Genome-wide transcriptional profiling of appressorium development by the rice blast fungus Magnaporthe oryzae

Abstract

The rice blast fungus Magnaporthe oryzae is one of the most significant pathogens affecting global food security. To cause rice blast disease the fungus elaborates a specialised infection structure called an appressorium. Here, we report genome wide transcriptional profile analysis of appressorium development using next generation sequencing (NGS). We performed both RNA-Seq and High-Throughput SuperSAGE analysis to compare the utility of these procedures for identifying differential gene expression in M. oryzae. We then analysed global patterns of gene expression during appressorium development. We show evidence for large-scale gene expression changes, highlighting the role of autophagy, lipid metabolism and melanin biosynthesis in appressorium differentiation. We reveal the role of the Pmk1 MAP kinase as a key global regulator of appressorium-associated gene expression. We also provide evidence for differential expression of transporter-encoding gene families and specific high level expression of genes involved in quinate uptake and utilization, consistent with pathogen-mediated perturbation of host metabolism during plant infection. When considered together, these data provide a comprehensive high-resolution analysis of gene expression changes associated with cellular differentiation that will provide a key resource for understanding the biology of rice blast disease.

Figure 1. Overall comparison of HT-SuperSAGE datasets.

A. Micrographs showing appressorium development at time-points used for HT-SuperSAGE analysis (scale bar = 10 µm). B. Micrographs comparing appresorium development at 4 h in wild-type Guy11 and Δpmk1 mutant backgrounds. C. Heatmap showing Euclidean distances between HT-SuperSAGE samples as calculated from a variance-stabilising transformation of the total count data. The darker the colour, the closer the two datasets are together. T4, T6, T8, T14 and T16 are time-points during appressorium development in Guy11, P4 is appresorium development at 4 in a Δpmk1 mutant. CM and MM are samples from Guy11 mycelium grown in complete medium and minimal medium respectively. D. Venn diagram illustrating overlaps between number of genes that are significantly up-regulated (P-value<0.05) in early appressorium development – 4 h to 8 h (vs mycelium grown in CM), significantly up-regulated (P-value<0.05) in mycelium grown in minimal medium (vs mycelium grown in CM) and significantly down-regulated (P-value<0.05) in a Δpmk1 mutant compared to Guy11 after 4 h E. Venn diagram illustrating overlaps between number of genes that are significantly up-regulated (P-value<0.05) in late appressorium development – 14 h to 16 h (vs mycelium grown in CM), significantly up-regulated (P-value<0.05) in mycelium grown in minimal medium (vs mycelium grown in CM) and significantly down-regulated (P-value<0.05) in a Δpmk1 mutant compared to Guy11 after 4 h. F. Venn diagram illustrating overlaps between number of genes that are significantly up-regulated (P-value<0.05) in early appressorium development – 4 h to 8 h (vs mycelium grown in CM) and late appressorium development – 14 h to 16 h (vs mycelium grown in CM).

Figure 2. Expression level of genes encoding enzymes that produce or utilise acetyl-CoA.

The diagrams illustrate metabolic pathways that produce (arrow pointing towards acetyl-CoA) or utilise (arrow pointing away from acetyl-CoA) acetyl-CoA. Transcript abundance was compared in those genes from each pathway that encode enzymes that directly utilise or produce acetyl-CoA A. Guy11 germinating conidia (4 h) and Guy11 mycelium grown in complete medium. B. Δpmk1 germinating conidia (4 h) and Guy11 germinating conidia (4 h). The diagrams also show which pathways contain genes significantly more highly expressed in one condition as compared to the other (see key for each diagram). These are based on adjusted P-value< = 0.05 for at least one of the genes in each pathway.

Figure 3. Heatmaps showing levels of transcript abundance during time course of appressorium development.

Levels of expression are represented as moderated log₂ ratio of transcript abundance compared to mycelium grown in complete medium. Values are from 4 h (T4) to 16 h (T16) after conidia were placed on a hydrophobic surface. Genes showing similar patterns of gene expression are clustered. A. Genes encoding enzymes from pathways that utilise malonyl-CoA. Specifically labelled are acetyl-CoA carboxylase (ACC), malonyl CoA-acyl carrier protein trans-acylase (MACT), fatty acid synthase (FAS), components of melanin biosynthesis pathway (ALB, BUF, RSY, THR) and the PKS-NRPS hybrid ACE1. B. Genes encoding enzymes involved in β-oxidation of fatty acids and the glyoxylate cycle. Genes are labelled according to pathways: fatty acid β-oxidation (BOX), glyoxylate cycle (GLY) and carnitine acetyl-transferases (CAT).

Figure 4. Expression of genes encoding enzymes from three metabolic pathways that affect the pool of acetyl-CoA.

A. The β-oxidation pathway, B. melanin biosynthesis, C. the glyoxylate shunt. For each enzyme, bar graphs show abundance of transcripts encoding this gene in: Guy11 mycelium grown in complete medium (green bar), Δpmk1 mutant conidia left to germinate for 4 h (red bar), Guy11 time course of appresorium development (4 h, 6 h, 8 h, 14 h, 16 h – black bars left to right).

Figure 5. Levels of transcript abundance from genes involved in quinate metabolism.

A. Heatmap showing levels of transcript abundance during time course of appressorium development. Levels of expression are represented as moderated log₂ ratio of transcript abundance as compared to mycelium grown in complete medium. Values are from 4 h (T4) to 16 h (T16) after conidia were incubated on a hydrophobic surface. Genes showing similar patterns of gene expression have been clustered. Homologues of genes involved in quinate metabolism in Neurospora crassa are labelled: quinate activator (qa-1F), quinate repressor (qa-1S), 3-dehydroquinase (qa-2), Quinate dehydrogenase (qa-3), 3-dehydroshikimate dehydratase (qa-4), function unknown (qa-x), quinate permeases (QP), shikimate pathway (S). B. Expression of genes involved in quinate metabolism. For each enzyme, bar graphs show abundance of transcripts encoding the gene in: Guy11 mycelium grown in complete medium (green bar), Δpmk1 mutant conidia left to germinate for 4 h (red bar), a Guy11 time course of appressorium development (4 h, 6 h, 8 h, 14 h, 16 h – black bars left to right).