Over 10 weeks this summer I carried out a research project investigating the role of chloroplasts in plant immunity at the University of Warwick with Dr Susan Breen. During this time, I had three avenues of investigation each of which allowed me to learn different techniques used in molecular plant pathology laboratories. These were cloning of Pseudomonas syringe effectors into expression vectors, screening of transgenic Arabidopsis thaliana and determining the effect of P. syringe on pep receptor knock out A. thaliana lines.
In my first project, I aimed to use the novel split GFP system recently developed by Park et al, (2017) to observe effector localisation in plant cells, specifically chloroplasts, and the effect on plant immunity. The GFP contains 11 β-sheets, which this new split GFP system breaks into two components; the first 10 sheets (GFP1-10) and last 11th sheet (GFP11). GFP1-10 is localised to specific organelles and transgenic A. thaliana lines have been generated. The GFP11 is tagged to the effector of interest, and when the effector localises to the organelle containing GFP1-10 the full-length protein reconstitutes and fluorescence occurs. This allows GFP tagging when investigating effectors secreted via the type 3 secretion system (T3SS) as the 11th GFP sheet is small enough to fit through the secretion machinery. The project outline was to clone three P. syringe effectors (HopN1, HopC1 or HopO1-2) into the GFP11 plasmid and subsequently transform these into a virulent strain of P. syringe. These strains would then be infiltrate into transgenic A. thaliana to assess the localisation of the effectors.
Gateway cloning was used to clone effectors into the GFP11 tagging plasmid. HopN1 and HopC1 had already been cloned into a gateway entry vector, so HopO1-2 was cloned from P. syringe gDNA and cloned into a gateway entry vector. HopO1-2, HopC1 and HopN1 were then cloned into the GFP11 tagging destination plasmid from Park et al, 2017. These plasmids were sequenced to ensure the correct gene was in our destination vector. Sadly, time didn’t allow for these plasmids to be transformed into P. syringe to assess the localise of the effectors within a plant cell.
My second project focused on a set of transgenic A. thaliana that had previously been generated. Previous analysis of a gene expression study highlighted a few plant genes that were induced in the presence of virulent P. syringe (DC3000) but not induced in the presence of non-virulent P. syringe (hrpA mutant). Therefore, in these transgenic plants GFP expression was driven by the promoters of these DC3000 induced gene. My role was to determine if this initial observation held true and screen for plants that showed high induction of GFP during DC3000 infection and no induction during hrpA mutant infection. A stereo fluorescence microscope was used to determine if GFP induction was occurring. Some plants that showed induction of GFP were also imaged using the confocal microscope to identify the GFP localisation. The plants with fluorescence were grown up to seed and harvested, whereas those that did not were discarded.
The last set of experiments I worked on was to investigate the effect on chlorophyll fluorescence of DC3000 and hrpA on Pep receptor (PepR) knock out plants. Pep1 is a plant derived peptide that is highly produced during wounding and plant infection. PepR1 and PepR2 are the receptors for Pep1 and detect the increased presence of Pep1, leading to induced immune response. A. thaliana PepR knock-out and Col-0 were infiltrated with DC3000 and hrpA mutant to determine what effect the loss of these receptors had on chlorophyll fluorescence and overall plant immunity. These plants were also tested to determine what pre-treatment with AtPep1 and Pep3 had on disease progression.
Over the course of the 10 weeks I carried out a lot of different techniques, including cloning, PCR bacterial infections, florescence microscopy, confocal microscopy and chlorophyll fluorescence analysis. Other techniques I also learn while in the laboratory were western blot and Dex-inducible experiments. Sadly, I did not have enough time to carry out everything outlined in my project, however I have learned a lot of incredibly valuable skills that I hope to use and improve upon in the future. I am very grateful to BSPP for providing me with this opportunity and thankful for Dr Susan Breen for being my supervisor and guiding me through my project.
University of Warwick
GFP fluorescence of transgenic A. thaliana 2267013_GFP strain imaged using confocal microscope.