Epistatic Relationship between MGV1 and TRI6 in the Regulation of Biosynthetic Gene Clusters in Fusarium graminearum.

Genetic studies have shown that the MAP kinase MGV1 and the transcriptional regulator TRI6 regulate many of the same biosynthetic gene clusters (BGCs) in Fusarium graminearum. This study sought to investigate the relationship between MGV1 and TRI6 in the regulatory hierarchy. Transgenic F. graminearum strains constitutively expressing MGV1 and TRI6 were generated to address both independent and epistatic regulation of BGCs by MGV1 and TRI6. We performed a comparative transcriptome analysis between axenic cultures grown in nutrient-rich and secondary metabolite-inducing conditions. The results indicated that BGCs regulated independently by Mgv1 included genes of BGC52, whereas genes uniquely regulated by TRI6 included the gene cluster (BGC49) that produces gramillin. To understand the epistatic relationship between MGV1 and TRI6, CRISPR/Cas9 was used to insert a constitutive promoter to drive TRI6 expression in the Δmgv1 strain. The results indicate that BGCs that produce deoxynivalenol and fusaoctaxin are co-regulated, with TRI6 being partially regulated by MGV1. Overall, the findings from this study indicate that MGV1 provides an articulation point to differentially regulate various BGCs. Moreover, TRI6, embedded in one of the BGCs provides specificity to regulate the expression of the genes in the BGC.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

A Bioinformatic Guide to Identify Protein Effectors from Phytopathogens.

Phytopathogenic fungi are a diverse and widespread group that has a significant detrimental impact on crops with an estimated annual average loss of 15% worldwide. Understanding the interaction between host plants and pathogenic fungi is critical to delineate underlying mechanisms of plant defense to mitigate agricultural losses. Fungal pathogens utilize suites of secreted molecules, called effectors, to modulate plant metabolism and immune response to overcome host defenses and promote colonization. Effectors come in many flavors including proteinaceous products, small RNAs, and metabolites such as mycotoxins. This review will focus on methods for identifying protein effectors from fungi. Excellent reviews have been published to identify secondary metabolites and small RNAs from fungi and therefore will not be part of this review.

Unraveling Plant-Pathogen Interactions in Cereals Using RNA-seq.

Over the past two decades, there have been significant advancements in the realm of transcriptomics, or the study of genes and their expression. Modern RNA sequencing technologies and high-performance computing are creating a "big data" revolution that provides new opportunities to explore the interactions between cereals and pathogens that affect grain yield and food safety. These data are being used to annotate genes and gene variants, as well as identify differentially expressed genes and create global gene co-expression networks. Moreover, these data can unravel the complex interactions between pathogen and host and identify genes and pathways involved in these interactions. This information can then be used for disease mitigation and the development of crops with superior resistance.

Fusarium graminearum Ste3 G-Protein Coupled Receptor: A Mediator of Hyphal Chemotropism and Pathogenesis.

Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Previous studies on Fusarium species have highlighted the involvement of the Ste2 G-protein-coupled receptor (GPCR) in mediating polarized hyphal growth toward host-released peroxidase. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to Fusarium graminearum chemotropism and pathogenicity. Fgste3Δ deletion strains were found to be compromised in the chemotropic response toward peroxidase, development of lesions on germinating wheat, and infection of Arabidopsis thaliana leaves. In the absence of FgSte3 or FgSte2, F. graminearum cells exposed to peroxidase showed no phosphorylation of the cell-wall integrity, mitogen-activated protein kinase pathway component Mgv1. In addition, transcriptomic gene expression profiling yielded a list of genes involved in cellular reorganization, cell wall remodeling, and infection-mediated responses that were differentially modulated by peroxidase when FgSte3 was present. Deletion of FgSte3 yielded the downregulation of genes associated with mycotoxin biosynthesis and appressorium development, compared to the wild-type strain, both in the presence of peroxidase. Together, these findings contribute to our understanding of the mechanism underlying fungal chemotropism and pathogenesis while raising the novel hypothesis that FgSte2 and FgSte3 are interdependent on each other for the mediation of the redirection of hyphal growth in response to host-derived peroxidase. IMPORTANCE Fusarium head blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to F. graminearum chemotropism and pathogenicity. These findings contribute to our understanding of the mechanisms underlying fungal chemotropism and pathogenesis.

Proximity-dependent biotinylation identifies a suite of candidate effector proteins from Fusarium graminearum.

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight in cereal crops. The identification of proteins secreted from pathogens to overcome plant defenses and cause disease, collectively known as effectors, can reveal the etiology of a disease process. Proximity-dependent biotin identification (BioID) was used to identify potential effector proteins secreted in planta by F. graminearum during the infection of Arabidopsis. Mass spectrometry analysis of streptavidin affinity-purified proteins revealed over 300 proteins from F. graminearum, of which 62 were candidate effector proteins (CEPs). An independent analysis of secreted proteins from axenic cultures of F. graminearum showed a 42% overlap with CEPs, thereby assuring confidence in the BioID methodology. The analysis also revealed that 19 out of 62 CEPs (approx. 30%) had been previously characterized with virulence function in fungi. The functional characterization of additional CEPs was undertaken through deletion analysis by the CRISPR/Cas9 method, and by overexpression into Triticum aestivum (wheat) leaves by the Ustilago hordei delivery system. Deletion studies of 12 CEPs confirmed the effector function of three previously characterized CEPs and validated the function of another four CEPs on wheat inflorescence or vegetative tissues. Lastly, overexpression in wheat showed that all seven CEPs enhanced resistance against the bacterial pathogen Pseudomonas syringae DC3000.

The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis.

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

MAMP and DAMP signaling contributes resistance to Fusarium graminearum in Arabidopsis.

Plants perceive externally produced microbe-associated molecular patterns (MAMPs) and endogenously produced danger-associated molecular patterns (DAMPs) to activate inducible immunity. While several inducible immune responses have been observed during Fusarium graminearum infection, the identity of the signaling pathways involved is only partly known. We screened 227 receptor kinase and innate immune response genes in Arabidopsis to identify nine genes with a role in F. graminearum resistance. Resistance-promoting genes included the chitin receptors LYK5 and CERK1, and the reactive oxygen species (ROS)-producing NADPH oxidase RbohF, which were required for full inducible immune responses during infection. Two of the genes identified in our screen, APEX and the PAMP-induced peptide 1 (PIP1) DAMP receptor RLK7, repressed F. graminearum resistance. Both RbohF and RLK7 were required for full chitin-induced immune responses and PIP1 precursor expression was induced by chitin and F. graminearum infection. Together, this indicates that F. graminearum resistance is mediated by MAMP and DAMP signaling pathways and that chitin-induced signaling is enhanced by PIP1 perception and ROS production.

The Canadian Fungal Research Network: current challenges and future opportunities.

Fungi critically impact the health and function of global ecosystems and economies. In Canada, fungal researchers often work within silos defined by subdiscipline and institutional type, complicating the collaborations necessary to understand the impacts fungi have on the environment, economy, and plant and animal health. Here, we announce the establishment of the Canadian Fungal Research Network (CanFunNet, https://fungalresearch.ca), whose mission is to strengthen and promote fungal research in Canada by facilitating dialogue among scientists. We summarize the challenges and opportunities for Canadian fungal research that were discussed at CanFunNet's inaugural meeting in 2019, and identify 4 priorities for our community: (i) increasing collaboration among scientists, (ii) studying diversity in the context of ecological disturbance, (iii) preserving culture collections in the absence of sustained funding, and (iv) leveraging diverse expertise to attract trainees. We have gathered additional information to support our recommendations, including a survey identifying underrepresentation of fungal-related courses at Canadian universities, a list of Canadian fungaria and culture collections, and a case study of a human fungal pathogen outbreak. We anticipate that these discussions will help prioritize fungal research in Canada, and we welcome all researchers to join this nationwide effort to enhance knowledge dissemination and funding advocacy.

Biotin-Based Proximity Labeling of Protein Complexes in Planta.

Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to the environment. However, a main limitation of traditional approaches to study protein interactions, such as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of interactions include many of the protein complexes that mediate the perception and response to cellular stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells, including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins (within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the study of proteomes in numerous organisms, its application to plant systems has only recently been realized. In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.

Ste2 receptor-mediated chemotropism of Fusarium graminearum contributes to its pathogenicity against wheat.

Fusarium Head Blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. While many studies have addressed the responses of both wheat and F. graminearum during their interaction, the possibility of fungal chemotropic sensing enabling pathogenicity remains unexplored. Based on recent findings linking the pheromone-sensing G-protein-coupled receptor Ste2 to host-directed chemotropism in Fusarium oxysporum, we investigated the role of the Ste2 receptor and its downstream signaling pathways in mediating chemotropism of F. graminearum. Interestingly, a chemotropic response of growing hyphae towards catalytically active Triticum aestivum 'Roblin' cultivar secreted peroxidases was detected, with deletion of STE2 in F. graminearum leading to loss of the observed response. At the same time, deletion of STE2 significantly decreased infection on germinating wheat coleoptiles, highlighting an association between Ste2, chemotropism and infection by F. graminearum. Further characterization revealed that the peroxidase-directed chemotropism is associated with stimulation of the fungal cell wall integrity mitogen-activated protein kinase signaling cascade. Altogether, this study demonstrates conservation of Ste2-mediated chemotropism by Fusarium species, and its important role in mediating pathogenicity.

Activation of biosynthetic gene clusters by the global transcriptional regulator TRI6 in Fusarium graminearum.

In F. graminearum, the transcription factor TRI6 positively regulates the trichothecene biosynthetic gene cluster (BGC) leading to the production of the secondary metabolite 15-acetyl deoxynivalenol. Secondary metabolites are not essential for survival, instead, they enable the pathogen to successfully infect its host. F. graminearum has the potential to produce a diverse array of secondary metabolites (SMs). However, given high functional specificity and energetic cost, most of these clusters remain silent, unless the organism is subjected to an environment conducive to SM production. Alternatively, secondary metabolite gene clusters (SMCs) can be activated by genetically manipulating their activators or repressors. In this study, a combination of transcriptomic and metabolomics analyses with a deletion and overexpressor mutants of TRI6 was used to establish the role of TRI6 in the regulation of several BGCs in F. graminearum. Evidence for direct and indirect regulation of BGCs by TRI6 was obtained by chromatin immunoprecipitation and yeast two-hybrid experiments. The results showed that the trichothecene genes are under direct control, while the gramillin gene cluster is indirectly controlled by TRI6 through its interaction with the pathway-specific transcription factor GRA2.

Regulation and Dynamics of Gene Expression During the Life Cycle of Fusarium graminearum.

Fungal pathogens survive harsh environments and overcome physical, temporal, and chemical barriers to colonize their hosts and reproduce. Fusarium graminearum was one of the first fungal plant pathogens for which transcriptomic tools were developed, making analysis of gene expression a cornerstone approach in studying its biology. The analysis of gene expression in diverse in vitro conditions and during infection of different cereal crops has revealed subsets of both unique and shared transcriptionally regulated genes. Together with genetic studies, these approaches have enhanced our understanding of the development and infection cycle of this economically important pathogen. Here, we will outline recent advances in transcriptional profiling during sporogenesis, spore germination, vegetative growth, and host infection. Several transcriptional regulators have been identified as essential components in these responses and the role of select transcription factors will be highlighted. Finally, we describe some of the gaps in our understanding of F. graminearum biology and how expression analysis could help to address these gaps.

DNA Methylation Is Responsive to the Environment and Regulates the Expression of Biosynthetic Gene Clusters, Metabolite Production, and Virulence in Fusarium graminearum.

Histone modifications play a significant role in the regulation of biosynthetic gene clusters (BGCs) in the phytopathogen Fusarium graminearum, by contrast, epigenetic regulation by DNA methyltransferases (DNMTs) is less documented. In this study, we characterized two DNMTs (FgDIM-2 and FgRID) in F. graminearum, with homologies to "Deficient in methylation" (DIM-2) and "Repeat-induced point (RIP) deficient" (RID) from Neurospora. The loss of DNMTs resulted in not only a decrease in average methylation density in the nutrient-poor, compared to nutrient-rich conditions, but also differences in the genes expressed between the WT and the DNMT mutant strains, implicating the external environment as an important trigger in altering DNA methylation patterns. Consequently, we observed significant changes in the regulation of multiple BGCs and alterations in the pathogenicity of the fungus.

Two 14-3-3 proteins contribute to nitrogen sensing through the TOR and glutamine synthetase-dependent pathways in Fusarium graminearum.

Fusarium graminearum responds to environmental cues to modulate its growth and metabolism during wheat pathogenesis. Nitrogen limitation activates virulence-associated behaviours in F. graminearum including mycotoxin production and penetrative growth. In other filamentous fungi, nitrogen sensing is mediated by both the Target of Rapamycin (TOR) and the glutamine synthetase (GS)-dependent signaling pathways. While TOR-dependent nitrogen responses have been demonstrated in F. graminearum, the involvement of GS remains unclear. Our study indicates that both the TOR and GS signalling pathways are involved in nitrogen sensing in F. graminearum and contribute to glutamine-induced mycelial growth. However, neither pathway is required for glutamine-induced repression of the mycotoxin deoxynivalenol (DON) indicating that an additional nitrogen sensing pathway must exist. Further, two genes FgBMH1 and FgBMH2 encoding 14-3-3 proteins regulate nitrogen responses with effects on gene expression, DON production and mycelial growth. Unlike yeast, where 14-3-3s function redundantly in regulating nitrogen sensing, the 14-3-3 proteins have differing functions in F. graminearum. While both FgBMH1 and FgBMH2 regulate early glutamine-induced DON repression, only FgBMH2 is involved in regulating reproduction, virulence and glutamine-induced AreA repression. Together, our findings help to clarify the nitrogen sensing pathways in F. graminearum and highlight the involvement of 14-3-3s in the nitrogen response of filamentous fungi.

Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to Fusarium graminearum in Wheat.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the Fusarium graminearum fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type F. graminearum as compared with a Δtri5 mutant strain that is unable to produce DON. Most of these genes were more induced by the DON-producing strain and included genes involved in secondary metabolism, signaling, transport, and stress responses. DON induction was confirmed for a subset of the genes, including TaNFXL1, by treating tissues with DON directly. Previous work indicates that the NFXL1 ortholog represses trichothecene-induced defense responses and bacterial resistance in Arabidopsis, but the role of the NFXL family has not been studied in wheat. We observed greater DON-induced TaNFXL1 gene expression in a susceptible wheat genotype relative to the F. graminearum-resistant genotype Wuhan 1. Functional testing using both virus-induced gene silencing and CRISPR-mediated genome editing indicated that TaNFXL1 represses F. graminearum resistance. Together, this suggests that targeting the TaNFXL1 gene may help to develop disease resistance in cultivated wheat.

An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat.

BACKGROUND: Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications. In addition, identification and characterization of edited events can be challenging in plants with complex genomes, such as wheat. RESULTS: In this study, we used the CRISPR/Cas9 system and developed a protocol that yielded high number of large deletions employing a pair of co-expressed sgRNA to target the same gene. The protocol was validated by targeting three genes, TaABCC6, TaNFXL1 and TansLTP9.4 in a wheat protoplast assay. Deletions of sequences located between the two sgRNA in each gene were the most frequent editing events observed for two of the three genes. A comparative assessment of editing frequencies between a codon-optimized Cas9 for expression in algae, crCas9, and a plant codon-optimized Cas9, pcoCas9, showed more consistent results with the vector expressing pcoCas9. Editing of TaNFXL1 by co-expression of sgRNA pair was investigated in transgenic wheat plants. Given the ploidy of bread wheat, a rapid, robust and inexpensive genotyping protocol was also adapted for hexaploid genomes and shown to be a useful tool to identify homoeolog-specific editing events in wheat. CONCLUSIONS: Co-expressed pairs of sgRNA targeting single genes in conjunction with the CRISPR/Cas9 system produced large deletions in wheat. In addition, a genotyping protocol to identify editing events in homoeologs of TaNFXL1 was successfully adapted.

Genomic Identification of the TOR Signaling Pathway as a Target of the Plant Alkaloid Antofine in the Phytopathogen Fusarium graminearum.

Antofine, a phenanthroindolizidine alkaloid, is a bioactive natural product isolated from milkweeds that exhibits numerous biological activities, including anticancer, antimicrobial, antiviral, and anti-inflammatory properties. However, the direct targets and mode of action of antofine have not been determined. In this report, we show that antofine displays antifungal properties against the phytopathogen Fusarium graminearum, the cause of Fusarium head blight disease (FHB). FHB does devastating damage to agriculture, causing billions of dollars in economic losses annually. We therefore sought to understand the mode of action of antofine in F. graminearum using insights from yeast chemical genomic screens. We used haploinsufficiency profiling (HIP) to identify putative targets of antofine in yeast and identified three candidate targets, two of which had homologs in F. graminearum The Fusarium homologues of two targets, glutamate dehydrogenase (FgGDH) and resistance to rapamycin deletion 2 (FgRRD2), can bind antofine. Of the two genes, only the Fgrrd2 knockout displayed a loss of virulence in wheat, indicating that RRD2 is an antivirulence target of antofine in F. graminearum Mechanistically, we demonstrate that antofine disrupts the interaction between FgRRD2 and FgTap42, which is part of the Tap42-phosphatase complex in the target of rapamycin (TOR) signaling pathway, a central regulator of cell growth in eukaryotes and a pathway of extensive study for controlling numerous pathologies.IMPORTANCEFusarium head blight caused by the fungal pathogen Fusarium graminearum is a devastating disease of cereal crops worldwide, with limited effective chemical treatments available. Here we show that the natural alkaloid compound antofine can inhibit fusarium head blight in wheat. Using yeast genomic screening, we identified the TOR pathway component RRD2 as a target of antofine that is also required for F. graminearum pathogenicity.

Clade I TGACG-Motif Binding Basic Leucine Zipper Transcription Factors Mediate BLADE-ON-PETIOLE-Dependent Regulation of Development.

Lateral organs formed by the shoot apical meristem (SAM) are separated from surrounding stem cells by regions of low growth called boundaries. Arabidopsis (Arabidopsis thaliana) BLADE-ON-PETIOLE1 (BOP1) and BOP2 represent a class of genes important for boundary patterning in land plants. Members of this family lack a DNA-binding domain and interact with TGACG-motif binding (TGA) basic Leu zipper (bZIP) transcription factors for recruitment to DNA. Here, we show that clade I bZIP transcription factors TGA1 and TGA4, previously associated with plant defense, are essential cofactors in BOP-dependent regulation of development. TGA1 and TGA4 are expressed at organ boundaries and function in the same genetic pathways as BOP1 and BOP2 required for SAM maintenance, flowering, and inflorescence architecture. Further, we show that clade I TGAs interact constitutively with BOP1 and BOP2, contributing to activation of ARABIDOPSIS THALIANA HOMEOBOX GENE1, which is needed for boundary establishment. These studies expand the functional repertoire of clade I TGA factors in development and defense.

Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum.

NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys325 ) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys325 to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta.