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

Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7

13 pages; 4 figuresThe colonization process of Olea europaea by the defoliating pathotype of Verticillium dahliae, and the in planta interaction with the endophytic, biocontrol strain Pseudomonas fluorescens PICF7 were determined. Differential fluorescent protein tagging was used for the simultaneous visualization of P. fluorescens PICF7 and V. dahliae in olive tissues. Olive plants were bacterized with PICF7 and then transferred to V. dahliae-infested soil. Monitoring olive colonization events by V. dahliae and its interaction with PICF7 was conducted using a non-gnotobiotic system, confocal laser scanner microscopy and tissue vibratoming sections. A yellow fluorescently tagged V. dahliae derivative (VDAT-36I) was obtained by Agrobacterium tumefaciens-mediated transformation. Isolate VDAT-36I quickly colonized olive root surface, successfully invaded root cortex and vascular tissues via macro- and micro-breakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site, and once established on/in root tissues, hindered pathogen colonization. For the first time using this approach, the entire colonization process of a woody plant by V. dahliae is reported. Early and localized root surface and root endophytic colonization by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt epidemics in olive.Junta de Andalucía, Spain. Grant P07-CVI- 02624 (Proyecto de Excelencia).Peer reviewe

Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens

The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual crop losses. The characteristic wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels, which undergo fluctuations in osmolarity. To gain insights into the mechanisms that confer the organisms’ pathogenicity and enable them to proliferate in the unique ecological niche of the plant vascular system, we sequenced the genomes of V. dahliae and V. albo-atrum and compared them to each other, and to the genome of Fusarium oxysporum, another fungal wilt pathogen. Our analyses identified a set of proteins that are shared among all three wilt pathogens, and present in few other fungal species. One of these is a homolog of a bacterial glucosyltransferase that synthesizes virulencerelated osmoregulated periplasmic glucans in bacteria. Pathogenicity tests of the corresponding V. dahliae glucosyltransferase gene deletion mutants indicate that the gene is required for full virulence in the Australian tobacco species Nicotiana benthamiana. Compared to other fungi, the two sequenced Verticillium genomes encode more pectindegrading enzymes and other carbohydrate-active enzymes, suggesting an extraordinary capacity to degrade plant pectin barricades. The high level of synteny between the two Verticillium assemblies highlighted four flexible genomic islands in V. dahliae that are enriched for transposable elements, and contain duplicated genes and genes that are important in signaling/transcriptional regulation and iron/lipid metabolism. Coupled with an enhanced capacity to degrade plant materials, these genomic islands may contribute to the expanded genetic diversity and virulence of V. dahliae, the primary causal agent of Verticillium wilts. Significantly, our study reveals insights into the genetic mechanisms of niche adaptation of fungal wilt pathogens, advances our understanding of the evolution and development of their pathogenesis, and sheds light on potential avenues for the development of novel disease management strategies to combat destructive wilt diseases

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

Symbiosis of meadow fescue with <i>Epichloë festucae</i>, a heritable symbiont.

<p>Single optical slice confocal micrographs of <i>E. festucae</i> expressing enhanced cyan-fluorescent protein were overlain with DIC bright field images of (A) ovules (bar = 100 µm), (B) embryos (bar = 200 µm), and (C) shoot apical meristem and surrounding new leaves (bar = 200 µm). (D) Asymptomatic (left) and “choked” (right) inflorescences simultaneously produced on a single grass plant infected with a single <i>E. festucae</i> genotype. Vertical (seed) transmission of the symbiont occurs via the asymptomatic inflorescence, whereas the choked inflorescence bears the <i>E. festucae</i> fruiting structure (stroma), which produces sexually derived spores (ascospores) that mediate horizontal transmission.</p