Published Protocols

Our lab members have varied backgrounds and regularly make use of innovative interdisciplinary techniques. In the past and in our ongoing research we have established and adapted a range of protocols for molecular plant pathology work, sometimes in collaboration with other research facilities such as OMCS, ISIS Neutron and Muon Source and Diamond Light Source. Please find below examples of some of our published protocols to date. 

• Detection of salicylic acid in vitro and in vivo: In collaborative work with Wei Huang we have shown that a luminescent SA biosensor can be used to monitor SA production in planta during bacterial and viral infection. Agroinfiltration reduces ABA levels and suppresses Pseudomonas syringae-elicited salicylic acid production in Nicotiana tabacum; Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1. 
• Studying nutrient assimilation in complex environments: We have used various approaches to study bacterial nutrient assimilation in complex environments. We have used Biolog Phenotype Microarray Plates in conjunction with pre-culture in selected environments and subsequent antibiotic treatment to demonstrate which nutrient assimilation pathways are active in that environment. Pseudomonas syringae pv. tomato DC3000 Uses Constitutive and Apoplast-Induced Nutrient Assimilation Pathways to Catabolize Nutrients That Are Abundant in the Tomato Apoplast (apsnet.org). We have also demonstrated the effectiveness of metabolite footprinting techniques in studying bacterial nutrient assimilation in the apoplast. Early changes in apoplast composition associated with defence and disease in interactions between Phaseolus vulgaris and the halo blight pathogen Pseudomonas syringae pv. phaseolicola.
• Apoplast extraction - JOVE: This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example. The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using Phaseolus vulgaris as an example
• Monitoring metabolite uptake and assimilation: Isotope-labelled metabolites can be used to study the uptake and metabolism of specific compounds, however such studies are often limited by access to labelled metabolites. Here we show that it is feasible to label whole cells rather than metabolites by culturing them on a labelled carbon source and then track the uptake and incorporation of unlabelled metabolites. GABA (γ-Aminobutyric Acid) uptake via the GABA permease GabP represses virulence gene expression in Pseudomonas syringae pv. tomato DC3000.
• Predicting metabolic networks. Our work on metabolism in plant-pathogenic bacteria necessitated the creation of tools and approaches for comparing predicted metabolic networks. In collaborative work with Jotun Hein, we created a software tool called Rahnuma along with approaches to predict and study metabolic networks, which we applied to plant pathogenic Pseudomonas. Aziz Mithani's research group has recently developed an exciting new web-based tool called MAPPS that builds on this work. MAPPS: A Web-Based Tool for Metabolic Pathway Prediction and Network Analysis in the Postgenomic Era. 
• Studying bacterial viability and metabolism at a single cell level using Raman-SIP. Raman microspectroscopy can be used to monitor the abundance of isotopes in cells. In collaborative work with Wei Huang we have shown that whole cell isotope labelling followed by Raman stable isotope probing (Raman-SIP) analyses using unlabelled metabolites or environments enables researchers to study bacterial metabolism and viability at a single cell level. Reverse and Multiple Stable Isotope Probing to Study Bacterial Metabolism and Interactions at the Single Cell Level | Analytical Chemistry (acs.org)
• pTRIC: RNA Interactome Capture (RIC) can systematically and comprehensively identify the proteins that interact with polyadenylated RNAs in living cells. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue using the model species Arabidopsis. Discovering the RNA-binding proteome of plant leaves with an improved RNA Interactome Capture method. 
• Macro to Micro imaging of bacteria in planta: Studying systemic colonisation of plants by bacteria requires both macro (whole plant/tissue) and microscopic imaging of bacterial localisation. This paper describes the development and use of dual bioluminescent and fluorescent reporter systems to monitor bacterial colonisation of plant tissues. From Macro to Micro: a combined bioluminescence‐fluorescence approach to monitor bacterial localization.
• Agromonas: This collaborative paper with Renier van der Hoorn's group shows how transient expression of immunity and infection-related proteins can be combined with P. syringae infection to investigate the role of these proteins in host-pathogen interactions. Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves.
• AgroLux: This joint paper with Renier van der Hoorn's group shows that bioluminescent agrobacteria can be used as indicators of whether conditions are favourable for protein expression by agroinfiltration and to study plant immune responses. AgroLux: bioluminescent Agrobacteria to improve molecular pharming and study plant immunity.
• High-throughput analysis of bacteria-nematode interactions: The model nematode Caenorhabditis elegans preferentially feeds on some bacteria over others, and finds the mushroom pathogenic bacterium Pseudomonas fluorescens NZI7 very unpalatable. Here we describe a high-throughput screen for mutants of NZI7 that have lost the ability to deter C. elegans from feeding. Pseudomonas fluorescens NZI7 repels grazing by C. elegans , a natural predator. 
• Quantifying biotic-induced stress in plants: We did not invent these techniques - but in this publication we provide protocols for a number of widely used approaches for studying plant immunity and plant-pathogen interactions, including detection of reactive oxygen species using DAB, luminol and NBT, measurement of callose deposition, trypan blue staining to detect cell death and electrolyte leakage to study loss of membrane integrity. Methods to quantify biotic-induced stress in plants.