Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction

Abstract

To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.

Figure 1.

Overlapping of unique rice EST sequences from control, resistant, and susceptible libraries at 24 h after inoculation with rice blast fungus using the advance search function under the RICE EST PAVE page on MGOS Web site.

Figure 2.

Functional categorization and percentage of rice ESTs based on their putative function using the KOGs protein database. A total of 68,920 EST sequences from 8 cDNA libraries were submitted to the KOG program to predict the putative functional classification of individual proteins.

Figure 3.

Northern-blot confirmation of 10 EST clones selected from the defense and signal transduction mechanism categories. About 10 μg of total RNA from susceptible and resistant reactions of Nipponbare plants at 6, 12, 24, and 72 h after inoculation were used in northern-blot analysis. For the susceptible reaction, Nipponbare plants were inoculated with rice blast strain Che86061. For the resistant reaction, Nipponbare plants were inoculated with rice blast strain C9240-1. RNA loading of each sample was verified by the intensity of ribosomal RNA bands on the agarose gel.

Figure 4.

Hierarchical clustering analysis of differentially expressed transcripts. Contigs with an R > 15 (434 in total) were used for hierarchical clustering analysis. A frequency of zero is indicated by black and a frequency increase is indicated by increasing intensities of red. The library information is summarized in Table I.