Abstract
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 CsKPI genes involved in biotic stresses in tea plant is not well known. Here, we identified three CsKPI genes from tea plant (Camellia sinensis) and characterized their expression and evolutionary patterns among plant species. We found that CsKPI1, CsKPI2, and CsKPI3 diverged from their common ancestor 72.94 million years ago (MYA), and the tandem duplication of CsKPI2 and CsKPI3 occurred 26.78 MYA. An in vitro protein assay showed that the three CsKPI proteins were functional and inhibited the production of p-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 CsKPI2 and CsKPI3 significantly accumulated after infection with Glomerella cingulata. The application of exogenous SA stimulated the high expression of both CsKPI2 and CsKPI3 by activating cis-elements within their promoters. Under Ectropis oblique attack, CsKPI1 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 CsKPI1, suggesting that CsKPI1 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 CsKPI1 in the physiological response to insect stress; the functional conservation between CsKPI2 and CsKPI3 confers resistance to pathogen infection in tea plant.
Highlights
The tea plant (Camellia sinensis) is an important economic woody crop that is cultivated worldwide[1]
The full-length sequence of CsKPI2 was obtained by rapid amplification of cDNA ends (RACE)
We further observed a high amino acid sequence similarity (75%) between CsKPI2 and CsKPI3, but CsKPI1 was less similar to CsKPI2 (53%) and CsKPI3 (51%); this suggested that CsKPI1 may have functionally diverged from CsKPI2 and CsKPI3
Summary
The tea plant (Camellia sinensis) is an important economic woody crop that is cultivated worldwide[1]. Protease inhibitors (PIs) are present in almost all organisms, including animals, plants, and microorganisms; they play key regulatory roles in a wide range of biological processes[8,9]. They function by directly or indirectly blocking active centers of target proteases to strictly control their activities[8,10]. Kunitz PIs (KPIs) are members of a subfamily of PIs involved in biotic defense; they function by repressing the digestive processes of insects and reducing fungal lesion development[9,14]. A serine PI (Kunitz-type inhibitor designated PKI1) isolated from potato showed direct inhibitory activity on Botrytis cinerea strains as reported by Hermosa et al.[16]
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