Comparative Transcriptomics of Fusarium graminearum and Magnaporthe oryzae Spore Germination Leading up To Infection

Abstract

For fungal plant pathogens, the germinating spore provides the first interaction with the host. Spore germlings move across the plant surface and use diverse penetration strategies for ingress into plant surfaces. Penetration strategies include pressurized melanized appressoria, which facilitate physically punching through the plant cuticle, and nonmelanized appressoria, which penetrate with the help of enzymes or cuticular damage to breach the plant surface. Two well-studied plant pathogens, Fusarium graminearum and Magnaporthe oryzae, are typical of these two modes of penetration. We applied comparative transcriptomics to Fusarium graminearum and Magnaporthe oryzae to characterize the genetic programming of the early host-pathogen interface. Four sequential stages of development following spore localization on the plant surface, from spore swelling to appressorium formation, were sampled for each species on culture medium and on barley sheaths, and transcriptomic analyses were performed. Gene expression in the prepenetration stages in both species and under both conditions was similar. In contrast, gene expression in the final stage was strongly influenced by the environment. Appressorium formation involved the greatest number of differentially expressed genes. Laser-dissection microscopy was used to perform detailed transcriptomics of initial infection points by F. graminearum. These analyses revealed new and important aspects of early fungal ingress in this species. Expression of the trichothecene genes involved in biosynthesis of deoxynivalenol by F. graminearum implies that toxisomes are not fully functional until after penetration and indicates that deoxynivalenol is not essential for penetration under our conditions. The use of comparative gene expression of divergent fungi promises to advance highly effective targets for antifungal strategies. IMPORTANCE Fusarium graminearum and Magnaporthe oryzae are two of the most important pathogens of cereal grains worldwide. Despite years of research, strong host resistance has not been identified for F. graminearum, so other methods of control are essential. The pathogen takes advantage of multiple entry points to infect the host, including breaches in the florets due to senescence of flower parts and penetration of the weakened trichome bases to breach the epidermis. In contrast, M. oryzae directly punctures leaves that it infects, and resistant cultivars have been characterized. The threat of either pathogen causing a major disease outbreak is ever present. Comparative transcriptomics demonstrated its potential to reveal novel and effective disease prevention strategies that affect the initial stages of disease. Shedding light on the basis of this diversity of infection strategies will result in development of increasingly specific control strategies.

Keywords: Fusarium graminearum; Magnaporthe oryzae; barley infection; infection process; secondary metabolites; spore germination; transcriptome.

FIG 1

(A and B) Stages of conidial germination and development for transcriptome analysis on (A) medium (A) and host (barley leaf sheaths) (B). Time indicates the point at which peak numbers of conidia reach the indicated stage; AP, appressorium; CN, conidium; BR, hyphal branch; H, hypha; stage 1, fresh conidia; stage 2, polar growth; stage 3, doubling of long axis; stage 4, first hyphal branching (Bird medium) or appressorium formation (host).

FIG 2

Multidimensional scaling (MDS) analysis plot applied to the transcriptomes of conidial germination on medium and on the host. (A) F. graminearum. (B) M. oryzae. Each point represents one sample, and the distance between two points reflects the leading log₂ fold change of the corresponding RNA-seq samples. Plot dimensions 1 and 2 show samples from medium and from the host at different stages, resulting in separate clusters.

FIG 3

Distribution of differentially expressed genes (DEGs; >3-fold; 5% false-discovery rate) with predicted and unknown functions in each stage on the host. (A) F. graminearum. (B) M. oryzae.

FIG 4

Comparison of functional enrichment of DEGs in the host for F. graminearum (A) and M. oryzae (B). DEGs (>3-fold for F. graminearum and > 2-fold for M. oryzae) were assessed for degree of functional enrichment and plotted in two-dimensional semantic spaces. Only GO terms with a P value of <0.05 are depicted in each panel. The size of the circles indicates frequency of GO term in the underlying Gene Ontology annotation database (circles of more general terms are larger). The color scale indicates the log₁₀ of the P value. See Table S3 for fully detailed data of GO terms.

FIG 5

Laser dissection of stage 5 F. graminearum infection sites on barley paleae and transcriptomics analysis. (A) Representative laser-captured infection sites stained with toluidine blue (hyphae) and Safranin O (host cell walls). Left, hyphal penetration and colonization of a single trichome. Right, infection and colonization of epidermal cells (surface) and reaction of sclerenchyma cells (below surface) to ingress of fungus. Lignified cell walls stained red with Safranin O; scale bar, 30 μm. (B) Multidimensional scaling (MDS) plot showing the expression correlation of the stage 1 and stage 5 samples; dim, dimension. (C) Gene Ontology (GO) enrichment analysis of biological processes for upregulated genes (purple) and downregulated genes (blue) of samples in B.

FIG 6

Expression profiles of secondary metabolite genes, presented as log₁₀ (fragments per kilobase per million [FPKM] + 1), during the stages of spore germination in F. graminearum on barley sheaths. Genes with similar expression patterns are grouped, with the final group composed of genes that have unique expression patterns.

FIG 7

Expression profiles of secondary metabolite genes, presented as log₁₀ (FPKM + 1), during the Stages of spore germination in M. oryzae on barley sheaths. Genes with similar expression patterns are grouped, with the final group composed of genes that have unique expression patterns.