This summer, I was given the opportunity by the BSPP to work in the laboratory of Dr Graeme Kettles at the University of Birmingham. This project investigated how oak tree seedlings respond to bacterial species associated with Acute Oak Decline (AOD). AOD is an emerging disease of native oaks in the UK, principally affecting Quercus robur and Quercus petraea. The main symptoms of infected oaks are bleeding fissures on the main trunk, which can lead to the death of the tree 4-6 years after infection. At least three different bacteria have been recognised to play a part in AOD, including Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana. Recently, several studies examined how oak trees respond to AOD infection at the transcriptomic level. To produce this data, researchers sampled from mature trees in nature, usually by way of removing tissues samples from disease lesions. This summer project aimed to reproduce these studies on young, glasshouse-grown oak seedlings. If results from field studies can be replicated in lab-grown plants, this would open the possibility to study this complex disease via more controlled and high-throughput experiments. The main objectives of this project were; (i) to develop a method for a glasshouse-based AOD bioassay, and (ii) to develop a protocol to detect oak seedling responses to microbe-associated molecular patterns (MAMPs).
For the first objective, 7-month-old saplings were inoculated with AOD-associated bacteria following artificial wounding to the main stem. At both 5-days and 6-weeks post inoculation, inoculated stem sections were harvested for RNA extraction. Stem samples were ground down using liquid nitrogen and an RNA extraction buffer previously used successfully with woody tissue. RNA was purified using an RNeasy kit and subsequently used as template for cDNA synthesis. Defence-related genes known to be transcriptionally responsive to AOD in natural infections were selected and primers designed for qRT-PCR analysis. For some of these genes it was not possible to accurately measure expression levels between mock- and bacterial infections. However, for some defence-related genes, expression was significantly higher in oak saplings exposed to higher concentrations of bacteria. This confirms the results from mature trees infected in nature.
For the second objective, reactive oxygen species (ROS) burst assays were developed using leaf tissue of Quercus robur. In model plants, such as Arabidopsis thaliana and Nicotiana tabacum, MAMPs can be detected using a luminescence-based assay when pathogen associated molecules are introduced to leaves. Such assays involve placing leaf discs into a 96-well plate and adding an assay mixture containing luminol, horseradish peroxidase (HRP) and MAMP molecules to induce ROS signalling. If the plant does respond, plant-produced ROS stimulate the oxidation of luminol by HRP, resulting in the emission of light, which can be quantified by a plate-reading luminometer, thus indicating that the relevant plant species is able to detect the MAMPs. Three common experimental MAMPs were selected to test the hypothesis, flg22, crude chitin polymers and chitin hexamers. Oak leaf discs were added to a 96-well plate and each column of discs exposed to a different MAMP. Water only controls were also included. The results from these experiments indicated that oak leaves do not respond as strongly as tobacco to the three MAMPs tested.
I would like to send my appreciation to the BSPP for helping fund my summer project. Working in Dr Kettles’ lab has provided me with an invaluable insight into the research environment. Understanding how such labs carry out research, develop ideas and overcome difficulties has inspired me apply for a PhD following graduation.
Chris Griffin
University of Birmingham
