ABSTRACT: Hpa1Xoo-Mediated Transcriptome in Transgenic Cotton Reveals the Constitutively Expressed Diverse Defense genes in Multiple Signaling Pathways Associated with Hypersensitive Cell Death Like other harpins, harpinXoo enables plants to acquire multiple resistance to pathogen and insect attack. However, the molecular model is not fully understood, especially in transgenic plant harpinXoo for genome-wide constitutive expression. Here, we showed that 530 cDNAs differentially expressed in transgenic hpa1Xoo-harboring cotton (T-34) compared to its receiptor (Z35), through analyzing the transcriptome profile in a customized cotton 12k cDNA microarray, which was enriched in 34 pathways. Among them, 123 genes were identified from T-34 as hypersensitive reaction-mediated defense genes, involved in reactive oxygen species-, salicylic acid-, jasmonates-, ethylene-, auxin-, abscisic acid-, and Ca2+-mediated signaling pathways, and we uncovered various components of defense responses associated with recognition of the pathogen-derived elicitor. Apart from elevated genes for basic defense, harpinXoo activated leucine-rich-repeat plant receptor kinases and mitogen-activated protein kinase cascade to strengthen downstream defense responses, including production of antimicrobial compounds in T-34. Defense genes from T-34 were expressed at a much higher level in response to pathogen infection compared with Z35. Simultaneous up- and down-regulation in differentially-expressed genes, and increased expression of genes encoding energy producing and consuming pathways, suggested that energy balance was maintained in the genetically modified cotton without biotic stress. High-energy demand only occurred following pathogen infection. Using kanamycin-resistance tests, positive plants were successfully screened from transgenic cotton T-34 T6 progenies; all the receiptor Z35 plants gave negative results. We randomly selected three positive plants for polymerase chain reaction (PCR) analysis; the different positive bands (420, 310 and 180 bp) were confirmed as harpinXoo, the 35s promoter, and the NOS terminator, respectively, by sequencing and BLAST searches (www.ncbi.nlm.nih.gov.). For further analysis, the positive results were confirmed by Southern and western blots. Three positive bands were obtained in each T-34 sample located at about 4, 6 and 10 kb in the Southern blot. This pattern suggests that T-34 incorporated the harpinXoo transgene at multiple chromosomal locations. Western blot analysis showed that the harpinXoo protein was constitutively expressed in T-34 transgenic lines, whereas it was barely detectable in Z35 (data not shown). The transgenic plants contained a selectable ‘marker’ gene, neomycin phosphotransferase II (nptII). Previous research showed that expression of marker genes (e.g. nptII, uidA, hph and uidA-gfp) did not contribute to visible phenotypes in transgenic plants. The non-targeted transgenic and non-transgenic plants were equivalent in their global patterns of transcription (El Ouakfaoui and Miki, 2005). Thus, the present study examined the many differences between T-34 and its receiptor Z35 in regard to the whole genome expression of G. hirsutum using microarrays to monitor changes in individual gene expression level effected by harpinXoo. All experiments were conducted using the 12k cotton cDNA microarray, and cotton true leaves and roots at the 4–5-true-leaf-stage. The use of this microarray enabled a quantitative approach to examine changes in transcript level for all genes within the G. hirsutum genome; as a result, inferences can be drawn regarding the molecular effects of transgenic cotton expressing harpinXoo. There were three biological replicates in the experiment for each organ.
