Duplication and transcriptional divergence of three Kunitz protease inhibitor genes that modulate insect and pathogen defenses in tea plant ().

Kunitz protease inhibitors (KPIs) are ubiquitous in plants and act as crucial compounds in defense responses against insect attack and pathogen infection. However, the influence of gene duplication on the postdivergence of the genes involved in biotic stresses in tea plant is not well known. Here, we identified three genes from tea plant () and characterized their expression and evolutionary patterns among plant species. We found that , , and diverged from their common ancestor 72.94 million years ago (MYA), and the tandem duplication of and occurred 26.78 MYA. An in vitro protein assay showed that the three CsKPI proteins were functional and inhibited the production of -nitroanilide (PNA) from an artificial substrate. The three CsKPI-GFP fusion proteins localized to the cytoplasm. We showed that salicylic acid (SA) and transcripts of and significantly accumulated after infection with . The application of exogenous SA stimulated the high expression of both and by activating -elements within their promoters. Under attack, expression and jasmonic acid (JA) levels were more abundant in both insect-damaged leaf tissues and undamaged neighboring leaves. The application of jasmonic acid methyl ester elicited high expression levels of , suggesting that accumulation requires JA production in tea plant. The overall findings suggest that the transcriptional divergence of KPI genes after duplication led to the specialized role of in the physiological response to insect stress; the functional conservation between and confers resistance to pathogen infection in tea plant.