Trichothecenes and Fumonisins: Key Players in Fusarium-Cereal Ecosystem Interactions.

Fusarium fungi produce a diverse array of mycotoxic metabolites during the pathogenesis of cereals. Some, such as the trichothecenes and fumonisins, are phytotoxic, acting as non-proteinaceous effectors that facilitate disease development in cereals. Over the last few decades, we have gained some depth of understanding as to how trichothecenes and fumonisins interact with plant cells and how plants deploy mycotoxin detoxification and resistance strategies to defend themselves against the producer fungi. The cereal-mycotoxin interaction is part of a co-evolutionary dance between Fusarium and cereals, as evidenced by a trichothecene-responsive, taxonomically restricted, cereal gene competing with a fungal effector protein and enhancing tolerance to the trichothecene and resistance to DON-producing F. graminearum. But the binary fungal-plant interaction is part of a bigger ecosystem wherein other microbes and insects have been shown to interact with fungal mycotoxins, directly or indirectly through host plants. We are only beginning to unravel the extent to which trichothecenes, fumonisins and other mycotoxins play a role in fungal-ecosystem interactions. We now have tools to determine how, when and where mycotoxins impact and are impacted by the microbiome and microfauna. As more mycotoxins are described, research into their individual and synergistic toxicity and their interactions with the crop ecosystem will give insights into how we can holistically breed for and cultivate healthy crops.

Transcriptional Profiling Reveals the Wheat Defences against Fusarium Head Blight Disease Regulated by a NAC Transcription Factor.

The wheat NAC transcription factor TaNACL-D1 enhances resistance to the economically devastating Fusarium head blight (FHB) disease. The objective of this study was to decipher the alterations in gene expression, pathways and biological processes that led to enhanced resistance as a result of the constitutive expression of TaNACL-D1 in wheat. Transcriptomic analysis was used to determine the genes and processes enhanced in wheat due to TaNACL-D1 overexpression, both in the presence and absence of the causal agent of FHB, Fusarium graminearum (0- and 1-day post-treatment). The overexpression of TaNACL-D1 resulted in more pronounced transcriptional reprogramming as a response to fungal infection, leading to the enhanced expression of genes involved in detoxification, immune responses, secondary metabolism, hormone biosynthesis, and signalling. The regulation and response to JA and ABA were differentially regulated between the OE and the WT. Furthermore, the results suggest that the OE may more efficiently: (i) regulate the oxidative burst; (ii) modulate cell death; and (iii) induce both the phenylpropanoid pathway and lignin synthesis. Thus, this study provides insights into the mode of action and downstream target pathways for this novel NAC transcription factor, further validating its potential as a gene to enhance FHB resistance in wheat.

Comprehensive analysis of pathogen-responsive wheat NAC transcription factors: new candidates for crop improvement.

Wheat NAC (TaNAC) transcription factors are important regulators of stress responses and developmental processes. This study proposes a new TaNAC nomenclature and identified defense-associated TaNACs based on the analysis of RNA-sequencing datasets of wheat tissue infected with major fungal pathogens. A total of 146 TaNACs were pathogen-responsive, of which 52 were orthologous with functionally characterized defense-associated NACs from barley, rice, and Arabidopsis, as deduced via phylogenetic analysis. Next, we focused on the phylogenetic relationship of the pathogen-responsive TaNACs and their expression profiles in healthy and diseased tissues. Three subfamilies ("a," "e," and "f") were significantly enriched in pathogen-responsive TaNACs, of which the majority were responsive to at least 2 pathogens (universal pathogen response). Uncharacterized TaNACs from subfamily "a" enriched with defense-associated NACs are promising candidates for functional characterization in pathogen defense. In general, pathogen-responsive TaNACs were expressed in at least 2 healthy organs. Lastly, we showed that the wheat NAM domain is significantly divergent in sequence in subfamilies "f," "g," and "h" based on HMMER and motif analysis. New protein motifs were identified in both the N- and C-terminal parts of TaNACs. Three of those identified in the C-terminal part were linked to pathogen responsiveness of the TaNACs and 2 were linked to expression in grain tissue. Future studies should benefit from this comprehensive in silico analysis of pathogen-responsive TaNACs as a basis for selecting the most promising candidates for functional validation and crop improvement.

Analysis of the chromosomal clustering of Fusarium-responsive wheat genes uncovers new players in the defence against head blight disease.

There is increasing evidence that some functionally related, co-expressed genes cluster within eukaryotic genomes. We present a novel pipeline that delineates such eukaryotic gene clusters. Using this tool for bread wheat, we uncovered 44 clusters of genes that are responsive to the fungal pathogen Fusarium graminearum. As expected, these Fusarium-responsive gene clusters (FRGCs) included metabolic gene clusters, many of which are associated with disease resistance, but hitherto not described for wheat. However, the majority of the FRGCs are non-metabolic, many of which contain clusters of paralogues, including those implicated in plant disease responses, such as glutathione transferases, MAP kinases, and germin-like proteins. 20 of the FRGCs encode nonhomologous, non-metabolic genes (including defence-related genes). One of these clusters includes the characterised Fusarium resistance orphan gene, TaFROG. Eight of the FRGCs map within 6 FHB resistance loci. One small QTL on chromosome 7D (4.7 Mb) encodes eight Fusarium-responsive genes, five of which are within a FRGC. This study provides a new tool to identify genomic regions enriched in genes responsive to specific traits of interest and applied herein it highlighted gene families, genetic loci and biological pathways of importance in the response of wheat to disease.

The wheat SnRK1α family and its contribution to Fusarium toxin tolerance.

Deoxynivalenol (DON) is a mycotoxin produced by phytopathogenic Fusarium fungi in cereal grain and plays a role as a disease virulence factor. TaFROG (Triticum aestivum Fusarium Resistance Orphan Gene) enhances wheat resistance to DON and it interacts with a sucrose non-fermenting-1 (SNF1)-related protein kinase 1 catalytic subunit α (SnRK1α). This protein kinase family is central integrator of stress and energy signalling, regulating plant metabolism and growth. Little is known regarding the role of SnRK1α in the biotic stress response, especially in wheat. In this study, 15 wheat (Triticum aestivum) SnRK1α genes (TaSnRK1αs) belonging to four homoeologous groups were identified in the wheat genome. TaSnRK1αs are expressed ubiquitously in all organs and developmental stages apart from two members predominantly detected in grain. While DON treatment had either no effect or downregulated the transcription of TaSnRK1αs, it increased both the kinase activity associated with SnRK1α and the level of active (phosphorylated) SnRK1α. Down-regulation of two TaSnRK1αs homoeolog groups using virus induced gene silencing (VIGS) increased the DON-induced damage of wheat spikelets. Thus, we demonstrate that TaSnRK1αs contribute positively to wheat tolerance of DON and conclude that this gene family may provide useful tools for the improvement of crop biotic stress resistance.

A wheat NAC interacts with an orphan protein and enhances resistance to Fusarium head blight disease.

Taxonomically-restricted orphan genes play an important role in environmental adaptation, as recently demonstrated by the fact that the Pooideae-specific orphan TaFROG (Triticum aestivum Fusarium Resistance Orphan Gene) enhanced wheat resistance to the economically devastating Fusarium head blight (FHB) disease. Like most orphan genes, little is known about the cellular function of the encoded protein TaFROG, other than it interacts with the central stress regulator TaSnRK1α. Here, we functionally characterized a wheat (T. aestivum) NAC-like transcription factor TaNACL-D1 that interacts with TaFROG and investigated its' role in FHB using studies to assess motif analyses, yeast transactivation, protein-protein interaction, gene expression and the disease response of wheat lines overexpressing TaNACL-D1. TaNACL-D1 is a Poaceae-divergent NAC transcription factor that encodes a Triticeae-specific protein C-terminal region with transcriptional activity and a nuclear localisation signal. The TaNACL-D1/TaFROG interaction was detected in yeast and confirmed in planta, within the nucleus. Analysis of multi-protein interactions indicated that TaFROG could form simultaneously distinct protein complexes with TaNACL-D1 and TaSnRK1α in planta. TaNACL-D1 and TaFROG are co-expressed as an early response to both the causal fungal agent of FHB, Fusarium graminearum and its virulence factor deoxynivalenol (DON). Wheat lines overexpressing TaNACL-D1 were more resistant to FHB disease than wild type plants. Thus, we conclude that the orphan protein TaFROG interacts with TaNACL-D1, a NAC transcription factor that forms part of the disease response evolved within the Triticeae.

Virus-Induced Gene Silencing (VIGS) for Functional Characterization of Disease Resistance Genes in Barley Seedlings.

With the recent advances in sequencing technologies, many studies are generating lists of candidate genes associated with specific traits. The major bottleneck in functional genomics is the validation of gene function. This is achieved by analyzing the effect of either gene silencing or overexpression on a specific phenotypic or biochemical trait. This usually requires the generation of stable transgenic plants and this can take considerable time. Therefore any technique that expedites the validation of gene function is of particular benefit in cereals, including barley. One such technique is Virus-Induced Gene Silencing (VIGS), which evokes a natural antiviral defense mechanism in plants. VIGS can be used to downregulate gene expression in a transient manner, but long enough to determine its effects on a specific phenotype. It is particularly useful for screening candidate genes and selecting those with potential for disease control. VIGS based on Barley Stripe Mosaic Virus (BSMV) is a powerful and efficient tool for the analysis of gene function in cereals. Here we present a BSMV VIGS protocol for simple and robust gene silencing in barley and describe it to evaluate the role of the hormone receptor BRI1 (Brassinosteroid Insensitive 1) in barley leaf resistance to Fusarium infection.

A wheat cytochrome P450 enhances both resistance to deoxynivalenol and grain yield.

The mycotoxin deoxynivalenol (DON) serves as a plant disease virulence factor for the fungi Fusarium graminearum and F. culmorum during the development of Fusarium head blight (FHB) disease on wheat. A wheat cytochrome P450 gene from the subfamily CYP72A, TaCYP72A, was cloned from wheat cultivar CM82036. TaCYP72A was located on chromosome 3A with homeologs present on 3B and 3D of the wheat genome. Using gene expression studies, we showed that TaCYP72A variants were activated in wheat spikelets as an early response to F. graminearum, and this activation was in response to the mycotoxic Fusarium virulence factor deoxynivalenol (DON). Virus induced gene silencing (VIGS) studies in wheat heads revealed that this gene family contributes to DON resistance. VIGS resulted in more DON-induced discoloration of spikelets, as compared to mock VIGS treatment. In addition to positively affecting DON resistance, TaCYP72A also had a positive effect on grain number. VIGS of TaCYP72A genes reduced grain number by more than 59%. Thus, we provide evidence that TaCYP72A contributes to host resistance to DON and conclude that this gene family warrants further assessment as positive contributors to both biotic stress resistance and grain development in wheat.

TaFROG Encodes a Pooideae Orphan Protein That Interacts with SnRK1 and Enhances Resistance to the Mycotoxigenic Fungus Fusarium graminearum.

All genomes encode taxonomically restricted orphan genes, and the vast majority are of unknown function. There is growing evidence that such genes play an important role in the environmental adaptation of taxa. We report the functional characterization of an orphan gene (Triticum aestivum Fusarium Resistance Orphan Gene [TaFROG]) as a component of resistance to the globally important wheat (T. aestivum) disease, Fusarium head blight. TaFROG is taxonomically restricted to the grass subfamily Pooideae. Gene expression studies showed that it is a component of the early wheat response to the mycotoxin deoxynivalenol (DON), which is a virulence factor produced by the causal fungal agent of Fusarium head blight, Fusarium graminearum. The temporal induction of TaFROG by F. graminearum in wheat spikelets correlated with the activation of the defense Triticum aestivum Pathogenesis-Related-1 (TaPR1) gene. But unlike TaPR1, TaFROG induction by F. graminearum was toxin dependent, as determined via comparative analysis of the effects of wild-type fungus and a DON minus mutant derivative. Using virus-induced gene silencing and overexpressing transgenic wheat lines, we present evidence that TaFROG contributes to host resistance to both DON and F. graminearum. TaFROG is an intrinsically disordered protein, and it localized to the nucleus. A wheat alpha subunit of the Sucrose Non-Fermenting1-Related Kinase1 was identified as a TaFROG-interacting protein based on a yeast two-hybrid study. In planta bimolecular fluorescence complementation assays confirmed the interaction. Thus, we conclude that TaFROG encodes a new Sucrose Non-Fermenting1-Related Kinase1-interacting protein and enhances biotic stress resistance.

A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance.

The mycotoxin deoxynivalenol (DON) acts as a disease virulence factor for Fusarium fungi, and tolerance of DON enhances wheat resistance to Fusarium head blight (FHB) disease. Two variants of an ATP-binding cassette (ABC) family C transporter gene were cloned from DON-treated wheat mRNA, namely TaABCC3.1 and TaABCC3.2. These represent two of three putative genes identified on chromosomes 3A, 3B, and 3D of the wheat genome sequence. Variant TaABCC3.1 represents the DON-responsive transcript previously associated with DON resistance in wheat. PCR-based mapping and in silico sequence analyses located TaABCC3.1 to the short arm of wheat chromosome 3B (not within the FHB resistance quantitative trait locus Fhb1). In silico analyses of microarray data indicated that TaABCC3 genes are expressed in reproductive tissue and roots, and in response to the DON producer Fusarium graminearum. Gene expression studies showed that TaABCC3.1 is activated as part of the early host response to DON and in response to the FHB defence hormone jasmonic acid. Virus-induced gene silencing (VIGS) confirmed that TaABCC3 genes contributed to DON tolerance. VIGS was performed using two independent viral construct applications: one specifically targeted TaABCC3.1 for silencing, while the other targeted this gene and the chromosome 3A homeologue. In both instances, VIGS resulted in more toxin-induced discoloration of spikelets, compared with the DON effects in non-silenced spikelets at 14 d after toxin treatment (≥2.2-fold increase, P<0.05). Silencing by both VIGS constructs enhanced head ripening, and especially so in DON-treated heads. VIGS of TaABCC3 genes also reduced the grain number by more than 28% (P<0.05), both with and without DON treatment, and the effects were greater for the construct that targeted the two homeologues. Hence, DON-responsive TaABCC3 genes warrant further study to determine their potential as disease resistance breeding targets and their function in grain formation and ripening.

CML9, a multifunctional Arabidopsis thaliana calmodulin-like protein involved in stress responses and plant growth?

Plants have evolved complex signaling networks to respond to their fluctuating environment and adapt their growth and development. Calcium-dependent signaling pathways play key role in the onset of these adaptive responses. In plant cells, the intracellular calcium transients are triggered by numerous stimuli and it is supposed that the large repertory of calcium sensors present in higher plants could contribute to integrate these signals in physiological responses. Here, we present data on CML9, a calmodulin-like protein that appears to be involved in plant responses to both biotic and abiotic stress. Using a reverse genetic approach based on gain and loss of function mutants, we present here data indicating that this CML might also be involved in root growth control in response to the flagellin, a pathogen-associated molecular pattern (PAMP) also involved in plant immunity.

Calmodulin and calmodulin-like proteins in plant calcium signaling.

Calmodulin (CaM) is a primary calcium sensor in all eukaryotes. It binds calcium and regulates the activity of a wide range of effector proteins in response to calcium signals. The list of CaM targets includes plant-specific proteins whose functions are progressively being elucidated. Plants also possess numerous calmodulin-like proteins (CMLs) that appear to have evolved unique functions. Functional studies of CaM and CMLs in plants highlight the importance of this protein family in the regulation of plant development and stress responses by converting calcium signals into transcriptional responses, protein phosphorylation or metabolic changes. This review summarizes some of the significant progress made by biochemical and genetic studies in identifying the properties and physiological functions of plant CaMs and CMLs. We discuss emerging paradigms in the field and highlight the areas that need further investigation.

Interaction of a plant pseudo-response regulator with a calmodulin-like protein.

Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.