Identification and analysis of in planta expressed genes of Magnaporthe oryzae

Abstract

Background: Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results: A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion: This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.

Figure 1

Genome wide distribution of infection ESTs. EST sequences were mapped to M. oryzae (A) or rice (B) chromosomes.

Figure 2

Gene Ontology (GO) annotation of fungal (A) and plant genes (B) expressed during their interaction. The ratio of hits to Biological Process is indicated. Fungal and rice ESTs were used to search matching ORFs from genome and full-length cDNA sequences, respectively. Translated ORF sequences were subjected to InterPro and GO analyses.

Figure 3

Process to search novel genes uniquely or preferentially identified during rice-blast fungus interactions.

Figure 4

Expression of infection EST specific fungal genes analyzed by real-time RT-PCR. Relative abundance and fold changes were calculated as described in Experimental Procedures. Cyclophillin was used as an internal control. (A) Fold changes during infectious growth were compared relative abundance during growth in complete medium. (B) Fold changes during infectious growth, conidiation, conidial germination, and nutrient starvation conditions were depicted as log scale.

Figure 5

Five types of errorneous gene model were illustrated. Orange bar represents exon region translated from cDNA sequences, blue bars represent exon region, black bars 5' and 3' untranslated region, and line intron region, respectively. Extremely long intron was described as dotted line in type III.

Figure 6

Intron length distribution and splicing site context. (A) Intron length distribution of 68 full-length cDNA sequences. (B) Splicing site consensus sequences for 5' exon-intron junctions (upper), branch point (middle), and 3' intron-exon junctions (lower) were calculated using WebLogo server at http://weblogo.berkely.edu. The general consensus sequences of each region were displayed in order of predominance from top to bottom at each position with large letter being higher frequency.