This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Zymoseptoria tritici (Z. tritici), a destructive wheat pathogen responsible for Septoria tritici blotch, poses a significant threat to European wheat production and causes annual losses ranging from €120-800 million. During infection, Z. tritici secretes small apoplastic effector proteins to aid its infection cycle. Investigating these effectors is crucial for understanding the interactions between the pathogen and the host plant’s immune system. In order to achieve this, techniques for the production of purified effectors are needed. Alternative techniques for producing purified proteins, like bacterial expression systems (e.g. Escherichia coli) are rapid and high-yield, but require optimisation and are challenging for proteins that require post-translational modifications in case of the cysteine-rich proteins. Eukaryotic expression systems (e.g. Pichia pastoris) allow post-translational modifications, but would require different conditions for each target protein which can prove as time-consuming and laborious approach. This study aims to investigate the feasibility of a technique known as apoplastic wash-fluid (AWF) extraction, to produce purified and biologically active Z. tritici cell-death inducing effector proteins, offering a potential solution for Z. tritici effector-related research. In brief, these effectors can be transiently expressed in the model plant Nicotiana benthamiana with signal peptides contained in the protein construct for secretion to the apoplast. Afterwards, AWF extraction involves the gentle washing of the plant apoplast with a suitable buffer and the collection of this fluid which contains the already in-planta processed effectors.
In order to achieve this, Agrobacterium tumefaciens strain cryX was electroporated with pEAQ-HT vectors harbouring our constructs of interest. The constructs of interest contained in our vectors were as follows: Zt12 (a Z. tritici effector), Zt12:3xHA (the same effector tagged with triple human haemagglutinin), MgNLP (a strong cell-death inducing effector; positive control) and empty vector (negative control).
Transformed agrobacteria was selected for by growth on kanamycin antibiotic plates and cross checked for constructs via colony PCR. The agrobacteria harbouring our constructs was whole-leaf infiltrated into 5-week-old N. benthamiana plants – using a total of 15 leaves per construct. AWF was extracted at 2-days post infiltration (dpi) – before cell-death could occur. The method involved leaves being severed and submerged into a beaker of ice-cold milli-Q water which was placed inside a large desiccator. Vacuum was applied briefly and then slowly released. In doing so, the apoplast was infiltrated with water, appearing dark upon completion. Leaves were blotted of excess water and sandwiched together, rolled in parafilm, then placed into a 50mL syringe inside a 50mL falcon tubes. Samples were centrifuged to collect AWF and the supernatant was aliquoted and re-centrifuged to remove plant debris. Raw AWF was spin-concentrated and a remainder of raw AWF was kept for further check-ups. Both raw and concentrated AWF were subjected to western-blotting assays to confirm the presence of our effector proteins.
Concentrated AWF samples containing the effectors were co-infiltrated with a 22 amino acid sequence of bacterial PAMP flagellin (flg22) in 4-week-old N. benthamiana plants in order to assess whether or not PAMP triggered immunity (PTI) is a prerequisite for effector triggered cell-death which was always observed during transient expression bioassays. By using this setup, we aimed to investigate whether the effector proteins alone are sufficient to elicit a hypersensitive response, or whether the initial activation of PTI is required to potentiate this response. Plants were observed under white and UV light for a period of 6 dpi. During the observations, only the positive control (MgNLP), as observed in the figure, seemed to have maintained its biological activity, even though very minimal in comparison to transient expression where strong cell-death was always observed. Based on these observations, it can be speculated that the transiently expressed effectors were already processed and recognised by N. benthamiana immune system, or that the effector proteins are not at the ideal concentration to trigger a cell-death response. For future investigations, would be interesting to have a higher recovery of effector proteins from AWF and infiltrate at a higher concentration.
This BSPP studentship has allowed me to gain invaluable laboratory experience from techniques to the working environment and I am extremely excited to pursue a career in plant pathology. I would like to thank the many people at Rothamsted Research who made it such a welcoming environment and my supervisor, Alina A Igna, not only for her support and guidance with my project, but for going even further to train me in many techniques that will be beneficial in my future pursuits.
David McLean
University of Dundee
TOP IMAGE: Leaves co-infiltrated with concentrated AWF containing tagged and untagged effectors, positive (MgNLP) and negative (EV) controls and mock-infiltrated leaves with the PAMP flg22 6 days post infiltration under both white and UV light.
