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.

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.

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.

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.

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.

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.

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.

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.

Oh, the places they'll go! A survey of phytopathogen effectors and their host targets.

Phytopathogens translocate effector proteins into plant cells where they sabotage the host cellular machinery to promote infection. An individual pathogen can translocate numerous distinct effectors during the infection process to target an array of host macromolecules (proteins, metabolites, DNA, etc.) and manipulate them using a variety of enzymatic activities. In this review, we have surveyed the literature for effector targets and curated them to convey the range of functions carried out by phytopathogenic proteins inside host cells. In particular, we have curated the locations of effector targets, as well as their biological and molecular functions and compared these properties across diverse phytopathogens. This analysis validates previous observations about effector functions (e.g. immunosuppression), and also highlights some interesting features regarding effector specificity as well as functional diversification of phytopathogen virulence strategies.

Whole genome sequencing and comparative genomics of closely related Fusarium Head Blight fungi: Fusarium graminearum, F. meridionale and F. asiaticum.

BACKGROUND: The Fusarium graminearum species complex is composed of many distinct fungal species that cause several diseases in economically important crops, including Fusarium Head Blight of wheat. Despite being closely related, these species and individuals within species have distinct phenotypic differences in toxin production and pathogenicity, with some isolates reported as non-pathogenic on certain hosts. In this report, we compare genomes and gene content of six new isolates from the species complex, including the first available genomes of F. asiaticum and F. meridionale, with four other genomes reported in previous studies. RESULTS: A comparison of genome structure and gene content revealed a 93-99% overlap across all ten genomes. We identified more than 700 k base pairs (kb) of single nucleotide polymorphisms (SNPs), insertions, and deletions (indels) within common regions of the genome, which validated the species and genetic populations reported within species. We constructed a non-redundant pan gene list containing 15,297 genes from the ten genomes and among them 1827 genes or 12% were absent in at least one genome. These genes were co-localized in telomeric regions and select regions within chromosomes with a corresponding increase in SNPs and indels. Many are also predicted to encode for proteins involved in secondary metabolism and other functions associated with disease. Genes that were common between isolates contained high levels of nucleotide variation and may be pseudogenes, allelic, or under diversifying selection. CONCLUSIONS: The genomic resources we have contributed will be useful for the identification of genes that contribute to the phenotypic variation and niche specialization that have been reported among members of the F. graminearum species complex.

Leucine metabolism regulates TRI6 expression and affects deoxynivalenol production and virulence in Fusarium graminearum.

TRI6 is a positive regulator of the trichothecene gene cluster and the production of trichothecene mycotoxins [deoxynivalenol (DON)] and acetylated forms such as 15-Acetyl-DON) in the cereal pathogen Fusarium graminearum. As a global transcriptional regulator, TRI6 expression is modulated by nitrogen-limiting conditions, sources of nitrogen and carbon, pH and light. However, the mechanism by which these diverse environmental factors affect TRI6 expression remains underexplored. In our effort to understand how nutrients affect TRI6 regulation, comparative digital expression profiling was performed with a wild-type F. graminearum and a Δtri6 mutant strain, grown in nutrient-rich conditions. Analysis showed that TRI6 negatively regulates genes of the branched-chain amino acid (BCAA) metabolic pathway. Feeding studies with deletion mutants of MCC, encoding methylcrotonyl-CoA-carboxylase, one of the key enzymes of leucine metabolism, showed that addition of leucine specifically down-regulated TRI6 expression and reduced 15-ADON accumulation. Constitutive expression of TRI6 in the Δmcc mutant strain restored 15-ADON production. A combination of cellophane breach assays and pathogenicity experiments on wheat demonstrated that disrupting the leucine metabolic pathway significantly reduced disease. These findings suggest a complex interaction between one of the primary metabolic pathways with a global regulator of mycotoxin biosynthesis and virulence in F. graminearum.

Antifungal Saponins from the Maya Medicinal Plant Cestrum schlechtendahlii G. Don (Solanaceae).

Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranoside (1) and new saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-ß-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 µM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 µM).

Intraspecies Interaction of Fusarium graminearum Contributes to Reduced Toxin Production and Virulence.

Fusarium graminearum is a pathogenic fungus that causes Fusarium head blight in wheat and lowers the yield and quality of grains by contamination with the trichothecene mycotoxin deoxynivalenol. The fungi coexist and interact with several different fusaria as well as other plant pathogenic fungi and bacteria in the field. In Canada, F. graminearum exists as two main trichothecene chemotypes: 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol. To understand the potential interactions between two isolates of these chemotypes, we conducted coinoculation studies both in culture and in planta. The studies showed that intraspecies interaction reduces trichothecene yield in culture and disease symptoms in wheat. To elucidate the genes involved in the intraspecies interaction, expression profiling was performed on RNA samples isolated from coinoculated cultures, and potential genes were identified by using the genome sequences of the respective isolates.

A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence.

Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.

Comparative secretome analysis of Fusarium graminearum and two of its non-pathogenic mutants upon deoxynivalenol induction in vitro.

To understand early events in plant-pathogen interactions, it is necessary to explore the pathogen secretome to identify secreted proteins that help orchestrate pathology. The secretome can be obtained from pathogens grown in vitro, and then characterized using standard proteomic approaches based on protein extraction and subsequent identification of tryptic peptides by LC-MS. A subset of the secretome is composed of proteins whose presence is required to initiate infection and their removal from the secretome would result in pathogens with reduced or no virulence. We present here comparative secretome from Fusarium graminearum. This filamentous fungus causes Fusarium head blight on wheat, a serious cereal disease found in many cereal-growing regions. Affected grain is contaminated with mycotoxins and cannot be used for food or feed. We used label-free quantitative MS to compare the secretomes of wild-type with two nonpathogenic deletion mutants of F. graminearum, Δtri6, and Δtri10. These mutations in mycotoxin-regulating transcription factors revealed a subset of 29 proteins whose relative abundance was affected in their secretomes, as measured by spectral counting. Proteins that decreased in abundance are potential candidate virulence factors and these included cell wall-degrading enzymes, metabolic enzymes, pathogenesis-related proteins, and proteins of unknown function.

Tri6 is a global transcription regulator in the phytopathogen Fusarium graminearum.

In F. graminearum, the transcriptional regulator Tri6 is encoded within the trichothecene gene cluster and regulates genes involved in the biosynthesis of the secondary metabolite deoxynivalenol (DON). The Tri6 protein with its Cys2His2 zinc-finger may also conform to the class of global transcription regulators. This class of global transcriptional regulators mediate various environmental cues and generally responds to the demands of cellular metabolism. To address this issue directly, we sought to find gene targets of Tri6 in F. graminearum grown in optimal nutrient conditions. Chromatin immunoprecipitation followed by Illumina sequencing (ChIP-Seq) revealed that in addition to identifying six genes within the trichothecene gene cluster, Tri1, Tri3, Tri6, Tri7, Tri12 and Tri14, the ChIP-Seq also identified 192 additional targets potentially regulated by Tri6. Functional classification revealed that, among the annotated genes, ∼40% are associated with cellular metabolism and transport and the rest of the target genes fall into the category of signal transduction and gene expression regulation. ChIP-Seq data also revealed Tri6 has the highest affinity toward its own promoter, suggesting that this gene could be subject to self-regulation. Electro mobility shift assays (EMSA) performed on the promoter of Tri6 with purified Tri6 protein identified a minimum binding motif of GTGA repeats as a consensus sequence. Finally, expression profiling of F. graminearum grown under nitrogen-limiting conditions revealed that 49 out of 198 target genes are differentially regulated by Tri6. The identification of potential new targets together with deciphering novel binding sites for Tri6, casts new light into the role of this transcriptional regulator in the overall growth and development of F. graminearum.

The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence.

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as "non-self" features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as "non-self" features or induce a "modified-self" state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2(Pto) also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2(Pto) were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2(Pto) interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2(Pto). In support of this hypothesis, HopF2 (Pto) interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2(Pto) did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2(Pto) and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.

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.