This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
This summer I joined Dr Phil Carella’s lab at the John Innes Centre, a part of the Norwich Biosciences Institute with the aim of establishing a cereal-Pseudomonas pathosystem for comparative phytopathology research. Pseudomonas syringae is a globally significant model plant pathogen which is responsible for a wide range of diseases particularly in agricultural crops; it was exciting to be a part of research on such a globally significant microbe. P. syringae is interesting for the huge diversity within this species complex, with different strains infect wildly different plant species, from early divergent plants such as liverworts to Nicotiana benthamiana and cereal crops. Pathovars of P. syringae wield varying host ranges from single host strains to broad-host strains such as P. syringae pv syringae (the causal pathogen of bacterial leaf blight in Pisum sativum and Triticum aestivum). The Carella lab group are interested in understanding the underlying mechanisms dictating generalised vs specific virulence factors within the species complex.
To undertake comparative phytopathology of P. syringae, pathosystems need to be established across a range of plant species. Previous model plants have been explored by the Carella lab, including N. benthamiana and Marchantia polymorpha, with the intention of developing pathosystems in representatives of every major clade of plants. My project during this 8-week summer internship was to expand on the group’s previous work by introducing cereal crops to the model.
Accessions of wheat were screened for susceptibility to P. syringae pv syringae, designated b728a. This strain was chosen for its broad host range and significance to other projects in the labs. Infection assays were performed in either one of two ways depending on the strain of P. syringae used. Leaves from two-week-old T. aestivum plants were inoculated with a 2×10^6 cfu/ml solution of b728a via syringe infiltration. At 3 days post-infection, leaf discs were taken of infected tissue and bacterial growth was quantified by either one of two ways. We performed serial dilution to manually count the concentration of CFUs in the leaf discs for the majority of P. syringae pathovars available. However, some strains had been transformed via electroporation to express the luciferase gene cassette known as lux genes. This method was used to measure relative bioluminescence between infected and uninfected tissue from P. syringae strains that have been transformed.
Unfortunately, there was no significant difference between the bacterial growth in any of the wheat accessions compared to a blank following a luminescence assay. The broad host range of b728a did not extend to wheat.
Following this, I screened different P. syringae pathovars against a single wheat accession – cadenza. P. syringae pv atrofaciens strains were selected for new infection assays, atrofaciens strains had been isolated from wheat and phenotype scans showed promising pathogenicity in comparison to the b728a strain. This was confirmed by serial dilution series where the atrofaciens pathovars had a higher colony count relative to b728a within the infected tissue.
By establishing a pathosystem we can study the genetics involved in Pseudomonas pathogenicity and plant immunity. Data gathered can be used to assess the contribution of known core virulence factors in cereal infection. Further investigation could include developing Type 3 secretion system (T3SS) mutants. T3SS injects effectors directly into the host cell to promote bacterial growth, so mutants can show us the contribution that effectors have to strain pathogenicity. From this research, the Pseudomonas-cereal pathosystem can be expanded to include other monocots, such as barley – which has the benefit of a smaller genome and higher transformation rates for genetic analysis.
I would like to thank the members of my lab group, particularly my supervisor Dr Khong-Sam Chia and group leader Dr Phil Carella, as well as BSPP for funding this summer placement. I have gained so many new skills in such a short period of time, which is incredibly valuable especially given recent setbacks with in-person lab experiences for all university students. This placement gave me a window into the day-to-day life of academic research and prepared me for a career in academic lab-based research.
Jacob Reeves
Imperial College London
TOP IMAGE : P. syringae pv atrofaciens (Paf 1852 and Paf 5011) and P. syringae pv syringae (b728a) phenotype scans for infection assays in T. aestivum cadenza strain.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
