Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

This protocol uses a streamlined approach for measuring plant gene expression in response to insect herbivory. By using detached leaves isolated in Petri dishes, the insects are easier to apply and monitor during the relatively short period of infestation contributing to reproducible gene expression data. This protocol may be adapted to many plant/insect interactions.

if baseline observations from whole plant systems are considered. The unpredictable nature of insect feeding can be challenging. Therefore, a dependable supply of properly staged larvae and healthy plants provide the best chance of success.

Visualizing how healthy plants and insects should appear and behave is instrumental to achieving optimal results especially for researchers new to the field of study. Demonstrating the procedure with me will be Frances Perez, a molecular biologist in the genetic improvement of fruits and vegetables lab. To prepare nodal propagated tissue culture potato plants, first obtain Kennebec plantlets grown from explant material with at least three to four nodes.

Use a sterile blade to remove the leaves cutting the branches close to the main stem and leaving about two millimeters of branch tissue per leaf. Cut approximately two millimeters above and below each node to remove the nodal sections from the stems and arrange the nodal cuttings in a sterile tissue culture vessel containing nodal transfer medium with the branch pointing up. Then transfer the nodal cuttings to a plant tissue culture chamber for to two to three weeks of growth at 24 degrees Celsius and a 16-hour light, eight-hour dark photo period.

To prepare the insects for feeding, obtain the desired larval stage of M.sexta and transfer one larva into each well of an appropriate containment vessel according to the size of the larva. Make placement templates for each harvest time point using five sturdy trays capable of holding a set of six appropriately sized Petri dishes lined with white paper. Trace a set of six circles using the appropriately sized Petri dish on the paper in each tray and label one set of circles control A, B, and C and the other infested A, B, and C.Then label each placement template with the appropriate harvest time.

Next, label one 1.7 milliliter microcentrifuge tube for each circle in the placement template to appropriately identify the perturbation, plant replication letter and harvest time point. When all of the tubes have been labeled, place a sterile filter paper disc into one Petri dish per template and moisten the discs with sterile water without letting excess liquid pool in each dish. Then place one dish into each circle in each placement template and place three potato plants of the same age and relative size next to each placement template.

To initiate the infestation, use sterile scissors to remove the top two size matched leaves from each plant and place one leaf in the control Petri dish and one in the infested Petri dish for each plant as quickly as possible. When all of the leaves have been placed, use soft touch forceps to transfer at least one larva into each infested dish as quickly as possible and set the timer for the desired infestation time. Observe the feeding to make sure that all of the larvae are eating, replacing any non-feeding larvae as necessary.

Remove all of the larvae from all of the leaves at the end of the infestation time and start the timer for the harvest. At the end of each harvest time point, transfer each leaf into the corresponding tube and immediately drop the tube into liquid nitrogen to freeze the leaf sample before storage at minus 80 degrees Celsius until RNA isolation. Healthy accurately staged larvae should begin feeding immediately after placement on the leaf surface and the feeding should continue in a fairly consistent manner throughout the infestation time period.

The larva at the bottom of this leaf did not consume any plant material and is an example of an unsuccessful infestation. These leaves were detached from two-week-old nodal propagated tissue culture plants and consumed at different rates and feeding styles for each larval stage. Based on these results, fourth instar larvae were selected to further assess damage to leaves for more mature soil grown potato plants that more closely approximate field grown potato plants.

In these gene expression studies, two new C2H2 zinc finger transcription factors that are robustly responsive to M.sexta herbivory were identified supporting the use of detached leaves for infestation assays. Remember to use age and size matched leaves and to properly stage the larvae for each experiment as uniformity results in more reproducible data. Leaf tissues can be assayed for stress-inducted reactive oxygen species and jasmonic acid hormone derivatives.

Whole transcriptome, proteome, or microbiome studies can also be performed on the leaf tissues.