Subtractive hybridization-assisted screening and characterization of genes involved in the rice-Magnaporthe oryzae interaction

Abstract

Abstract Transcription profiling assays have revealed substantial changes in gene expression during plant-microbe interactions, but it is often time- and labor-consuming to define the causative roles of the differentially expressed genes that finetune the plant responses to diverse pathogens. We improved the duplex-specific nuclease-mediated transcriptome subtraction method and integrated it with SMART cDNA library construction technology to generate normalized libraries consisting of full-length cDNAs derived from transcripts upregulated in the rice-Magnaporthe oryzae interaction. By adding BP recombination sites to the sequence of primers used for the synthesis of full-length cDNAs, we were able to transfer the full-length cDNAs of the library to an expression binary vector with CaMV 35S promoter, which allowed a subsequent screening for candidate genes potentially involved in the disease process by Agrobacterium-mediated transient assay. Results showed that the newly established approach preferentially suppressed the cDNA abundance of most constitutive genes and enriched or normalized that of the upregulated ones. Subsequent screening with transient expression in planta isolated 61 clones, which carry full-length cDNAs of 32 distinct genes, were capable of causing cell death on Nicotiana benthamiana. When transiently expressed in barley leaf, five of the identified genes were able to induce chlorosis, and additional 11 genes were found to promote disease symptoms caused by infection with the blast pathogen while the others did not enhance the symptoms of pathogen infection. These observations demonstrate that the subtractive hybridization-assisted functional screening is an efficient approach that provides initial leads to the role of candidate genes in the complex process of plant-microbe interactions.

Keywords

• Duplex-specific nuclease
• Subtractive hybridization
• Pathogenesis-related genes
• Functional screening
• Transient expression
• Cell death