Characterization of Defense-Associated Gene Expression in Phytolacca Americana Using Novel Transcriptomic and Genomic Resources
Neller, Kira Catherine Maxine
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Phytolacca americana (pokeweed) is a non-model plant with agriculturally relevant characteristics, being a heavy metal hyperaccumulator resistant to virus infection. Pokeweed synthesizes pokeweed antiviral protein (PAP), an RNA glycosidase thought to function in defense. Study of this plant, and of the biological relevance of PAP, has been hindered by a lack of reference sequences. Here, the first mRNA transcriptome, miRNA transcriptome, and de novo genome assembly of pokeweed are reported. The mRNA transcriptome was initially built using plants subjected to jasmonic acid (JA), a hormone that mediates resistance to pathogens. Using this resource, ~59,000 transcripts and ~52,000 natural antisense transcript (NAT) pairs were annotated. Differential expression analysis revealed that many transcripts were JA-responsive, including several PAP isoforms and PAP-specific NAT pairs. The annotated mRNA transcriptome was also used to investigate miRNA control of JA response. Novel miRNA/target pairs involved in hormone biosynthesis, signal transduction, and pathogen defense were predicted. One of these comprised a transcript encoding a JA biosynthesis enzyme, which was validated, representing the first reported miRNA-induced cleavage event of a non-transcription factor target of the JA-response pathway. For approximately half of the pairs, expression of the miRNA and target was positively correlated, suggesting a biologically relevant regulatory dynamic. To gain additional knowledge of the pokeweed defense response, a comprehensive mRNA transcriptome was obtained from plants subjected to different stresses. This transcriptome was used to annotate the first pokeweed de novo genome assembly, revealing the structures of ~30,000 genes. Several known PAP isoform genes were identified, as well as a putative novel isoform and PAP pseudogenes. The gene model of each PAP isoform contained a long intron in the 5 untranslated region, and preliminary evidence suggests it regulates gene expression. Differential expression analysis confirmed that in addition to JA, several PAP isoforms responded to other stresses. PAP isoforms were co-expressed with genes involved in terpenoid biosynthesis, JA-mediated signalling, and metabolism of amino acids and carbohydrates. Taken together, this study generated the first large-scale sequence references for pokeweed and provided insight into the regulation of defense-associated gene expression in this non-model plant.