Novel-iridoviral kinase induces mortality and reduces performance of green peach aphids (Myzus persicae) in transgenic Arabidopsis plants

Abstract

Insect pests are a serious threat to agricultural productivity. Genetically-modified crops offer a promising alternative, and Bacillus thuringiensis endotoxin genes have played a major role in this respect. However, to overcome insect tolerance issues and to broaden the target range, it is critical to identify alternative insecticidal toxins working through novel mechanisms. In our previous study, a kinase from Chilo iridescent virus (CIV) that has insecticidal activity was identified and designated as Iridovirus Serine/Threonine Kinase (ISTK). A 35 kDa truncated form of ISTK, designated iridoptin, was obtained during expression and purification of ISTK in the yeast system. This yeast-expressed CIV toxin induced 50% mortality in cotton aphids and 100% mortality in green peach aphids (GPA). In this study, codon optimized ISTK gene and iridoptin fragment (designated oISTK and oIRI, respectively) were designed by altering the codon usage features that are seldom present in plant exons. Codon-optimized gene(s) cloned into plant expression vectors were used to stably transform Arabidopsis plants. PCR analysis of genomic DNA of transformed plants confirmed the presence of the DNA insert (oISTK/oIRI) in selected transgenic lines. Further screening was performed by selecting the PCR positive lines, which showed expression of respective toxins at the polypeptide level, using Western blot analyses. Codon-optimized gene constructs resulted in significant improvement in levels of expression of ISTK and iridoptin polypeptide and confirmed its stability in planta. The stable lines expressing either of the two toxin forms induced moderate to very high mortality in GPAs and significantly affected GPA development and fecundity.