ABSTRACT
Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.
Subject(s)
DNA Transposable Elements , Eukaryota , DNA Transposable Elements/genetics , Eukaryota/genetics , Gene Transfer, Horizontal , Recombinases/genetics , Tyrosine/genetics , Evolution, MolecularABSTRACT
BACKGROUND: Fusarium graminearum and Fusarium avenaceum are two of the most important causal agents of Fusarium head blight (FHB) of wheat. They can produce mycotoxins that accumulate in infected wheat heads, including deoxynivalenol (DON) and enniatins (ENNs), produced by F. graminearum and F. avenaceum, respectively. While the role of DON as a virulence factor in F. graminearum toward wheat is well known, ENNs in F. avenaceum has been poorly explored. Results obtained to-date indicate that ENNs may confer an advantage to F. avenaceum only on particular hosts. RESULTS: In this study, with the use of ENN-producing and ENN non-producing F. avenaceum strains, the role of ENNs on F. avenaceum virulence was investigated on the root, stem base and head of common wheat, and compared with the role of DON, using DON-producing and DON non-producing F. graminearum strains. The DON-producing F. graminearum strain showed a significantly higher ability to cause symptoms and colonise each of the tested tissues than the non-producing strain. On the other hand, the ability to produce ENNs increased initial symptoms of the disease and fungal biomass accumulation, measured by qPCR, only in wheat heads, and not in roots or stem bases. LC-MS/MS analysis was used to confirm the presence of ENNs and DON in the different strains, and results, both in vitro and in wheat heads, were consistent with the genetics of each strain. CONCLUSION: While the key role of DON on F. graminearum virulence towards three different wheat tissues was noticeable, ENNs seemed to have a role only in influencing F. avenaceum virulence on common wheat heads probably due to an initial delay in the appearance of symptoms.
Subject(s)
Fusarium , Plant Diseases , Trichothecenes , Triticum , Triticum/microbiology , Triticum/metabolism , Fusarium/pathogenicity , Fusarium/genetics , Fusarium/metabolism , Trichothecenes/metabolism , Virulence , Plant Diseases/microbiology , Mycotoxins/metabolism , DepsipeptidesABSTRACT
The rhizosphere interaction between plant roots or pathogenic microbes is initiated by mutual exchange of signals. However, how soil pathogens sense host signals is largely unknown. Here, we studied early molecular events associated with host recognition in Fusarium graminearum, an economically important fungal pathogen that can infect both roots and heads of cereal crops. We found that host sensing prior to physical contact with plant roots radically alters the transcriptome and triggers nitric oxide (NO) production in F. graminearum We identified an ankyrin-repeat domain containing protein (FgANK1) required for host-mediated NO production and virulence in F. graminearum In the absence of host plant, FgANK1 resides in the cytoplasm. In response to host signals, FgANK1 translocates to the nucleus and interacts with a zinc finger transcription factor (FgZC1), also required for specific binding to the nitrate reductase (NR) promoter, NO production, and virulence in F. graminearum Our results reveal mechanistic insights into host-recognition strategies employed by soil pathogens.
Subject(s)
Fusarium/metabolism , Host-Pathogen Interactions/physiology , Nitric Oxide/metabolism , Plant Diseases/immunology , Signal Transduction , Ankyrins/metabolism , Crops, Agricultural/metabolism , Edible Grain/metabolism , Fungal Proteins , Fusarium/pathogenicity , Plant Diseases/microbiology , Plant Roots/metabolism , Transcription Factors/metabolism , TranscriptomeABSTRACT
Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.
Subject(s)
Biological Control Agents , Pesticides , Ecosystem , RNA Interference , RNA, Small Interfering/genetics , Crops, Agricultural/genetics , Plant Diseases/genetics , Plant Diseases/prevention & control , Plant Diseases/microbiologyABSTRACT
One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.
Subject(s)
Crop Protection , Solanum lycopersicum , RNA Interference , Botrytis , Plant Diseases/prevention & control , Plant Diseases/microbiology , RNA, Double-Stranded/genetics , RNA, Small Interfering/genetics , Solanum lycopersicum/genetics , Plants/geneticsABSTRACT
Plant root-produced constitutive and inducible defences inhibit pathogenic microorganisms within roots and in the rhizosphere. However, regulatory mechanisms underlying host responses during root-pathogen interactions are largely unexplored. Using the model species Brachypodium distachyon (Bd), we studied transcriptional and metabolic responses altered in Bd roots following challenge with Fusarium graminearum (Fg), a fungal pathogen that causes diseases in diverse organs of cereal crops. Shared gene expression patterns were found between Bd roots and spikes during Fg infection associated with the mycotoxin deoxynivalenol (DON). Overexpression of BdMYB78, an up-regulated transcription factor, significantly increased root resistance during Fg infection. We show that Bd roots recognize encroaching Fg prior to physical contact by altering transcription of genes associated with multiple cellular processes such as reactive oxygen species and cell development. These changes coincide with altered levels of secreted host metabolites detected by an untargeted metabolomic approach. The secretion of Bd metabolites was suppressed by Fg as enhanced levels of defence-associated metabolites were found in roots during pre-contact with a Fg mutant defective in host perception and the ability to cause disease. Our results help to understand root defence strategies employed by plants, with potential implications for improving the resistance of cereal crops to soil pathogens.
Subject(s)
Brachypodium/microbiology , Fusarium/physiology , Metabolome , Mycotoxins/metabolism , Transcriptome , Trichothecenes/metabolism , Adaptation, Biological , Brachypodium/genetics , Brachypodium/immunology , Brachypodium/metabolism , Host Microbial Interactions , Plant Immunity/physiology , Plant Roots/microbiology , Signal Transduction/immunologyABSTRACT
Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.
Subject(s)
Fusarium , Fusarium/genetics , Phylogeny , Plant Diseases , PlantsABSTRACT
Fusarium pseudograminearum (Fp), the causative fungal pathogen of the diseases Fusarium crown rot, is an important constraint to cereals production in many countries including Australia. Fp produces a number of secondary metabolites throughout its life cycle. One of these metabolites, the cyclic lipopeptide fusaristatin A, is encoded by a specific gene cluster containing a polyketide synthase and a three-module non-ribosomal peptide synthetase. However, a recent survey of Fp populations across Australia suggests that this cluster may only be present in a subset of isolates from Western Australia (WA). In this study, we screened 319 Fp isolates from WA and 110 Fp isolates from the Australian eastern states of New South Wales, Victoria, Queensland and South Australia to examine the distribution of this gene cluster among Australian Fp populations. The fusaristatin A gene cluster was found to be present in ~50% of Fp isolates from WA but completely absent in Fp isolates from eastern states. To determine its potential function, mutants of the fusaristatin A gene cluster were generated by disrupting the non-ribosomal peptide synthetase and polyketide synthase genes simultaneously in two different parental backgrounds. The mutants showed increased growth rates and were significantly more aggressive than their respective parental strains on wheat in crown rot pathogenicity assays. This suggested that fusaristatin A has a negative effect on fungal development and aggressiveness. The possible reasons for the geographically restricted presence of the fusaristatin A gene cluster and its role in fungal biology are discussed.
Subject(s)
Depsipeptides/biosynthesis , Fusarium/growth & development , Fusarium/genetics , Triticum/microbiology , Australia , DNA, Fungal , Edible Grain/microbiology , Fungal Proteins , Fusarium/pathogenicity , Gene Knockout Techniques , Host Microbial Interactions , Multigene Family , Peptide Synthases/genetics , Plant Diseases/microbiology , Polyketide Synthases/geneticsABSTRACT
Globally, fungal pathogens cause enormous crop losses and current control practices are not always effective, economical or environmentally sustainable. Tools enabling genetic management of wild pathogen populations could potentially solve many problems associated with plant diseases. A natural gene drive from a heterologous species can be used in the globally important cereal pathogen Fusarium graminearum to remove pathogenic traits from contained populations of the fungus. The gene drive element became fixed in a freely crossing population in only three generations. Repeat-induced point mutation (RIP), a natural genome defence mechanism in fungi that causes C to T mutations during meiosis in highly similar sequences, may be useful to recall the gene drive following release, should a failsafe mechanism be required. We propose that gene drive technology is a potential tool to control plant pathogens once its efficacy is demonstrated under natural settings.
Subject(s)
Fusarium , Gene Drive Technology , Fusarium/genetics , Plant Diseases , Triticum/geneticsABSTRACT
Translation is a highly dynamic cellular process whereby genetic information residing in an mRNA molecule is converted into a protein that in turn executes specific functions. However, pre-synthesized mRNA levels do not always correlate with corresponding protein levels, suggesting that translational control plays an essential role in gene regulation. A better understanding of how gene expression is regulated during translation will enable the discovery of new genes and mechanisms that control important traits in plants. Therefore, in recent years, several methods have been developed to analyse the translatome; that is, all mRNAs being actively translated at a given time, tissue, and/or developmental stage. Ribosome profiling or ribo-seq is one such technology revolutionizing our ability to analyse the translatome and in turn understand translational control of gene expression. Ribo-seq involves isolating mRNA-ribosome complexes, treating them with a RNase, and then identifying ribosome-protected mRNA regions by deep sequencing. Here, we briefly review recent ribosome profiling studies that revealed new insights into plant biology. Manipulation of novel genes identified using ribosome profiling could prove useful for increasing yield through improved biotic and abiotic stress tolerance.
Subject(s)
Protein Biosynthesis , Ribosomes , Gene Expression Profiling , Plants/genetics , Plants/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Stress, PhysiologicalABSTRACT
Plant species, populations and communities are under threat from climate change, invasive pathogens, weeds and habitat fragmentation. Despite considerable research effort invested in genome engineering for crop improvement, the development of genetic tools for the management of wild plant populations has rarely been given detailed consideration. Gene drive systems that allow direct genetic management of plant populations via the spread of fitness-altering genetic modifications could be of great utility. However, despite the rapid development of synthetic tools and their enormous promise, little explicit consideration has been given to their application in plants and, to date, they remain untested. This article considers the potential utility of gene drives for the management of wild plant populations, and examines the factors that might influence the design, spread and efficacy of synthetic drives. To gain insight into optimal ways to design and deploy synthetic drive systems, we investigate the diversity of mechanisms underlying natural gene drives and their dynamics within plant populations and species. We also review potential approaches for engineering gene drives and discuss their potential application to plant genomes. We highlight the importance of considering the impact of plant life-history and genetic architecture on the dynamics of drive, investigate the potential for different types of resistance evolution, and touch on the ethical, regulatory and social challenges ahead.
Subject(s)
Gene Drive Technology , Plant Weeds , Weed Control/methodsABSTRACT
Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500â¯nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms.
Subject(s)
Carbon-Carbon Lyases/metabolism , Fusarium/enzymology , Cytosol/metabolism , Endoplasmic Reticulum, Smooth/metabolism , Endoplasmic Reticulum, Smooth/ultrastructure , Fusarium/ultrastructure , Metabolic Networks and Pathways , Microscopy/methods , Mycotoxins/metabolism , NanoparticlesABSTRACT
Fusarium is a genus of filamentous fungi that contains many agronomically important plant pathogens, mycotoxin producers, and opportunistic human pathogens. Comparative analyses have revealed that the Fusarium genome is compartmentalized into regions responsible for primary metabolism and reproduction (core genome), and pathogen virulence, host specialization, and possibly other functions (adaptive genome). Genes involved in virulence and host specialization are located on pathogenicity chromosomes within strains pathogenic to tomato (Fusarium oxysporum f. sp. lycopersici) and pea (Fusarium 'solani' f. sp. pisi). The experimental transfer of pathogenicity chromosomes from F. oxysporum f. sp. lycopersici into a nonpathogen transformed the latter into a tomato pathogen. Thus, horizontal transfer may explain the polyphyletic origins of host specificity within the genus. Additional genome-scale comparative and functional studies are needed to elucidate the evolution and diversity of pathogenicity mechanisms, which may help inform novel disease management strategies against fusarial pathogens.
Subject(s)
Fungal Proteins/genetics , Fusarium/genetics , Fusarium/pathogenicity , Genome, Fungal , Plant Diseases/microbiology , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Fusarium/classification , Fusarium/metabolism , Phylogeny , VirulenceABSTRACT
KEY MESSAGE: This study demonstrates how identification of genes underpinning disease-resistance QTL based on differential expression and SNPs can be improved by performing transcriptomic analysis on multiple near isogenic lines. Transcriptomic analysis has been widely used to understand the genetic basis of a trait of interest by comparing genotypes with contrasting phenotypes. However, these approaches identify such large sets of differentially expressed genes that it proves difficult to isolate which genes underpin the phenotype of interest. This study tests whether using multiple near isogenic lines (NILs) can improve the resolution of RNA-seq-based approaches to identify genes underpinning disease-resistance QTL. A set of NILs for a major effect Fusarium crown rot-resistance QTL in barley on the 4HL chromosome arm were analysed under Fusarium crown rot using RNA-seq. Differential gene expression and single nucleotide polymorphism detection analyses reduced the number of putative candidates from thousands within individual NIL pairs to only one hundred and two genes, which were differentially expressed or contained SNPs in common across NIL pairs and occurred on 4HL. Our findings support the value of performing RNA-seq analysis using multiple NILs to remove genetic background effects. The enrichment analyses indicated conserved differences in the response to infection between resistant and sensitive isolines suggesting that sensitive isolines are impaired in systemic defence response to Fusarium pseudograminearum.
Subject(s)
Disease Resistance/genetics , Hordeum/genetics , Plant Diseases/genetics , Quantitative Trait Loci , Sequence Analysis, RNA , Fusarium , Gene Expression Profiling , Gene Expression Regulation, Plant , Genotype , Hordeum/microbiology , Phenotype , Plant Diseases/microbiology , Polymorphism, Single NucleotideABSTRACT
During their interactions with plants, fungal pathogens employ large numbers of pathogenesis-associated molecules including secreted effectors and enzymes that can degrade various defence compounds. However, in many cases, in planta targets of pathogen-produced enzymes remain unknown. We identified a gene in the wheat pathogen Fusarium graminearum, encoding a putative enzyme that shows 84% sequence identity to FoTom1, a tomatinase produced by the tomato pathogen Fusarium oxysporum f. sp. lycopersici. In F. oxysporum f. sp. lycopersici, FoTom1 is a virulence factor involved in the degradation of tomato defence compound tomatine, a saponin compound. Given that wheat is unknown to produce tomatine, we tested the ability of F. graminearum to degrade tomatine and found that F. graminearum was unable to degrade tomatine in culture. However, FgTom1 degraded tomatine in vitro when heterologously expressed. To determine the possible function of FgTom1 in pathogen virulence, we generated FgTom1 knockout mutants (ΔTom1). ΔTom1 mutants were not different from wild type when grown in culture but showed significant reduction in pathogen virulence in root rot and head blight assays. In an attempt to identify possible in planta targets of FgTom1, the metabolomes of wheat heads infected with wildtype pathogen and ΔTom1 were compared and several peaks differentially abundant between treatments identified. Although the exact identity of these peaks is currently unknown, this result suggested that FgTom1 may have in planta targets in wheat, possibly tomatine-like saponin compounds. Overall, our results presented here show that FgTom1 is a new virulence factor in F. graminearum.
Subject(s)
Disease Resistance/genetics , Fusarium/enzymology , Glycoside Hydrolases/metabolism , Triticum/microbiology , Fusarium/pathogenicity , Glycoside Hydrolases/genetics , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Roots/metabolism , Plant Roots/microbiology , Tomatine/chemistry , Tomatine/metabolism , Virulence Factors/chemistry , Virulence Factors/metabolismABSTRACT
Bread wheat (Triticum aestivum L.) is an allopolyploid species containing three ancestral genomes. Therefore, three homoeologous copies exist for the majority of genes in the wheat genome. Whether different homoeologs are differentially expressed (homoeolog expression bias) in response to biotic and abiotic stresses is poorly understood. In this study, we applied a RNA-seq approach to analyse homoeolog-specific global gene expression patterns in wheat during infection by the fungal pathogen Fusarium pseudograminearum, which causes crown rot disease in cereals. To ensure specific detection of homoeologs, we first optimized read alignment methods and validated the results experimentally on genes with known patterns of subgenome-specific expression. Our global analysis identified widespread patterns of differential expression among homoeologs, indicating homoeolog expression bias underpins a large proportion of the wheat transcriptome. In particular, genes differentially expressed in response to Fusarium infection were found to be disproportionately contributed from B and D subgenomes. In addition, we found differences in the degree of responsiveness to pathogen infection among homoeologous genes with B and D homoeologs exhibiting stronger responses to pathogen infection than A genome copies. We call this latter phenomenon as 'homoeolog induction bias'. Understanding how homoeolog expression and induction biases operate may assist the improvement of biotic stress tolerance in wheat and other polyploid crop species.
Subject(s)
Polyploidy , Transcriptome/genetics , Triticum/genetics , Chromosomes, Plant/genetics , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiologyABSTRACT
Background and Aims: Fusarium crown rot caused by the fungal pathogen Fusarium pseudograminearum is a disease of wheat and barley, bearing significant economic cost. Efforts to develop effective resistance to this disease have been hampered by the quantitative nature of resistance and a lack of understanding of the factors associated with resistance and susceptibility. Here, we aimed to dissect transcriptional responses triggered in wheat by F. pseudograminearum infection. Methods: We used an RNA-seq approach to analyse host responses during a compatible interaction and identified >2700 wheat genes differentially regulated after inoculation with F. pseudograminearum . The production of a few key metabolites and plant hormones in the host during the interaction was also analysed. Key Results: Analysis of gene ontology enrichment showed that a disproportionate number of genes involved in primary and secondary metabolism, signalling and transport were differentially expressed in infected seedlings. A number of genes encoding pathogen-responsive uridine-diphosphate glycosyltransferases (UGTs) potentially involved in detoxification of the Fusarium mycotoxin deoxynivalenol (DON) were differentially expressed. Using a F. pseudograminearum DON-non-producing mutant, DON was shown to play an important role in virulence during Fusarium crown rot. An over-representation of genes involved in the phenylalanine, tryptophan and tyrosine biosynthesis pathways was observed. This was confirmed through metabolite analyses that demonstrated tryptamine and serotonin levels are induced after F. pseudograminearum inoculation. Conclusions: Overall, the observed host response in bread wheat to F. pseudograminearum during early infection exhibited enrichment of processes related to pathogen perception, defence signalling, transport and metabolism and deployment of chemical and enzymatic defences. Additional functional analyses of candidate genes should reveal their roles in disease resistance or susceptibility. Better understanding of host responses contributing to resistance and/or susceptibility will aid the development of future disease improvement strategies against this important plant pathogen.
Subject(s)
Fusarium/physiology , Gene Expression Regulation, Plant , Plant Diseases/microbiology , Trichothecenes/metabolism , Triticum/genetics , Triticum/microbiology , High-Throughput Nucleotide Sequencing , Host-Pathogen Interactions , Sequence Analysis, DNAABSTRACT
Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes.
Subject(s)
Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Alleles , Fusarium/enzymology , Fusarium/genetics , Mutation , Trichothecenes/biosynthesis , Adenosine Monophosphate/metabolism , DNA, Fungal , Flow Cytometry/methods , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fusarium/metabolism , High-Throughput Screening Assays/methods , Mycotoxins/metabolism , Phenotype , Plant Diseases/microbiology , Trichothecenes/metabolism , Triticum/microbiologyABSTRACT
A number of cereals produce the benzoxazolinone class of phytoalexins. Fusarium species pathogenic towards these hosts can typically degrade these compounds via an aminophenol intermediate, and the ability to do so is encoded by a group of genes found in the Fusarium Detoxification of Benzoxazolinone (FDB) cluster. A zinc finger transcription factor encoded by one of the FDB cluster genes (FDB3) has been proposed to regulate the expression of other genes in the cluster and hence is potentially involved in benzoxazolinone degradation. Herein we show that Fdb3 is essential for the ability of Fusarium pseudograminearum to efficiently detoxify the predominant wheat benzoxazolinone, 6-methoxy-benzoxazolin-2-one (MBOA), but not benzoxazoline-2-one (BOA). Furthermore, additional genes thought to be part of the FDB gene cluster, based upon transcriptional response to benzoxazolinones, are regulated by Fdb3. However, deletion mutants for these latter genes remain capable of benzoxazolinone degradation, suggesting that they are not essential for this process.
Subject(s)
Benzoxazoles/metabolism , Fusarium/genetics , Genes, Fungal , Multigene Family , Transcription Factors/genetics , Transcription Factors/metabolism , Triticum/microbiology , Benzoxazoles/pharmacology , Fusarium/drug effects , Fusarium/growth & development , Fusarium/metabolism , Gene Expression Regulation, Fungal , Inactivation, Metabolic , Plant Diseases , Triticum/metabolismABSTRACT
Plants produce a variety of secondary metabolites to defend themselves from pathogen attack, while pathogens have evolved to overcome plant defences by producing enzymes that degrade or modify these defence compounds. However, many compounds targeted by pathogen enzymes currently remain enigmatic. Identifying host compounds targeted by pathogen enzymes would enable us to understand the potential importance of such compounds in plant defence and modify them to make them insensitive to pathogen enzymes. Here, a proof of concept metabolomics-based method was developed to discover plant defence compounds modified by pathogens using two pathogen enzymes with known targets in wheat and tomato. Plant extracts treated with purified pathogen enzymes were subjected to LC-MS, and the relative abundance of metabolites before and after treatment were comparatively analysed. Using two enzymes from different pathogens the in planta targets could be found by combining relatively simple enzymology with the power of untargeted metabolomics. Key to the method is dataset simplification based on natural isotope occurrence and statistical filtering, which can be scripted. The method presented here will aid in our understanding of plant-pathogen interactions and may lead to the development of new plant protection strategies.