Functional analysis of soybean proteinase inhibitor genes and cyst nematode-inducible synthetic promoters for insects and nematode-resistance in plants

Proteinase inhibitors (PIs) from legumes have the potential for use as protectants in response to pests and pathogens. Soybean (Glycine max) contains two trypsin inhibitors (TIs): Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. In addition to the three known TIs (KTI1, KTI2 and KTI3), novel inhibitors KTI5, KTI7, and BBI5 were identified in soybean. Their functional role was further examined by overexpression in soybean and Arabidopsis. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf feeding bioassay against corn earworm (Helicoverpa zea) showed significant reduction in larval weight in transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with corn earworm on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, soybean cyst nematode (SCN) bioassay of KTI7 and BBI5 overexpressing lines showed no effect between transgenic and non-transgenic plants. Furthermore, the growth and productivity study showed no significant differences between transgenic and non-transgenic plants under greenhouse conditions. The present study provides further insights into the potential applications of TI genes for insect resistance improvement in plants.

The functionality of two SCN (Heterodera glycines)-inducible synthetic promoters (4×M1.1 and 4×M2.3) was studied in whole transgenic soybean. Transgenic soybean plants containing the individual synthetic promoter::GUS construct revealed no GUS activity in leaves and roots under unstressed condition. While upon nematode infection, the synthetic promoters direct GUS expression to roots predominantly in the nematode feeding structures induced by the SCN and by the root-knot nematode (RKN), Meloidogyne incognita. Furthermore, this study examined the specificity of the synthetic promoters in response to various biotic (insect, and three bacterial species), abiotic (dehydration, salt, cold, wounding), and phytohormones (salicylic acid, methyl jasmonate, and abscisic acid) stresses in the transgenic plants. These wide-range analyses provide insights into the potential applications of synthetic promoter engineering for conditional expression of transgenes for resistance improvement in plants.