Functional Analysis of Mating Type Genes and Transcriptome Analysis during Fruiting Body Development of Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction

Sterile plantlets of <i>Hemerocallis</i> ‘Jurassic Spider’ inoculated with <i>Botrytis deweyae</i> and <i>Botrytis elliptica</i> isolates.

<p>Plantlets were grown in vermiculite with growth medium and are shown from above, 10 days after inoculations. A. <i>B. deweyae</i> B1 isolate. B. <i>B. deweyae</i> B2 isolate. C. <i>B. deweyae</i> B4 isolate. D. <i>B. elliptica</i>. E. control (no fungal inoculation).</p

Effect of inoculation of <i>Hemerocallis</i> plantlets with <i>Botrytis deweyae</i>.

<p>A.Control <i>Hemerocallis</i> ‘Jurassic Spider’ plantlets (uninoculated with <i>Botrytis</i>) (left) and <i>Hemerocallis</i> ‘Jurassic Spider’ plantlets infected with <i>B. deweyae</i> (right) after inoculation with B1 isolate, shown 10 days after inoculation. The plant material was excised from sterile vermiculite. Scale indicates 10 cm. B. Close-up of tissue necrosis on the basal portion of a <i>Hemerocallis</i> ‘Jurassic Spider’ plantlet following <i>B. deweyae</i> infection, following colonisation of leaf bases. Scale indicates 5 mm.</p

Phylogeny of <i>Botrytis</i> based on the combined analysis of 5 different genes.

<p>Sequences of <i>G3PDH</i>, <i>HSP60</i>, <i>RPB2</i>, <i>NEP1</i> and <i>NEP2</i> were used. The phylogenetic position of <i>B. deweyae</i> (B1 isolate, type) is underlined. The phylogeny was constructed using the genus <i>Sclerotinia</i> as the outgroup.</p

Table of fungal isolates identified in this study.

<p>Host name, host symptoms and the date of isolation are described. The post-isolation fungal culture signal intensity (S.I.), from tests of PBST suface washings of fungal cultures, with EnviroLogix Botrytis QuickStix using the anti-<i>Botrytis</i>-monoclonal antibody BC-12.CA4, is shown.</p

Alignment of a region of partial <i>ITS</i> sequence from various <i>Botrytis/Botryotinia</i> species.

<p>The aligned region shows the indel identified in all 6 examined isolates of <i>B. deweyae</i>.</p

Development after isolation of <i>Botrytis deweyae</i> cultures from surface-sterilised <i>Hemerocallis</i> material.

<p>The initial development of multiple melanised sclerotia distributed across the colony surface can be seen in this maturing culture. Scale bar indicates 1</p

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper