Fusarium head blight (FHB) is a destructive disease of wheat and other cereals. FHB occurs in Europe, North America and around the world causing significant losses in production and endangers human and animal health. In this article, we provide the strategic steps for the specific target selection for the phytopathogen system wheatFusarium graminearum. The economic impact of FHB leads to the need for innovation. Currently used fungicides have been shown to be effective over the years, but recently cereal infecting Fusaria have developed resistance. Our work presents a new perspective on target selection to allow the development of new fungicides. We developed an innovative approach combining both genomic analysis and molecular modeling to increase the discovery for new chemical compounds with both safety and low environmental impact. Our protein targets selection revealed 13 candidates with high specificity, essentiality and potentially assayable with a favorable accessibility to drug activity. Among them, three proteins: trichodiene synthase, endoglucanase-5 and ERG6 were selected for deeper structural analyses to identify new putative fungicides. Overall, the bioinformatics filtering for novel protein targets applied for agricultural purposes is a response to the demand for chemical crop protection. The availability of the genome, secretome and PHI-base allowed the enrichment of the search that combined experimental data in planta. The homology modeling and molecular dynamics simulations allowed the acquisition of three robust and stable conformers. From this step, approximately ten thousand compounds have been virtually screened against three candidates. Forty-five top-ranked compounds were selected from docking results as presenting better interactions and energy at the binding pockets and no toxicity. These compounds may act as inhibitors and lead to the development of new fungicides.

The fungus Fusarium graminearum forms an intimate association with the host species wheat whilst infecting the floral tissues at anthesis. During the prolonged latent period of infection, extracellular communication between live pathogen and host cells must occur, implying a role for secreted fungal proteins. The wheat cells in contact with fungal hyphae subsequently die and intracellular hyphal colonisation results in the development of visible disease symptoms. Since the original genome annotation analysis was done in 2007, which predicted the secretome using TargetP, the F. graminearum gene call has changed considerably through the combined efforts of the BROAD and MIPS institutes. As a result of the modifications to the genome and the recent findings that suggested a role for secreted proteins in virulence, the F. graminearum secretome was revisited. In the current study, a refined F. graminearum secretome was predicted by combining several bioinformatic approaches. This strategy increased the probability of identifying truly secreted proteins. A secretome of 574 proteins was predicted of which 99% was supported by transcriptional evidence. The function of the annotated and unannotated secreted proteins was explored. The potential role(s) of the annotated proteins including, putative enzymes, phytotoxins and antifungals are discussed. Characterisation of the unannotated proteins included the analysis of Pfam domains and features associated with known fungal effectors, for example, small size, cysteine-rich and containing internal amino acid repeats. A comprehensive comparative genomic analysis involving 57 fungal and oomycete genomes revealed that only a small number of the predicted F. graminearum secreted proteins can be considered to be either species or sequenced strain specific.

The Dothideomycete fungus Mycosphaerella graminicola is the causal agent of Septoria tritici blotch, a devastating disease of wheat leaves that causes dramatic decreases in yield. Infection involves an initial extended period of symptomless intercellular colonisation prior to the development of visible necrotic disease lesions. Previous functional genomics and gene expression profiling studies have implicated the production of secreted virulence effector proteins as key facilitators of the initial symptomless growth phase. In order to identify additional candidate virulence effectors, we re-analysed and catalogued the predicted protein secretome of M. graminicola isolate IPO323, which is currently regarded as the reference strain for this species. We combined several bioinformatic approaches in order to increase the probability of identifying truly secreted proteins with either a predicted enzymatic function or an as yet unknown function. An initial secretome of 970 proteins was predicted, whilst further stringent selection criteria predicted 492 proteins. Of these, 321 possess some functional annotation, the composition of which may reflect the strictly intercellular growth habit of this pathogen, leaving 171 with no functional annotation. This analysis identified a protein family encoding secreted peroxidases/chloroperoxidases (PF01328) which is expanded within all members of the family Mycosphaerellaceae. Further analyses were done on the non-annotated proteins for size and cysteine content (effector protein hallmarks), and then by studying the distribution of homologues in 17 other sequenced Dothideomycete fungi within an overall total of 91 predicted proteomes from fungal, oomycete and nematode species. This detailed M. graminicola secretome analysis provides the basis for further functional and comparative genomics studies.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

Acquiring and exploring whole genome sequence information for a species under investigation is now a routine experimental approach. On most genome browsers, typically, only the DNA sequence, EST support, motif search results, and GO annotations are displayed. However, for many species, a growing volume of additional experimental information is available but this is rarely searchable within the landscape of the entire genome.

RNA interference is a natural defense against viruses in plants. To date, the only viral siRNAs characterized have been those for positive-sense RNA viruses with one or two genome components. Here, we characterized siRNAs derived from rice stripe virus (RSV), a member of the genus Tenuivirus with four genomic RNAs and an ambisense coding strategy. Deep sequencing of small RNAs from infected rice leaves showed that siRNAs were derived almost equally from virion and complementary RNA strands and were mostly 20–22 nucleotides long. Most viral siRNAs were produced within the coding sequences and 5′ termini of the RSV genome. RSV siRNAs had a higher G and lower C content than the viral genome but a strong A/U bias at the first nucleotide and a U bias at the final one, suggesting preferential targeting of such sequences by rice Dicer-like proteins.

Fusarium graminearum (teleomorph: Gibberella zeae) is an Ascomycete fungal plant pathogen which infects a range of agriculturally important crops, including wheat, barley, and maize. A random plasmid insertion mutagenesis approach was used to analyze the pathogenicity of the PH-1 strain, for which full genomic information is available. Fungal transformants were initially screened for their ability to infect wheat ears. From a total of 1,170 transformants screened, eight were confirmed to be highly reduced in pathogenicity toward wheat ears and roots. These were designated disease-attenuated F. graminearum (daf) mutants. The in vitro growth rate and appearance of each daf mutant was equivalent to the parental strain. Deoxynivalenol (DON) was not detected in threshed grain recovered from ears inoculated with the daf10 mutant. Plasmid rescue and sequencing of the mutant daf10 revealed a deletion of approximately 350 kb from one end of chromosome 1. This chromosome segment is predicted to contain 146 genes. Microarray analysis of daf10 gene expression during growth in DON-inducing conditions confirmed the large deletion. The identities of the genes deleted and their potential role in DON production, pathogenesis, and other life processes are discussed.

Pathogenic micro-organisms have been suggested to vary the number of intragenic repeats present within secreted or cell membrane/cell wall-associated proteins in order to manipulate host immune responses. We have identified a number of genes predicted to encode secreted proteins possessing internal tandem repeats in the genome sequence of Mycosphaerella graminicola (isolate IPO323), a wheat leaf-specific fungal pathogen and causal agent of Septoria tritici blotch disease. Twenty-three M. graminicola Tandem Repeat Proteins (MgTRPs) were subject to further analysis. Many MgTRPs varied in the number of intragenic repeats between isolates and almost all were expressed. Peak gene expression was frequently observed towards the end of the symptomless phase of wheat leaf colonisation which typically lasts for 8–10 days after inoculation. In contrast, with one exception, increased expression of the majority of MgTRPs was not detected during interactions with resistant host genotypes. Repeat number differences detected in genomic DNA were retained in different transcript sizes produced during plant infection by different isolates. One in planta expressed MgTRP was found to reside within a ∼6 kb region that appears to be absent from a number of tested isolates and also from individual members of a modern field population. Sequence analysis of another in planta expressed MgTRP from six isolates highlighted the potential for structural changes which may occur as a consequence of varying internal repeat numbers and provided support for repeat variation occurring as a consequence of intragenic recombination. These data provide new insights into the genetic variation which exists within M. graminicola populations at the level of in planta expressed secreted/surface-associated proteins which are candidate effectors in the host–pathogen interaction.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

The new plant virus family Virgaviridae is described. The family is named because its members have rod-shaped virions (from the Latin virga = rod), and it includes the genera Furovirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus and Tobravirus. The chief characteristics of members of the family are presented with phylogenetic analyses of selected genes to support the creation of the family. Species demarcation criteria within the genera are examined and discussed.

Plant viruses transmitted by the obligate root-infecting plasmodiophorid parasites Polymyxa graminis and Polymyxa betae cause devastating yield losses to cereal and sugar beet crops worldwide. Barley is a non-host for P. betae but is a host for P. graminis. Using the Barley1 GeneChip® microarray we have investigated the transcriptional re-programming of barley roots during the earliest non-host and host interactions with zoospores of these protist species. At high confidence levels we detected 20 and 13 genes with increased transcriptional activity in response to P. betae and P. graminis, respectively, compared to unchallenged barley roots. Functional classification of the induced genes showed that a majority of the genes from both responses were associated with a classic defence response. Validation by quantitative RT-PCR analysis indicated that all of the genes examined were induced to comparable levels in both non-host and host responses. Our results also demonstrated that the barley defence-associated genes, RAR1, ROR1 or ROR2 were not essential for limiting the establishment of P. betae infection in barley. These data suggest that in barley roots the Polymyxa species induce a similar basal defence response whether the interaction is with a non-host or host. Thus, the early response to protist plant parasites appears to be part of the general ‘frontline’ defence against invading microbes.

from the EFGC meeting (4/5-8/08) that mentioned Fusarium. Novel ways to explore sequenced fungal genomes Kim Hammond-Kosack, Andrew Beacham, Thomas Baldwin, Martin Urban, Salvador Gazan, John Antoniw Rothamsted Research, Harpenden, United Kingdom Complete genomic sequence information is now readily available for many pathogenic and non-pathogenic fungal species and in the near future this will extend to different strains of a single species. For some species this new sequence information has been aligned to an existing genetic map. By exploring and visualising gene content and gene type within the chromosome landscape new information on the evolution of fungal genomes can be obtained. The sequenced genome of the cereal attacking Ascomycete pathogen Fusarium graminearum (teleomorph Gibberella zeae) has been explored at various scales using a new visualisation tool in combination with statistics, Affymetrix gene expression data, gene function categories and recombination frequencies. These combined analyses have revealed that genes coding for specific protein types are non randomly distributed over the four F. graminearum chromosomes. Some gene types are located only in centromeric regions, whereas others only reside in sub-telomeric regions, for example in planta expressed genes. In addition, regions of high recombination tend to harbour specific protein classes, for example polyketide synthases, whereas other gene types are solely located in regions of low /no recombination. These analyses have also revealed the existence on chromosome 1 of a ~25 kb micro-region rich in homologues of pathogenicity genes and that most of the experimentally verified F.graminearum pathogenicity genes do not reside in either sub-telomeric regions or regions with a high recombination frequency. The four large F. graminearum chromosomes are hypothesised to have arisen from a series of earlier chromosome fusion events. The gene content of these genomic sub-regions is unique and reflects this suspected evolutionary path. Msb2, a putative membrane mucin functioning in signalling and pathogenesis of Fusarium oxysporum Elena Perez-Nadales, Antonio Di Pietro University of Cordoba, Cordoba, Spain The soilborne vascular wilt fungus Fusarium oxysporum infects a wide variety of plant species by directly penetrating roots, invading the cortex and colonizing the vascular tissue. The mitogen activated protein kinase (MAPK) Fmk1 is essential for plant infection. The signalling components upstream of the Fmk1 cascade are currently unknown. We have identified a gene from F. oxysporum whose predicted product shows homology with Msb2, a mucin functioning at the head of the filamentation MAPK cascade in yeast. To test whether F. oxysporum Msb2 is involved in signalling through the Fmk1 pathway, we produced targeted knockout mutants both in the wild type and the Δfmk1 background. The ∆msb2 strains were still able to grow invasively on fruit tissue and across cellophane sheets, but are less virulent on tomato plants than the wild type strain. Similar to Δfmk1 strains, Δmsb2 mutants also showed reduced secretion of pectinolytic enzymes. In contrast to Δfmk1 mutants, growth of Δmsb2 strains was specifically affected by the cell wall-targeting compounds Congo Red (CR) and Calcofluor White (CFW). Interestingly, a Δfmk1Δmsb2 double mutant had dramatically impaired growth in the presence of CR and CFW, suggesting that Fmk1 and Msb2 interact genetically to regulate cell integrity in F. oxysporum. Western analysis with phospho-ERK antibody revealed similarities in the phosphorylation pattern between Δfmk1 and Δmsb2 strains. These results suggest that F. oxysporum Msb2 interacts with the Fmk1 cascade to regulate a number of virulence-related functions as well as response to cell wall stress. Hybrid polyketide synthase non-ribosomal peptide synthetases (PKS-NRPS) in fungi Russell Cox, Colin Lazarus, Andrew Bailey, Thomas Simpson, Zhongshu Song, Laura Halo

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue, followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke or tolerate it. We are studying a major host-specific fungal pathogen of cultivated wheat, Septoria tritici (teleomorph Mycosphaerella graminicola). Here, we describe the host responses of wheat leaves infected with M. graminicola during the development of disease symptoms and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response, a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease-resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell-membrane integrity and dramatic increases in apoplastic metabolites and the rate of fungal growth. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms, and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.

DPVweb (http://www.dpvweb.net/) provides a central source of information about viruses, viroids and satellites of plants, fungi and protozoa. Comprehensive taxonomic information, including brief descriptions of each family and genus, and classified lists of virus sequences are provided. The database also holds detailed, curated, information for all sequences of viruses, viroids and satellites of plants, fungi and protozoa that are complete or that contain at least one complete gene (currently, n approximately 9000). For comparative purposes, it also contains a single representative sequence of all other fully sequenced virus species with an RNA or single-stranded DNA genome. The start and end positions of each feature (gene, non-translated region and the like) have been recorded and checked for accuracy. As far as possible, nomenclature for genes and proteins are standardized within genera and families. Sequences of features (either as DNA or amino acid sequences) can be directly downloaded from the website in FASTA format. The sequence information can also be accessed via client software for PC computers (freely downloadable from the website) that enable users to make an easy selection of sequences and features of a chosen virus for further analyses.

To reduce reliance on fungicides for disease control on oilseed rape, Decision Support Systems (DSS) are being developed to guide fungicide applications as part of an integrated pest and disease management strategy. This paper reports the development and uptake of an empirical model for prediction of risk of severe light leaf spot using disease survey and meteorological data to produce autumn regional and crop-specific prediction of epidemic risk for the following spring. Following initial delivery of the light leaf spot forecast via the farming press, an Internet-based interactive website was developed to provide crop-specific predictions. Uptake by growers, advisors and other members of the arable industry has been encouraging. There is evidence to suggest that growers and their advisors visit the website for an up-to-date prediction at the time when they are making fungicide application decisions.

It is clear that MPs play an essential role in the pathogenesis and movement within the plant of many plant viruses. However, studies of the structure and function of such proteins are still in their infancy. Substantial progress may be expected in the next few years, particularly in the area of cell-to-cell movement where viruses are proving useful tools to study the basic processes of macromolecular trafficking between adjacent plant cells.

THE earlier investigations of virus diseases of plants were attempted by the standard methods of the plant pathologists, and failed to reveal the cause of the disease, or to elucidate the nature of the infective agents. These methods have been supplemented by a technique of the widest biological basis, including that used by biochemists and physicists to investigate proteins and other compounds of high molecular weight. The author, with his colleagues, has played a prominent part in the isolation, purification and chemical recognition of nucleoproteins obtained only from infected plants. His hypothesis, freely accepted to-day, considers the nucleoproteins to be the viruses; this forms the theme of his book, which might with advantage have been entitled “Recent Research on the Nature of Plant Viruses”. Thus it differs, particularly in outlook but also in treatment, from other recent text-books of virus diseases which are primarily concerned with the economic host plants; this one especially considers the infective agents.Plant Viruses and Virus DiseasesBy F. C. Bawden. (A New Series of Plant Science Books, Vol. 5.) Pp. xii + 272. (Leiden: Chronica Botanica Co.; London: Wm. Dawson and Sons, Ltd., 1939.) 7 guilders.

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.