Identification of genes up-regulated in bacterial-blight-resistant upland cotton in response to inoculation with Xanthomonas campestris pv. malvacearum

In this first transcriptome study of the cotton-Xanthomonas campestris pv. malvacearum interaction, clones from a cDNA library were used to identify host genes expressed in upland cotton leaves following inoculation. cDNA was prepared from inoculated and non-inoculated leaves of cotton line Im216, which has exceptionally high, broad and stable resistance to bacterial blight, expressed as a hypersensitive-resistant response. Suppression subtractive hybridization was used to make a normalized cDNA library that is differential between transcripts of inoculated and non-inoculated leaves. More than 2000 clones were generated. They yielded 121 unique non-redundant sequences, consisting of 97 with similarity to sequences submitted to GenBank and 24 without good matches. PCR-amplified cDNAs of the 121 genes, as well as of 13 previously identified genes, were arrayed onto glass slides. These microarrays were used to analyze transcripts in Im216 leaves over the extended period of 8-60 h after inoculation. Microarray analysis indicated that 98% of the genes were significantly up-regulated at one or more of the sampling times. Of these, 63% had sequence similarity to plant genes associated with defense responses, i.e., to genes that function in disease/defense, protein synthesis/turnover, secondary metabolism, signaling, stress/programmed cell death, or code for pathogenesis-related proteins or retrotransposon-like proteins. Seventeen percent of the genes showed highest differential abundance during the first day after inoculation, prior to when the microscopic hypersensitive response is first seen. A majority had their highest differential expression at the latest time observed, 60 h, when healthy cells neighboring dying or dead cells are likely to be the major participants in the response.