Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Abstract

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Keywords: Magnaporthe oryzae; MapMan analysis; defense response; rice; transcriptomics.

Fig. 1

Validation of samples used for the microarray experiment. (A) Expression profiles of defense marker genes in rice leaves infected with rice blast fungus at indicated time points. (B) Accumulation of phytoalexins, sakuranetin and momilactone A in fungal infected leaves were detected by HPLC.

Fig. 2

Analysis of rice transcriptome in response to incompatible (R) and compatible (S) blast fungus infection. (A) Hierarchical clustering and heat-map of gene expression in incompatible and compatible M. oryzae-infected leaf tissues at 12 and 48 hpi. (B) K-mean clustering analysis of gene expression profiles of 608 genes induced by M. oryzae. Acronyms stand for rice gene expression under the following conditions: R/C_12, incompatible sample at 12 hpi; R/C_48, incompatible sample at 48 hpi; S/C_12, compatible sample at 12 hpi; S/C_48, compatible sample at 48 hpi.

Fig. 3

Heat-Map and Gene Ontology (GO) enrichment analysis of highly expressed genes in incompatible type interaction at 12 and 48 hpi. (A) Heat-Map of 261 highly induced genes, which were classified into four sub-groups based on their expression profiles by K-mean clustering analysis. (B) Number of enriched GO in four sub-groups.

Fig. 4

Gene Ontology (GO) enrichment and MapMan analysis of up-regulated genes in incompatible type fungal infection. Number of enriched GO at 12 (A) and 48 hpi (B). Mapman analysis of up-regulated genes related with biotic stresses at 12 (C) and 48 hpi (D). Regulation overview (E and F) and cellular response overview (G and H) of up-regulated genes at 12 and 48 hpi.

Fig. 5

Up-regulation of receptor-like genes in incompatible type interaction. (A) Heat-map of receptor-like genes identified from microarray analysis. (B) Transcriptional expression of receptor-like genes in response to compatible and incompatible type fungal infections at 12, 24, 48, and 72 hpi were confirmed by RT-PCR.