Transgenic sugarcane overexpressing CaneCPI-1 negatively affects the growth and development of the sugarcane weevil Sphenophorus levis.

KEY MESSAGE: Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification.

Transgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large-scale production of proteins. In this work, we present the establishment of transgenic sugarcane expressing a His-tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of (HIS)CaneCPI-1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified (HIS)CaneCPI-1 was immunodetected using a polyclonal antibody, which was also able to detect the (HIS)CaneCPI-1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified (HIS)CaneCPI-1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production.

Mechanisms of sugarcane response to herbivory

Deciphering plant-insect interactions at the molecular level is one of the major topics of interest in contemporary plant biology research. In the last few years, various aspects of the plant response to insect damage have been investigated, including the characterization of direct and indirect responses, the regulation of gene expression resulting from insect attack and the signal transduction pathways. Such research has resulted in the proposal of new methods to enhance host resistance to insect pests, including the use of insecticidal genes that can be transferred by genetic engineering into target crops. By integrating the understanding of how plants react to insect damage with the techniques of molecular biology researchers should be able to increase the wide range of methods available for the control of insect pests. The sugarcane transcriptome project (SUCEST) has allowed the identification of several orthologues genes involved in the plant response to insect damage. In this paper we summarize several aspects of the complex interaction between plants and insects and describe the use of in silica analysis to provide information about gene expression in different sugarcane tissues in response to insect attack.