADON less efficiently than Atdtx3 and Atabcb4 mutants. Therefore, the A. thaliana Col-0 mutant Atdtx1 line was transformed and expressed FgTRI101. The Atdtx1 mutant lines expressing FgTRI101 showed resistance to DON but had significantly shorter roots than the FgTRI101 Col-0 transgenic line. Furthermore, significantly less 3-ADON was detected in the media used to grow the transgenic Atdtx1 mutant seedlings expressing FgTRI101 than the Col-0 seedlings expressing FgTRI101. These data indicate that AtDTX1 is involved in efflux of 3-ADON and that at least another transporter or other mechanism is associated with 3-ADON transport.

Fusarium graminearum (Fg) is the primary causal agent of Fusarium head blight (FHB) in wheat, barley, and other small grains in North America and worldwide. FHB results in yield reduction and contaminates grain with mycotoxins that pose threats to human and livestock health. Three genetically distinct North American (NA) populations of Fg have been characterized, which are generally associated with differences in their predominant trichothecene chemotype: NA1/15-acetyl-deoxynivalenol (15-ADON), NA2/3-acetyl-deoxynivalenol (3-ADON), and NA3/3α-acetoxy, 7,15-dihydroxy-12,13-epoxytrichothec-9-ene (NX-2). Recent studies found that the NA3 population had significantly less spread on point-inoculated wheat spikes than the NA1 and NA2 populations, and NX toxins are important for Fg spread and initial infection in wheat. In this follow-up study, to compare the effect of the three populations on initial infection and mycotoxin production on different hosts, we dip-inoculated spikes of the moderately resistant wheat cultivar Alsen and the susceptible barley cultivar Voyager using five strains from each population to evaluate disease, trichothecene mycotoxin accumulation, and trichothecene production per unit of fungal biomass. In dip-inoculated wheat spikes, the NA3 population produced significantly more trichothecene per unit of fungal biomass and accumulated higher levels of trichothecene per plant biomass than the NA1 and NA2 populations, regardless of the disease levels caused by the three populations. In contrast to its critical role during wheat infection, NX toxins had no effect on barley infection. In dip-inoculated barley, the NA1 population was more infectious and caused more severe FHB symptoms than the NA2 and NA3 populations; however, the NA3 population produced significantly higher toxin per unit of fungal biomass in infected barley tissues than the NA1 population. This study provides critical information on the emerging NA3 population, which produces high levels of NX toxin and poses a potential food safety concern.

Trichothecenes are sesquiterpenoid toxins produced by diverse ascomycetes, including Fusarium. The trichothecene analog deoxynivalenol (DON) produced by the Fusarium head blight (FHB) pathogen Fusarium graminearum is a virulence factor on wheat and a major food and feed safety concern. In Fusarium, the trichothecene biosynthetic gene (TRI) cluster consists of 7–14 genes. Most TRI cluster genes are conserved and their specific roles in trichothecene biosynthesis have been determined. An exception is TRI14, which is not required for DON synthesis in vitro but is required for spread of F. graminearum in wheat heads. In the current study, gene expression analyses revealed that TRI14 was highly induced in infected wheat heads. We demonstrated that TRI14 was not only required for F. graminearum spread but also important for initial infection in wheat. Although a prior study did not detect DON in infected seeds, our analyses showed significantly less DON and fungal biomass in TRI14-mutant (designated ∆tri14)-inoculated heads than wild-type-inoculated heads. Gene expression comparison showed that the level of expression of TRI genes was similar in the wheat tissues infected with ∆tri14 or the wild type, indicating the reduced toxin levels caused by ∆tri14 may be due to less fungal growth. ∆tri14 also caused less lesion and grew less in wheat coleoptiles than the wild type. The growth of ∆tri14 in carboxymethylcellulose medium was more sensitive to hydrogen peroxide than the wild type. The data suggest that TRI14 plays a critical role in F. graminearum growth, and potentially protects the fungus from plant defense compounds.

Fusarium graminearum, the causal agent of Fusarium head blight (FHB), produces various mycotoxins that contaminate wheat grains and cause profound health problems in humans and animals. Deoxynivalenol (DON) is the most common trichothecene found in contaminated grains. Our previous study showed that Arabidopsis-expressing F. graminearum trichothecene 3-O-acetyltransferase (FgTRI101) converted DON to 3-acetyldeoxynivalenol (3-ADON) and excreted it outside of Arabidopsis cells. To determine if wheat can convert and excrete 3-ADON and reduce FHB and DON contamination, FgTRI101 was cloned and introduced into wheat cv Bobwhite. Four independent transgenic lines containing FgTRI101 were identified. Gene expression studies showed that FgTRI101 was highly expressed in wheat leaf and spike tissues in the transgenic line FgTri101-1606. The seedlings of two FgTri101 transgenic wheat lines (FgTri101-1606 and 1651) grew significantly longer roots than the controls on media containing 5 µg/mL DON; however, the 3-ADON conversion and excretion was detected inconsistently in the seedlings of FgTri101-1606. Further analyses did not detect 3-ADON or other possible DON-related products in FgTri101-1606 seedlings after adding deuterium-labeled DON into the growth media. FgTri101-transgenic wheat plants showed significantly enhanced FHB resistance and lower DON content after they were infected with F. graminearum, but 3-ADON was not detected. Our study suggests that it is promising to utilize FgTRI101, a gene that the fungus uses for self-protection, for managing FHB and mycotoxin in wheat production.

Domain of unknown function 26 (DUF26) is a non-catalytic protein domain found only in land plant proteins. These proteins are often associated with defense. Cotton (Gossypium hirsutum L) CRR1 is a secreted protein consisting of two DUF26 domains connected by a linker. In this study we report that CRR1 is cleaved by an alkaline subtilase secreted by Verticillium dahliae, a hemi-biotrophic fungal pathogen. Recombinant CRR1 was converted from a 30 kDa glycoprotein into products of similar to 15 kDa (CRR1-P) when incubated with secreted protein extracts from V. dahliae cultures. Using this activity as a guide, the protease was purified, and its tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. It was identified as alkaline subtilase G2X826_VERDV, which we named Vd-DUMP for V. dahliae DUF26 modifying protein. This identification was confirmed by producing active recombinant Vd-DUMP in the yeast Pichia pastoris. Biochemical analysis indicated that Vd-DUMP cleaves CRR1 between the DUF26 domains. Infiltration of Vd-DUMP into Arabidopsis (Arabidopsis thaliana) leaves or cotton cotyledons induced cell death, a response that was absent when Vd-DUMP was chemically inactivated prior to infiltration. This study identifies plant DUF26 proteins, associated with defense, as substrates for alkaline subtilases secreted by fungi that are known to function as elicitors and effectors.

In the United States and Canada, Fusarium graminearum (Fg) is the predominant etiological agent of Fusarium head blight (FHB), an economically devastating fungal disease of wheat and other small grains. Besides yield losses, FHB leads to grain contamination with trichothecene mycotoxins that are harmful to plant, human, and livestock health. Three genetic North American populations of Fg, differing in their predominant trichothecene chemotype (i.e., NA1/15ADON, NA2/3ADON, and NA3/NX-2), have been identified. To improve our understanding of the newly discovered population NA3 and how population-level diversity influences FHB outcomes, we inoculated heads of the moderately resistant wheat cultivar Alsen with 15 representative strains from each population and evaluated disease progression, mycotoxin accumulation, and mycotoxin production per unit Fg biomass. Additionally, we evaluated population-specific differences in induced host defense responses. The NA3 population was significantly less aggressive than the NA1 and NA2 populations but posed a similar mycotoxigenic potential. Multiomics analyses revealed patterns in mycotoxin production per unit Fg biomass, expression of Fg aggressiveness-associated genes, and host defense responses that did not always correlate with the NA3-specific severity difference. Our comparative disease assay of NA3/NX-2 and admixed NA1/NX-2 strains indicated that the reduced NA3 aggressiveness is not due solely to the NX-2 chemotype. Notably, the NA1 and NA2 populations did not show a significant advantage over NA3 in perithecia production, a fitness-related trait. Together, our data highlight that the disease outcomes were not due to mycotoxin production or host defense alone, indicating that other virulence factors and/or host defense mechanisms are likely involved.

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most devastating diseases of wheat and barley worldwide. Effectors suppress host immunity and promote disease development. The genome of F. graminearum contains hundreds of effectors with unknown function. Therefore, investigations of the functions of these effectors will facilitate developing novel strategies to enhance wheat resistance to FHB. We characterized a F. graminearum effector, FgNls1, containing a signal peptide and multiple eukaryotic nuclear localization signals. A fusion protein of green fluorescent protein and FgNls1 accumulated in plant cell nuclei when transiently expressed in Nicotiana benthamiana. FgNls1 suppressed Bax-induced cell death when co-expressed in N. benthamiana. We revealed that the expression of FgNLS1 was induced in wheat spikes infected with F. graminearum. The Fgnls1 mutants significantly reduced initial infection and FHB spread within a spike. The function of FgNLS1 was restored in the Fgnls1-complemented strains. Wheat histone 2B was identified as an interacting protein by FgNls1-affinity chromatography. Furthermore, transgenic wheat plants that silence FgNLS1 expression had significantly lower FHB severity than control plants. This study demonstrates a critical role of FgNls1 in F. graminearum pathogenesis and indicates that host-induced gene silencing targeting F. graminearum effectors is a promising approach to enhance FHB resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Plants respond to fungal infections by activating defense genes including producing reactive oxygen species (ROS). The fungus Fusarium graminearum causes Fusarium head blight (FHB), a serious disease of wheat and barley. FHB results in crop yield loss and contaminates grain with mycotoxins. In a prior study, we discovered that chitin induces tissue-specific ROS burst in wheat. However, it is unknown whether other fungal cell wall components could induce defense response in wheat. Therefore, we evaluated ROS and defense gene responses in different wheat tissues that had been treated with chitin, laminarin, or both. Different ROS patterns were induced in wheat treated with laminarin or chitin. Furthermore, we found that ROS were enhanced in wheat tissues treated with both chitin and laminarin. This study provides novel information for enhancing plat immunity to increase plant resistance.

Fusarium graminearum causes Fusarium head blight (FHB) on wheat and barley and contaminates grains with various mycotoxins that are toxic to humans and animals. Deoxynivalenol (DON), a type B trichothecene, is an essential virulence factor that is required for F. graminearum to spread within a wheat head. Recently, novel type A trichothecenes NX-2 and NX-3 (NX) have been found in F. graminearum. NX trichothecenes lack a keto group at the C8 position. To determine if NX trichothecenes play a role similar to that of DON during F. graminearum infection, deletion mutants of TRI5, the first gene for trichothecene biosynthesis, were generated from strains PH-1, NRRL46422, and NRRL44211 (hereafter 44211) representing the 15-acetyl-DON, 3-acetyl-DON, and NX chemotypes. No trichothecene production was detected in any of the Δ tri5 mutants in cultures or inoculated wheat heads. FHB symptoms were restricted to the inoculated wheat spikelets when point-inoculated with the Δ tri5 mutants, confirming the necessity of NX and DON for FHB spread. Furthermore, whole-head dip inoculations revealed significant reductions in disease and fungal biomass in wheat heads inoculated with 44211Δ tri5 compared with 44211. Introduction of the native 44211 TRI5 and a Trichoderma arundinaceum TRI5 ortholog in the 44211Δ tri5 mutant complemented trichothecene production in vitro; however, introducing both TRI5 partially restored wild-type levels of NX in infected heads. Our results demonstrate that NX trichothecenes play an important role in Fusarium graminearum initial infection as well as FHB spread. Thus, TRI5 may serve as an ideal target to control plant infection, FHB spread, and mycotoxin production simultaneously. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Fusarium trichothecenes are among the mycotoxins of most concern to food and feed safety. Production of these mycotoxins and presence of the trichothecene biosynthetic gene (TRI) cluster have been confirmed in only two multispecies lineages of Fusarium: the Fusarium incarnatum-equiseti (Incarnatum) and F. sambucinum (Sambucinum) species complexes. Here, we identified and characterized a TRI cluster in a species that has not been formally described and is represented by Fusarium sp. NRRL 66739. This fungus is reported to be a member of a third Fusarium lineage: the F. buharicum species complex. Cultures of NRRL 66739 accumulated only two trichothecenes, 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol. Although these are not novel trichothecenes, the production profile of NRRL 66739 is novel, because in previous reports 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol were components of mixtures of 6–8 trichothecenes produced by several Fusarium species in Sambucinum. Heterologous expression analysis indicated that the TRI13 gene in NRRL 66739 confers trichothecene 7-hydroxylation. This contrasts the trichothecene 4-hydroxylation function of TRI13 in other Fusarium species. Phylogenetic analyses suggest that NRRL 66739 acquired the TRI cluster via horizontal gene transfer from a close relative of Incarnatum and Sambucinum. These findings provide insights into evolutionary processes that have shaped the distribution of trichothecene production among Fusarium species and the structural diversity of the toxins.

A wild-type allele of TaHRC suppresses the calcium-mediated immune response to Fusarium graminearum infection and facilitates the spread of Fusarium head blight disease symptoms within a wheat spike.

SummaryFusarium head blight (FHB) is one of the most destructive diseases of wheat worldwide. Epidemics of FHB cause a serious reduction in grain yield and quality of wheat and result in significant economic losses to wheat producers. Recently, we have cloned a histidine-rich calcium-binding protein gene (TaHRC) as the causal gene for Fhb1 and demonstrated that the wild-type allele of TaHRC conditions FHB susceptibility and a large deletion including the start codon resulted in FHB resistance. However, the molecular mechanisms on how TaHRC regulating FHB susceptibility remains unknown. In this study, we conducted yeast two-hybrid screening (Y2H) against the wheat cDNA expression libraries using TaHRC as bait and identified a cation exchanger (CAX)-interacting protein 4 (TaCAXIP4) as the candidate protein that interacts with TaHRC to affect calcium transport activity. The strong interaction was further confirmed by Bimolecular fluorescence complementation (BiFC) assays. Using gene editing, we edited three different sites (one before and one within and one after the NLS domain) of TaHRC in a susceptible wheat cultivar ‘Bobwhite’ using the CRISPR/Cas9 gene editing technology and demonstrated the N-terminus carrying NLS domain of TaHRC plays a critical role for the interaction and conditions TaHRC function on FHB susceptibility. We determined that the interaction between TaCAXIP4 and TaHRC occurs in the nuclei of cells by subcellular colocalization assay. Intriguingly, we found TaHRC can sequester TaCAXIP4 to suppress the Ca2+ transporting activity of TaCAX1 (a H+/Ca2+ antiporter) through yeast calcium suppression assay and suggested wild-type TaHRC may hijack TaCAXIP4 to suppresses calcium-mediated plant immune response during Fusarium infection in wheat. Furthermore, we performed the reactive oxygen species (ROS) assays and further showed that TaHRC might suppress the chitin-triggered plant immune responses during Fusarium infection by sequestering TaCAXIP4 to trigger FHB susceptibility, which facilitates the pathogen spread within a wheat spike. This work provides first line of evidence to support wild type Fhb1 is a susceptible gene and how Fhb1 wild type allele regulate FHB susceptibility.

14 15 The genus Fusarium is one of the most economically important groups of fungal plant pathogens 16 that cause serious diseases of many crops worldwide (Leslie and Summerell, 2013). The diseases 17 reduce yield, thereby resulting in economic losses (Nganje et al., 2004;Viljoen et al., 2020). In 18 addition, some Fusarium spp. can produce mycotoxins that contaminate infected grains and pose a 19 threat to human and animal health (Escrivá et al., 2015). Fusarium spp. employ intricate mechanisms 20 to overcome plant defenses. The fungus invades the host and colonizes it utilizing various infection 21 strategies. In the Fusarium genome, in addition to a region responsible for primary metabolism (core 22 genome), there are regions responsible for pathogen virulence (adaptive genome) (Ma et al., 2013).The understanding of the mechanisms that Fusarium use to overcome host defenses will provide 24 novel targets to control diseases. Therefore, this Research Topic aimed to highlight the recent works 25 on economically important species of Fusarium and their interactions with their hosts. This Research 26 Topic attracted 16 manuscripts, of which 8 were accepted and published. The articles cover 27 important outcomes of Fusarium pathogenesis, and some key aspects are summarized below.

Producing recombinant proteins with incorporated selenomethionine (SeMet) facilitates solving X-ray crystallographic structures of novel proteins. Production of SeMet labeled proteins in the yeast Pichia pastoris (syn. Komagataella phaffii) is difficult because SeMet is mildly toxic, reducing protein expression levels. To counteract this yield loss for a novel protease, Epicoccum sorghi chitinase modifying protein (Es-cmp), a novel disease promoting protease secreted by these plant pathogenic fungi, we isolated a yeast strain that secreted more protein. By comparing the expression level of 48 strains we isolated one that produced significantly more protein. This strain was found to be gene dosed, having four copies of the expression cassette. After optimization the strain produced Es-cmp in defined media with SeMet at levels nearly equal to that of the original strain in complex media. Also, we produced SeMet labeled protein for a homologous protease from the fungus Fusarium vanettenii, Fvan-cmp, by directly selecting a gene dosed strain on agar plates with increased zeocin. Linearization of plasmid with PmeI before electroporation led to high numbers of 1 mg/mL zeocin resistant clones with significantly increased expression compared to those selected on 0.1 mg/mL. The gene dosed strains expressing Es-cmp and Fvan-cmp allowed production of 8.5 and 16.8 mg of SeMet labeled protein from 500 mL shake flask cultures. The results demonstrate that selection of P. pastoris expression strains by plating after transformation on agar with 1 mg/mL zeocin rather than the standard 0.1 mg/mL directly selects gene dosed strains that can facilitate production of selenomethionine labeled proteins.