Identification of Defense-Related Rice Genes by Suppression Subtractive Hybridization and Differential Screening

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Abstract
Identification of host genes involved in defense responses is one of most critical steps leading to the elucidation of disease resistance mechanisms in plants. In this study, two different cloning strategies were employed to identify defense-related genes from a tropical japonica rice cultivar (Oryza sativa cv. Drew). With the use of bacterial colony arrays, differential screening of a blast fungus (Pyricularia grisea)-induced rice cDNA library led to the isolation of 22 distinct rice genes that are expressed differentially in response to blast infection. Sequence analysis indicates that most of them are full-length cDNAs encoding pathogenesis-related proteins or other relatively abundant proteins. In combination with treatments of cycloheximide plus jasmonic acid (JA) or benzothiadiazole (BTH) in rice seedlings, the polymerase chain reaction-based suppression subtractive hybridization also was conducted to search for immediate early (IE) defense-related genes whose transcription is independent of de novo protein synthesis. The initial screening of only 768 subtracted clones resulted in the identification of 34 distinct IE genes that are induced by JA, BTH, and/or blast infection. Database searches revealed that these IE genes encode putative mitogen-activated protein kinase, diacylglycerol kinase, zinc finger protein, RelA-SpoT protein, ankyrin-containing protein, ABC transporter, beta-ketoacyl-CoA synthase, and other potential defense-signaling components. Further characterization of these novel IE genes will likely facilitate the elucidation of defense signal transduction in rice plants.