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Transcriptome Analysis Reveals New Insight Into Appressorium Formation and Function in the Rice Blast Fungus Magnaporthe Oryzae

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Transcriptome Analysis Reveals New Insight Into Appressorium Formation and Function in the Rice Blast Fungus Magnaporthe Oryzae

Yeonyee Oh et al. Genome Biol.

Abstract

Background: Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. The molecular processes regulating appressorium formation are incompletely understood.

Results: We analyzed genome-wide gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore germination, 2,154 (approximately 21%) genes showed differential expression, with the majority being up-regulated. During appressorium formation, 357 genes were differentially expressed in response to both stimuli. These genes, which we refer to as appressorium consensus genes, were functionally grouped into Gene Ontology categories. Overall, we found a significant decrease in expression of genes involved in protein synthesis. Conversely, expression of genes associated with protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular transportation exhibited a dramatic increase. We functionally characterized several differentially regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown findings that protein degradation and amino acid metabolism are essential for appressorium formation and subsequent infection.

Conclusion: We present the first comprehensive genome-wide transcript profile study and functional analysis of infection structure formation by a fungal plant pathogen. Our data provide novel insight into the underlying molecular mechanisms that will directly benefit efforts to identify fungal pathogenicity factors and aid the development of new disease management strategies.

Figures

Figure 1
Figure 1
Experimental and microarray design for spore germination and appressorium induction. (a) Spores were placed on the hydrophilic (Phil) and hydrophobic (Pho) surfaces of GelBond and incubated for 7 and 12 h. For induction of appressoria by cAMP, spores were placed on the hydrophilic surface of GelBond with (cAMP9) and without (Phil9) cAMP and incubated for 9 h. (b) Diagrams show microarray design. Arrows connect samples directly compared on two channel Agilent M. oryzae oligonucleotide microarrays. Arrow heads = Cy5, arrow tails = Cy3.
Figure 2
Figure 2
Gene expression profile clustering and correlation analysis. (a) Hierarchical clustering analysis of gene expression profiless for spore germination (SG), germ tube elongation (GE), appressorium initiation (AI), appressorium maturation (AM) and cAMP-induced appressoria (CI). Differential expression of each gene is indicated in color (red shows induced, green shows repressed, and numbers next to scale indicate fold change (log2)). (b) Correlation coefficient for pairwise gene expression profiles shown in (a).
Figure 3
Figure 3
RT-PCR and qRT-PCR analysis of gene expression. (a) RT-PCR using RNA isolated from spores germinated on the hydrophobic (Pho12) and hydrophilic (Phil12) surfaces of GelBond after 12 h incubation compared to expression fold change (FC) derived from microarray data from the same time point. (b) qRT-PCR analysis of MPG1 and PTH11 using RNA from appressoria induced by cAMP (+cAMP) and germinating spores (-cAMP) after 9 h incubation on the hydrophilic surface of GelBond. Gene expression fold changes for MPG1 and PTH11 were 0.2 and 0.2, respectively, in our cAMP microarray study.
Figure 4
Figure 4
Functional categorization of appressorium consensus genes. (a) Appressorium associated expression profiles were combined and 240 up-regulated and 117 down-regulated genes were designated as appressorium consensus genes (in italics). Abbreviations are same as in Figure 2. (b) Up-regulated (in pink) and down-regulated (in blue) genes were grouped according to their putative function.
Figure 5
Figure 5
Vegetative growth of the SPM1 deletion mutant on various nutrient sources. SPM1 deletion mutant (Δspm1), ectopic strain and wild type 70-15 (WT) were incubated on solidified complete media (CM), oatmeal media (OA), V8 media (V8) and minimal media (MM) for seven days. Results shown are typical for all four independent SPM1 deletion mutants.
Figure 6
Figure 6
Appressorium formation and pathogenicity of targeted gene deletion mutants.
Figure 7
Figure 7
Vegetative growth of Mgd1 deletion mutants on various nutrient sources. (a-h) Wild type 70-15 (a-d) and Mgd1 deletion mutant (e-h) were grown on minimal media (a,e), complete media (b,f), minimal media with Tween 20 (c,g) or polyethylene glycol (d,h) as carbon source for seven days. Results shown are typical of all four independent Mgd1 deletion mutants. Results for ectopic strains were similar to wild type (data not shown). (i-k) Mgd1 deleted mutants (ΔMgd1a, ΔMgd1b), ectopic strains (ectopic a, ectopic b) and wild type 70-15 (WT) were grown for seven days on minimal media (0.125% glucose) with glutamine (i) and glutamic acid (j) as nitrogen source and minimal media (1% glucose) (k) as depicted in (l). Photographs in (i-k) were taken on a light box to highlight differences in mycelial density.

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