| 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. To date, this is the largest EST collection generated from a single plant-pathogen interaction in plants. Therefore, the sequences reported in this study provide a significant improvement in our understanding of the rice defense mechanism to the rice blast fungus and will streamline the community effort in elucidating the functions of many defense response genes in rice.; Ubiquitination-regulated protein degradation is a common regulatory mechanism that controls a range of cellular processes in eukaryotes. Recent studies showed that ubiquitination might also play an important role in plant disease resistance. We have recently identified 43 rice ubiquitination-related E2 and E3 genes in our microarray analysis. These genes were induced or suppressed in Pi2 and Pi9 resistant plants after rice blast inoculation. The expression patterns of these E2 and E3 genes were confirmed using northern blot and RT-PCR methods. Among these E2 and E3 genes, we chose the homologue (OsSINAT5) of the Arabidopsis SINAT5 for a detailed functional analysis. Northern blot hybridization and RT-PCR confirmed that OsSINAT5 was induced by rice blast infection as early as 6 hr after inoculation. Functional analysis of this gene in resistance to rice pathogens was conducted using reverse genetics and biochemical approaches. |
