Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2018
Publication Date: 5/15/2019
Citation: Novak, N.G., Perez, F.G., Jones, R.W., Lawrence, S.D. 2019. Detached leaf assays: A simplified approach to study gene expression in potato during infestation by the chewing insect Manduca sexta. Journal of Visualized Experiments. 147:e59153.
Interpretive Summary: Insect damage to crops is a leading cause of yield loss to agriculture in the US. Discovery of genes that can enhance resistance to insects is key for agricultural crop improvement. This work describes a detached leaf assay that can quickly identify new genes that respond to infestation. The assay requires less space, resources and personal than the typical whole plant assay. Six newly discovered regulatory genes were tested to determine their response to insect feeding. These genes belong to a family with several members known to enhance tolerance to specific stresses such as cold, drought or high salt soils. The goal of this work was to test whether the 6 newly identified members of this family responded to feeding by Manduca sexta. Indeed 2 new candidates were identified for further study using this assay. This assay will be beneficial to researchers interested in understanding the plants natural tolerance to insect pests and breeding plants with more resistance.
Technical Abstract: The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression. The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified. Potato variety Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation specific C2H2 transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.