| This project going to be… | Experimental (lab/field based) |
| Email | alina.igna@rothamsted.ac.uk |
| Position held | PhD Student |
| Title of Project | Production of biologically active fungal effector proteins utilising Apoplastic Wash Fluid from Nicotiana benthamiana |
| Institution Department and Address | Department of Protecting Crops and the Environment, Rothamsted Research, West Common |
| Harpenden, Hertfordshire AL5 2JQ |
| United Kingdom |
| Full Name of Supervisor | Alina A Igna |
| Date of Project Commencement | 19/06/2023 |
| Duration (weeks) | 6 |
| Brief Description of Project | Project background: The fungus Zymoseptoria tritici is the causal agent of the foliar disease Septoria tritici blotch of wheat, which is considered the greatest threat to European and UK wheat production. Similar to other fungal pathogens, Z. tritici produces small secreted apoplastic effector proteins which facilitate the successful colonization of wheat during infection. In a previous study by Kettles and colleagues (2017), the non-host plant Nicotiana benthamiana was used to investigate its immune responses upon treatment with Z. tritici derived effectors via Agrobacterium-mediated infiltration. This study unexpectedly revealed that several fungal effectors were recognised in this non-host plant, leading to plant cell death. Furthermore, in my PhD project I have investigated the early defence signalling events, such as Mitogen Activated Protein-like Kinases (MAPKs). The results so far show that transient expression of three Z. tritici effectors in N. benthamiana leads to MAPKs activation prior to cell-death, suggesting a role of MAPKs in the N. benthamiana immune system response to Z. tritici effectors. However, the transient expression system has a major drawback – some plant species are not amenable to this expression system (e.g. monocotyledons). Therefore, the need of producing purified form of effector proteins is ideal to further being able to functionally test these effectors on other plant species. To tackle this aspect, secretion pathway of model plant N. benthamiana was explored in our lab to produce mature active form of Z. tritici effector proteins – a rapid method known as Apoplastic Wash Fluid (AWF) extraction. Briefly, the Z. tritici effectors are transiently expressed and secreted into the apoplast of N. benthamiana. At 2 days post-agroinfiltration the AWF from leaves expressing the effectors is extracted. Despite preliminary results obtained so far, the optimisation and replication of this method is still required to prove whether this could be a viable choice to potentially produce biological active form of Z. tritici effector proteins. Project Aim: Explore AWF extraction for production of biologically active Z. tritici effector proteins and potentially test these on different plant species (e.g. wheat) The student will: -Use model plant N. benthamiana and glass house facilities -Transform Agrobacterium tumefaciens with the constructs of interest -Work in sterile environment to grow/maintain bacterial media/cultures -Perform needless syringe infiltration with (1) agrobacteria-suspended solutions; (2) AWF extract -Assess the progress of leaf cell-death development -Extract AWF via vacuum infiltration and centrifugation steps -Extract total plant leaf protein -Perform Western Blots (anti-MAPK; anti-HA) -Record and report key results In addition to molecular biology and proteomics skills, the student will experience life as a scientist by taking part in seminars, journal clubs and internal lab meetings |
| Attach the recommended reading for the project | Torriani SF, Melichar JP, Mills C, Pain N, Sierotzki H, Courbot M. Zymoseptoria tritici: a major threat to wheat production, integrated approaches to control. Fungal Genetics and Biology. (2015) 1;79:8-12. doi.org/10.1016/j.fgb.2015.04.010 |
| Kettles GJ, Bayon C, Canning G, Rudd JJ, Kanyuka K. Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana. New Phytol. 2017 Jan;213(1):338-350. doi: 10.1111/nph.14215. |
| Dagvadorj B, Solomon PS. Simple and efficient heterologous expression of necrosis-inducing effectors using the model plant Nicotiana benthamiana. Plant Direct. 2021 Aug 23;5(8):e341. doi: 10.1002/pld3.341. |
