Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants.

MAIN CONCLUSION: Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins. A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling pathways.

Isolation of defense-related genes differentially expressed in the resistance interaction between pepper fruits and the anthracnose fungus Colletotrichum gloeosporioides.

Unripe mature green fruits of pepper (Capsicum annuum) are susceptible to Colletotrichum gloeosporioides, whereas ripe red fruits are not. We established this pepper-C. gloeosporioides interaction as a model system to study the fungal resistance that develops during ripening of nonclimacteric fruit. Histochemical examination of transverse sections suggested that fungal invasion 24 h after inoculation (HAI) and colonization 48 HAI are critical events that differentiate between resistant and susceptible interactions. Based on this observation, we used messenger RNA differential display to isolate defense-related genes differentially expressed at 24 and 48 HAI. RNA gel blot analysis showed that six out of eighty cloned cDNAs were differentially expressed after infection of ripe fruit. The proteins encoded by these six clones, ddP1, ddP3, ddP4, ddP6, ddP13, and ddP47, showed significant homology to aldehyde dehydrogenase, P23 protein, NP24 protein, cytochrome P450 protein, esterase, and MADS-box protein, respectively, and may be involved in the resistance of ripe fruit to C. gloeosporioides infection.