For Blighted Waves of Grain: Fusarium graminearum in the Postgenomics Era

Fungi display astounding diversity in their pathogenicity. We categorize them as biotrophs or necrotrophs and as obligate or facultative pathogens, but these categories do not reflect their polymorphic nature. Our approach to controlling disease would be better served by improving our understanding of the complete life cycle of the fungal pathogen, from infection and colonization to overwintering, using all of the tools at our disposal. A handful of fungal plant pathogens are now being studied at this level, including Fusarium graminearum (sexual stage, Gibberella zeae), the causal agent of head blight of wheat (Triticum aestivum), oat (Avena sativa), and barley (Hordeum vulgare) and ear and stalk rot of maize (Zea mays). Although F. graminearum is arguably one of the best studied fungal plant pathogens, the genetic bases of its life cycle and pathogenicity are poorly defined. This article focuses on the contributions that genomics and postgenomic studies are making to our understanding of the entire life cycle of this important pathogen. The genome of this fungal pathogen cannot be viewed in isolation, just as the fungus does not exist in isolation. Genomics can be used to elucidate F. graminearum's interaction with its hosts, leading to a clearer picture of its ecological niche. Fusarium head blight is a global problem. F. graminearum is the most prominent causal agent of head blight in the United States, Canada, and Europe (McMullen et al., 1997). A study of a worldwide collection of F. graminearum isolates has resulted in the proposed division of the original species into 11 distinct species based on phylogenetic analyses of DNA sequences (O'Donnell et al., 2004; Starkey et al., 2007). The name F. graminearum was retained for the proposed species predominantly causing head blight in the United States and Europe. The proposed division of F. graminearum has not been accepted by all Fusarium researchers (Leslie and Bowden, 2008). Other Fusarium species, including F. culmorum, F. poae, F. pseudograminearum, and F. avenaceum, may also be associated with kernel blight and related diseases throughout the world. Interestingly, individual species may predominate in specific grain hosts, regions of the world, or under certain climatic conditions. F. graminearum produces several mycotoxins, including deoxynivalenol (DON) and derivatives, zearalenone, fusarin C, and aurofusarin. Although head blight causes low grain weight, the primary economic and health consequences of the disease are due to mycotoxin contamination, primarily DON. DON is a potent protein biosynthesis inhibitor affecting the digestive system and major organ function in humans and animals when ingested in sufficient quantities (Council for Agricultural Science and Technology, 2003). DON levels are regulated in food supplies of many countries; for example, the European Community limits DON levels to 0.5 μg g−1 for cereals and the United States limits DON levels to 1 μg g−1 for finished products for human consumption (Council for Agricultural Science and Technology, 2003). In the upper midwestern United States, DON levels frequently exceed this limit. In barley, the presence of F. graminearum can also lead to uncontrolled foaming (gushing) of beer caused by an unknown fungal component. Recently, the presence of F. graminearum in maize has resulted in high levels of zearalenone and other mycotoxins in the “distillers' dried grains with solids,” remnants from maize-based ethanol production fed to cattle and pigs (Wu and Munkvold, 2008). Zearalenone causes estrogenic effects in animals, including humans, and is regulated in some countries. Although zearalenone is of concern to the U.S. Food and Drug Administration, there are currently no regulatory standards limiting its levels in grain (Council for Agricultural Science and Technology, 2003). The genome sequence was publicly released by the Broad Institute in 2003 (www.broad.mit.edu/annotation/genome/fusarium_group/MultiHome.html). The genome sequence release has greatly stimulated research activity on F. graminearum. An average of 50 articles per year were published between 2004 and 2007, compared with an average of 20 from 1999 through 2002, prior to the genome's release (ISI Web of Science; search criterion: Fusarium graminearum in the title). The genome size (36.1 Mb) is typical for a filamentous fungus. It contains genes encoding 13,937 predicted proteins (mips.gsf.de/genre/proj/FGDB/) distributed over four chromosomes. Of these genes, 2,001 are not similar to those of any other sequenced organism (orphans) and 5,812 have homology to proteins of unknown function. There are fewer repeat sequences and an unusually low number of paralogous genes in comparison with genome sequences of other filamentous ascomycetes (Cuomo et al., 2007). Some fungi retain the capability to render duplicated sequences nonfunctional by “repeat induced point mutation” (RIP; Galagan and Selker, 2004). RIP, which is active during meiosis, riddles both copies of duplicated genes with point mutations. The existence of a RIPing mechanism has been demonstrated in F. graminearum and may explain the lack of multicopy sequences in the genome (Cuomo et al., 2007). The availability of the F. graminearum genome sequence provides the opportunity to understand how this pathogen has evolved to occupy its niche. The first step in this process was to identify regions of the genome with high sequence variability among strains. Single nucleotide polymorphism (SNP) analysis of the F. graminearum sequence was conducted using the two major U.S. experimental strains (PH-1, nearly complete sequence; GZ3639, 0.4× coverage) and resulted in the identification of regions of high SNP density that correspond with telomeric regions and central chromosomal regions (centromeres have not been identified; Cuomo et al., 2007). Genes associated with basal metabolism and those highly conserved are more amply represented in the low SNP regions. As has been noted in other organisms, subtelomeric regions appear to harbor a larger fraction of the genes necessary for niche specificity (Farman, 2007). In F. graminearum, high SNP regions are enriched for genes of pathogenicity, genes unique to F. graminearum, secreted proteins, and gene clusters involved in secondary metabolism (Cuomo et al., 2007). However, the telomeric regions are not well represented in the current genome sequence. More advanced sequencing methods, such as adaptations of the pyrosequencing procedure (i.e. 454 sequencing), which eliminate cloning as a step in sequencing, would be useful to completing these regions. One of the most interesting outcomes of genome sequencing is the identification of genes that have limited phylogenetic distribution: the orphan genes. The 2,001 orphan genes identified in the genome sequence of F. graminearum are concentrated in the high SNP regions (Cuomo et al., 2007). A recent study of 122 bacterial genomes indicated that about 1.9% of the genes from any single genome are orphan genes (Wilson et al., 2005). A similar study has not been conducted for fungi, but it would be useful, considering the more complex life cycles of fungi compared with bacteria. However, the orphan genes of F. graminearum remain functionally elusive. Will genes for pathogenicity be well represented in these sequences? How many orphan genes will be related to other life cycle stages? The life cycle of F. graminearum (sexual phase, G. zeae), causal agent of Fusarium head blight on wheat. Details of specific aspects of the cycle are discussed in the text. Following infection of wheat florets, the fungus expresses genes for DON biosynthesis almost immediately (Jansen et al., 2005). DON is a virulence factor in wheat, causing tissue necrosis (Proctor et al., 1995; Desjardins et al., 1996) and allows the fungus to spread into the rachis from florets in wheat (Jansen et al., 2005). Interestingly, DON is the only mycotoxin shown to be a virulence factor. Colonization of developing seeds is accompanied by DON accumulation, resulting in shriveled, undersized grain referred to as tombstones. There is some evidence that DON also functions as a virulence factor in maize (Harris et al., 1999), but in barley, spread of the disease is limited and virulence does not appear to be due to the presence of the toxin (Jansen et al., 2005). A detailed understanding of the interactions of F. graminearum with maize and barley is lacking. F. graminearum can readily complete its life cycle in culture or in association with its host. As with the vast majority of fungi, F. graminearum is haploid for most of its life cycle. For this fungus, sexual development begins with the formation of hyphae with binucleate cells. An extended binucleate phase (called the dikaryotic phase when two genetically distinct nuclei remain paired as new cells form) is a hallmark of the phylum Ascomycota (to which F. graminearum belongs). In the Ascomycota, this phase is the initial step in sexual development. F. graminearum is homothallic, meaning that it does not need a sexually distinct partner to develop sexual spores (ascospores), and as a result, the two nuclei of the binucleate cells are genetically identical. Homothallism in F. graminearum is due to the presence of genes associated with both mating types (Mat1-1 and Mat1-2) in the haploid genome (Yun et al., 2000). The binucleate cells of F. graminearum develop small coiled cells, which are the fruiting body initials (Trail and Common, 2000). In culture, the initials develop uninterrupted into flask-shaped perithecia that are filled with asci. Asci are tubular sacs containing the ascospores, which are the products of meiosis. Asci extend up to the mouth of the perithecium and forcibly discharge their ascospores into the air (Fig. 1). The mechanism of ascospore discharge is under study (Trail et al., 2002, 2005; Hallen and Trail, 2008). In the laboratory, the entire life cycle takes about 2 weeks, with asci maturing and firing sequentially for about the last 4 d (Bowden and Leslie, 1999; Trail et al., 2002). Sexual development is a critical part of the disease cycle. In infected wheat, perithecium initials develop in association with the plant's stomates and silica cells, and, together with the binucleate hyphae from which they arise, are the overwintering structures (Guenther and Trail, 2005). In the field, the perithecia are ephemeral. The airborne ascospores are the primary inoculum of the disease, which is considered to be a monocyclic disease. In one study, elimination of the sexual stage by deletion of the mating-type locus resulted in substantial disease reduction in field trials compared with plots inoculated with a strain complemented back to wild-type ascospore production (Desjardin et al., 2006). Copious asexual spores (conidia) are produced on the surface of infected plants or on crop residue during damp periods. Conidia are produced in slimy masses borne on sporodochia (cushion-shaped hyphal structures). The fusiform shape of the conidia and their formation in slimy masses have been associated with rain-splash dispersal (Deacon, 2006). Conidia are considered to serve predominantly in short-distance dispersal (Shaner, 2003). However, the relative contributions of conidia versus ascospores to disease epidemiology remain unresolved. We hypothesize that during vegetative growth within the host, the acquisition of carbon resources is the most important activity. Lipid bodies can be seen in the binucleate hyphae associated with the perithecium initials. These resources would be necessary for overwinter survival and sexual reproduction, and their acquisition appears to occur during the initial colonization of the host's stalk (Guenther and Trail, 2005). Through gene expression analysis of colonized stalk tissue and functional analysis of individual genes involved in lipid biosynthesis and degradation, we are exploring these aspects of the life cycle. Once the stalk senesces, the resources must be protected from other microorganisms that may compete for host residues in the soil or may attack the fungus. Little is known of the microbial interactions in the crop debris and soil that affect the overwinter survival of this pathogen. The head blight pathogen has been studied for well over a century, beginning with the first report of the disease in England by Smith (1884). As in many organisms, research has been revitalized by the availability of genome sequence. The focus has largely shifted to gene function analyses, which can be performed using a variety of “omics” approaches. Following the sequencing of the genome, assigning function to the predicted proteins has been a challenge. Yet, the efforts of many laboratories worldwide have made the F. graminearum genome one of the best annotated of the genome sequences of filamentous fungi. The hand annotation effort has been spearheaded by the Munich Information Center for Protein Sequences (mips.gsf.de/genre/proj/FGDB). Following the release of the genome, an Affymetrix GeneChip was designed and produced for expression analyses (Gueldener et al., 2006). To date, data from over 100 chip analyses have been posted on www.plexdb.org, including time courses of inoculated barley heads (Gueldener et al., 2006), sexual development (Hallen et al., 2007), and conidial germination (Seong et al., 2008), as well as analyses of mutants (Hallen and Trail, 2008) and mycelia in culture under starvation conditions (Gueldener et al., 2006). Coexpression analyses based on these arrays can be used for assignment of gene function based on the association of expression patterns across a variety of conditions. Coexpression of sets of genes in filamentous fungi will be an more for assigning functions to genes are not is the study of can be used to the of expression studies and to proteins in specific or analyses have been conducted in F. graminearum to identify proteins regulated by the mating-type gene in a of et al., 2008) and those in culture on plant et al., 2005). To identify genes associated with mycotoxin analyses were also performed for F. graminearum under conditions et al., 2008). et conducted an analysis of proteins secreted by F. graminearum on a variety of including plant to identify proteins important to the The in was compared with F. graminearum proteins from the of infected Interestingly, the proteins identified from the from those identified in in with the majority of the genes in the to regions of the genome containing a high of the genes of a fraction of secreted proteins identified in the no which that this may not exist in all secreted The genes identified in this study are during a of growth that the fungus prior to and a unique of to our understanding of the pathogenicity of F. graminearum. The to the study of produced by an organism under a of conditions or over a of studies of F. graminearum are just being the of produced during the of the life cycle will to our understanding of disease. In of the of secondary will be useful for ecological studies these in the interaction of F. graminearum with its and for secondary metabolism has been the of studies in fungi but has been limited to individual and products A effort on understanding the associated with DON biosynthesis is studies of the of products produced by F. graminearum will greatly our current of these in this fungus. The genome sequence provides a from which to individual genes or of genes for functional for genetic are well for F. graminearum and have greatly the to the genome for functional F. graminearum has highly that can be used for gene Although it is homothallic, F. graminearum can which it to genetic analyses (Bowden and Leslie, has been used to which can be for such as of virulence (Seong et al., et al., 2007), of mycotoxin or perithecium development et al., 2007). The availability of the genome the process of gene identification by which identification of a gene from a DNA sequence the A gene can be to the Recently, was to genes in F. graminearum et al., 2005). This will be useful for the expression of genes that may when In several genes can be in one to several genes that have of for these and several of them are for in fungi et al., et al., 2005). the genome sequence of the focus of research has been on genes that function in pathogenicity. have been to identify these genes, including major proteins et al., et al., et al., et al., 2008), mutants for of the to disease (Seong et al., et al., 2007), and genes to pathogenicity genes from other fungi et al., et al., et al., 2006). of these can also that genes for pathogenicity in one fungus are not for pathogenicity in other fungi. genes may serve in fungi, a pathogen to disease affecting other in a In a single the gene may be for growth both in and in the between is a pathogenicity factor and is important to function. The specific between host and pathogen known as has not been identified in the F. graminearum interaction with its A approach was to identify a involved in The gene was identified from analysis of a culture on wheat et al., 2005). this gene was the resulting both its to on wheat and, more its to disease on wheat. However, activity of the extended in culture, that were An of the annotated genome sequence of which are annotated as of expression data in this researchers to the of genes to identify the the genome sequence was the of F. graminearum was poorly In fungi, genes for secondary metabolism and some genes for primary metabolism are and 2008). These clusters from bacterial in that the genes within the have regulatory sequences and can be of genes involved in together with regulatory genes and genes for or of the The gene for fungal secondary genes for and These of are and appear to be in most both and but they are associated in many fungi with virulence and with to to sequencing of the genome, F. graminearum was known to four fusarin C, a identified as and The sequence an of genes not known to and the availability of the genome sequence the entire of genes to be identified et al., et al., 2005). of gene in F. graminearum was and the mutants were et al., 2005). Of the genes functions were to products of the remain genes, which were the zearalenone et al., and Trail, et al., 2006). In genes for the of et al., 2005; et al., et al., the of the perithecium and fusarin were identified et al., 2005). Interestingly, of the genes no effects on pathogenicity et al., 2005). Of the predicted genes, have been functionally and and to be involved in the of which are involved in sexual development and pathogenicity et al., et al., et al., 2008). The functions of the genes have not been studies of the predicted have not been The of the mycotoxins other DON in the life cycle is not It is that the expression of these genes is important for interactions with other For evidence that zearalenone sexual development and vegetative growth of other fungi and 2007). In a of is a agent et al., 2004; et al., 2006), has not been for similar activity. analyses have been performed on of development during the of the perithecia (Hallen et al., 2007). These are the first expression analyses of sexual development in any filamentous fungus. The strain used for sequencing was of its to a of developing perithecia on the surface of in a The to the of tissue in the binucleate perithecium hyphae that the central and genes were identified as only during sexual of were orphan genes, unique to F. graminearum (Hallen et al., 2007). analysis of these genes will to an understanding of the process of sexual development in the fungi. germination of F. graminearum was both and through gene expression using the Affymetrix GeneChip (Seong et al., 2008). The study genes associated with specific of development during the germination analysis also genes associated with the For example, genes associated with development were identified as the development in and the of of types identified in the studies of conidial germination and perithecium development about that can be used to understand in fungi in In studies of plant disease, the frequently is on the host, in the that understanding the host will lead to the on disease This approach has been largely by the approach to for and in many been approach to the of disease has been to but with only of the specific life cycles of the The pathogen F. graminearum the of developing by of these for has not been in wheat. has been associated with the which under high disease have not been of the grain head also has been for These an to develop The approach is to the pathogen life cycle. An understanding of that life cycle the of a approach to The have to identify those genes that can be used to the life cycle. sequencing has that of the predicted genes in filamentous fungi are unique to fungi. This number of genes provides the for that fungi. These genes need to be functionally to of their in the life cycle. This can be used to develop culture or genetically for disease The field is an by one plant species and by and However, a microbial as well as other and are of this interactions is important for the of The genome sequence of F. graminearum is one that can elucidate the ecological of this of several other Fusarium species stage, Gibberella F. and F. stage, have also been These species the diversity of within this of To understand the genomes of the fungi, they must also be studied in the of genomes of the host and fungi and that may also the host and soil that will be in crop The release of the maize and wheat genome sequences in the will be a critical step this studies of the of wheat and barley to F. graminearum have been et al., et al., 2006). in one species in an can affect other species in the In the of and functional analyses for fungi of plant pathogens, and will affect our understanding of the and among all of these and 2008) and of such as F. graminearum to among Genes that affect interactions with other fungi pathogens, and and are important for the The of researchers on F. graminearum has to and the genome sequence. and at the Broad Institute performed the genome sequencing and and are involved in genome analysis and the Affymetrix The of an are 1 was by The sequencing of the genome was by the of the U.S. of and number to and