Neil Moore

Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Genetics, Genomics and Evolution of Ergot Alkaloid Diversity

The ergot alkaloid biosynthesis system has become an excellent model to study evolutionary diversification of specialized (secondary) metabolites. This is a very diverse class of alkaloids with various neurotropic activities, produced by fungi in several orders of the phylum Ascomycota, including plant pathogens and protective plant symbionts in the family Clavicipitaceae. Results of comparative genomics and phylogenomic analyses reveal multiple examples of three evolutionary processes that have generated ergot-alkaloid diversity: gene gains, gene losses, and gene sequence changes that have led to altered substrates or product specificities of the enzymes that they encode (neofunctionalization). The chromosome ends appear to be particularly effective engines for gene gains, losses and rearrangements, but not necessarily for neofunctionalization. Changes in gene expression could lead to accumulation of various pathway intermediates and affect levels of different ergot alkaloids. Genetic alterations associated with interspecific hybrids of Epichloë species suggest that such variation is also selectively favored. The huge structural diversity of ergot alkaloids probably represents adaptations to a wide variety of ecological situations by affecting the biological spectra and mechanisms of defense against herbivores, as evidenced by the diverse pharmacological effects of ergot alkaloids used in medicine.

Support for XML markup of image-based electronic editions

Image-based electronic editions enable researchers to view and study in an electronic environment historical manuscript images intricately linked to edition, transcript, glossary and apparatus files. Building image-based electronic editions poses a two-fold challenge. For humanities scholars, it is important to be able to use image and text to successfully encode the desired features of the manuscripts. Computer Scientists must find mechanisms for representing markup in its association both with the images, text and other auxiliary files and for making the representation available for efficient querying. This paper addresses the architecture of one such solution, that uses efficient data structures to store image-based encodings in main memory and on disk.

Genomes of Plant-Associated Clavicipitaceae

Abstract Fungi of family Clavicipitaceae serve as models for evolution on the symbiotic continuum from pathogenic to mutualistic. Clavicipitaceous fungi associate with plants, invertebrates, and other fungi. Most plant-associated Clavicipitaceae systemically colonize shoots, but the fungal fruiting structures are localized to inflorescences, florets, buds, leaves, or nodes. Many Clavicipitaceae decrease or eliminate host seed production, but some have evolved such intimate symbioses with plant hosts that they disseminate clonally in seeds (vertical transmission) without damage or any reduction in plant fertility. In such cases, the fungi dramatically enhance host fitness by producing defensive alkaloids and through other mechanisms. To date, sequences have been assembled for 26 Clavicipitaceae representing 21 species in seven genera. These include three Claviceps species that fruit on and replace host ovaries, two Metarhizium species that parasitize insects and associate with plant roots, and 21 strains of systemic plant parasites or symbionts. Of the latter, 14 are capable of vertical transmission, and of those, 7 are strictly seed-borne mutualists in genera Epichloe and Periglandula . Alkaloid biosynthetic genes are widely distributed among these fungi. Gene clusters for ergot alkaloids and indole-diterpenes, both of which are neurotoxins in vertebrates and invertebrates, are present in members of all seven genera. The genes for anti-insect loline alkaloids and peramine have a more restricted distribution, but are present in many of the vertically transmissible Epichloe species. The availability of these genome sequences will facilitate studies of the evolution and mechanisms underlying the diversity of metabolism, host interactions, and niche adaptation of plant-associated Clavicipitaceae.

Computational complexity of the problem of tree generation under fine-grained access control policies

We consider the problem of deciding whether a fine-grained access control policy for tree updates allows a particular document to be constructed. This problem arises from a number of natural questions related to document security, authenticity, and verifiability. Fine-grained access control is the problem of specifying the set of operations that may be performed on a complex structure. For tree-structured databases and documents, particularly XML, a rule-based approach is most common. In this model, access control policies consist of rules that select the allowed or disallowed targets of queries or updates based on their hierarchical relationships to other nodes.We show that, for a typical form of rule-based fine-grained access control policies based on a simple fragment of XPath, this problem is undecidable. We also prove lower bounds on the complexity of various restrictions of this problem, and demonstrate deterministic and nondeterministic polynomial-time algorithms for two restrictions in particular.These results show that, for sufficiently complex access control languages, certain forms of analysis are very difficult or even impossible, limiting the ability to verify documents, audit existing policies, and evaluate new policies. Thus rule-based access control policies based on XPath are, in some sense, too powerful, demonstrating the need for a model of access control of tree updates that bridges the gap between expressive and analyzable policies.

Visualizing and sharing results in bioinformatics projects: GBrowse and GenBank exports

Background Effective tools for presenting and sharing data are necessary for collaborative projects, typical for bioinformatics. In order to facilitate sharing our data with other genomics, molecular biology, and bioinformatics researchers, we have developed software to export our data to GenBank and combined the export utilities with our customized and augmented version of GBrowse. The GBrowse bioinformatics software provides a way for users to graphically view genomic feature data stored in a back-end database. GenBank is an open-access database, maintained by the National Center for Biotechnology Information (NCBI), containing annotated versions of all publicly available DNA sequences. To support exports relevant to our data, we have developed a tool that processes a list of expressed sequence tags (ESTs), correlates these tags with information from our database, and constructs a set of files for batch submission to the GenBank EST database (dbEST).

A Colletotrichum graminicola mutant deficient in the establishment of biotrophy reveals early transcriptional events in the maize anthracnose disease interaction

BackgroundColletotrichum graminicola is a hemibiotrophic fungal pathogen that causes maize anthracnose disease. It progresses through three recognizable phases of pathogenic development in planta: melanized appressoria on the host surface prior to penetration; biotrophy, characterized by intracellular colonization of living host cells; and necrotrophy, characterized by host cell death and symptom development. A “Mixed Effects” Generalized Linear Model (GLM) was developed and applied to an existing Illumina transcriptome dataset, substantially increasing the statistical power of the analysis of C. graminicola gene expression during infection and colonization. Additionally, the in planta transcriptome of the wild-type was compared with that of a mutant strain impaired in the establishment of biotrophy, allowing detailed dissection of events occurring specifically during penetration, and during early versus late biotrophy.ResultsMore than 2000 fungal genes were differentially transcribed during appressorial maturation, penetration, and colonization. Secreted proteins, secondary metabolism genes, and membrane receptors were over-represented among the differentially expressed genes, suggesting that the fungus engages in an intimate and dynamic conversation with the host, beginning prior to penetration. This communication process probably involves reception of plant signals triggering subsequent developmental progress in the fungus, as well as production of signals that induce responses in the host. Later phases of biotrophy were more similar to necrotrophy, with increased production of secreted proteases, inducers of plant cell death, hydrolases, and membrane bound transporters for the uptake and egress of potential toxins, signals, and nutrients.ConclusionsThis approach revealed, in unprecedented detail, fungal genes specifically expressed during critical phases of host penetration and biotrophic establishment. Many encoded secreted proteins, secondary metabolism enzymes, and receptors that may play roles in host-pathogen communication necessary to promote susceptibility, and thus may provide targets for chemical or biological controls to manage this important disease. The differentially expressed genes could be used as ‘landmarks’ to more accurately identify developmental progress in compatible versus incompatible interactions involving genetic variants of both host and pathogen.

Host Tissue Environment Directs Activities of an Epichloë Endophyte, While It Induces Systemic Hormone and Defense Responses in Its Native Perennial Ryegrass Host

Increased resilience of pasture grasses mediated by fungal Epichloë endophytes is crucial to pastoral industries. The underlying mechanisms are only partially understood and likely involve very different activities of the endophyte in different plant tissues and responses of the plant to these. We analyzed the transcriptomes of Epichloë festucae and its host, Lolium perenne, in host tissues of different function and developmental stages. The endophyte contributed approximately 10× more to the transcriptomes than to the biomass of infected tissues. Proliferating mycelium in growing host tissues highly expressed genes involved in hyphal growth. Nonproliferating mycelium in mature plant tissues, transcriptionally equally active, highly expressed genes involved in synthesizing antiherbivore compounds. Transcripts from the latter accounted for 4% of fungal transcripts. Endophyte infection systemically but moderately increased transcription of L. perenne genes with roles in hormone biosynthesis and perception as well as stress and pathogen resistance while reducing expression of genes involved in photosynthesis. There was a good correlation between transcriptome-based observations and physiological observations. Our data indicate that the fitness-enhancing effects of the endophyte are based both on its biosynthetic activities, predominantly in mature host tissues, and also on systemic alteration of the hosts hormonal responses and induction of stress response genes. [Formula: see text] Copyright

A comparative genomic analysis of putative pathogenicity genes in the host-specific sibling species Colletotrichum graminicola and Colletotrichum sublineola

BackgroundColletotrichum graminicola and C. sublineola cause anthracnose leaf and stalk diseases of maize and sorghum, respectively. In spite of their close evolutionary relationship, the two species are completely host-specific. Host specificity is often attributed to pathogen virulence factors, including specialized secondary metabolites (SSM), and small-secreted protein (SSP) effectors. Genes relevant to these categories were manually annotated in two co-occurring, contemporaneous strains of C. graminicola and C. sublineola. A comparative genomic and phylogenetic analysis was performed to address the evolutionary relationships among these and other divergent gene families in the two strains.ResultsInoculation of maize with C. sublineola, or of sorghum with C. graminicola, resulted in rapid plant cell death at, or just after, the point of penetration. The two fungal genomes were very similar. More than 50% of the assemblies could be directly aligned, and more than 80% of the gene models were syntenous. More than 90% of the predicted proteins had orthologs in both species. Genes lacking orthologs in the other species (non-conserved genes) included many predicted to encode SSM-associated proteins and SSPs. Other common groups of non-conserved proteins included transporters, transcription factors, and CAZymes. Only 32 SSP genes appeared to be specific to C. graminicola, and 21 to C. sublineola. None of the SSM-associated genes were lineage-specific. Two different strains of C. graminicola, and three strains of C. sublineola, differed in no more than 1% percent of gene sequences from one another.ConclusionsEfficient non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, was observed in epidermal cells as a rapid deployment of visible resistance responses and plant cell death. Numerous non-conserved SSP and SSM-associated predicted proteins that could play a role in this non-host recognition were identified. Additional categories of genes that were also highly divergent suggested an important role for co-evolutionary adaptation to specific host environmental factors, in addition to aspects of initial recognition, in host specificity. This work provides a foundation for future functional studies aimed at clarifying the roles of these proteins, and the possibility of manipulating them to improve management of these two economically important diseases.