This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
This summer, I worked with Dr Pierre Buscaill at the University of Bristol to explore the effects of glutamate (Glu) on plant immunity in Nicotiana benthamiana (Nb). I employed various techniques, including in vitro microbiology experiments, in planta phytopathology assays, and bioinformatic analyses.
Glutamate, an amino acid present in all plant cells, is released into the extracellular space (the apoplast) upon cell damage caused by environmental stress or pathogen infection. There, it acts as a signalling molecule recognised by transmembrane glutamate-like receptors (GLRs), which activate defence-related pathways. Exogenous Glu treatment enhances pathogen resistance in several plants, but its role in Nb immunity remains understudied. This project aimed to understand how Glu affects the defence responses of Nb against the model pathogen Pseudomonas syringae (Ps).
To determine whether GLRs are conserved in Nb and to compare them with GLRs in other plants, we conducted phylogenetic analyses on 19 GLR protein sequences from Arabidopsis and 12 from tomato, as well as 13 sequences from Nb. The protein sequences were aligned, and phylogenetic trees were constructed using the Maximum Likelihood and Neighbour-Joining methods, rooted with prokaryotic glutamate receptor GluR0 and bacterial periplasmic amino acid-binding protein GlnH. The results showed that GLRs are conserved in Nb, and likely involved in Glu signalling.
We assessed the effect of Glu-induced signalling on local and systemic resistance in Nb by evaluating defence responses to Ps infection and its flagellin-derived peptide, flg22. This was done by syringe-infiltrating leaves with Glu or soaking plant pots in Glu. Control plants were treated with water.
To investigate the effect of Glu on intracellular immunity, we measured ion leakage, an indicator of hypersensitive response (HR), following Ps infection. Leaves treated with bacteria showed significantly more ion leakage than untreated leaves, indicating that the differences were due to Ps. At 50 mM Glu, significant differences between water- and Glu-treated plants were observed in leaf-treated but not root-treated plants. At 200 mM Glu, treated leaves showed higher conductivity, indicating that higher Glu concentrations have significant effects on cell death.
To study the effect of Glu-induced signalling on cell surface immunity, we measured reactive oxygen species (ROS) burst using a luminol-based assay. Leaf discs from Glu- and water-treated plants were exposed to varying concentrations of flg22, and chemiluminescence was measured using a microplate reader. Glu treatment caused a more pronounced ROS burst compared to water-treated leaves when flg22 was present, while no ROS burst was observed in the absence of flg22.
Finally, we examined whether Glu could affect Ps colonisation. Leaves infiltrated with 50 mM Glu or water were inoculated with bacteria at six spots. Infected sites were examined using a fluorescence microscope. Glu-treated leaves had slightly more infected sites and larger lesions, indicating that Glu might enhance pathogen spread through the vascular system.
In summary, Glu influences both extracellular and intracellular immunity and affects Ps spread. Future research will focus on further understanding these molecular mechanisms and their correlation with resistance.
This internship enhanced my laboratory skills and provided valuable experience in plant pathology research. I am grateful to Dr Pierre Buscaill, the Plant Pathology Lab, and BSPP for this opportunity.
Katy Cheung
University of Bristol
Cover image: Effect of Glu on Ps colonisation of distant tissue. Nb leaves were infiltrated with 50 mM Glu or sterilised water, left for 24 hours, and then inoculated with Ps using pipette tips. Leaves were examined under a fluorescence microscope 7 days post-infection.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
