The roles of extracellular vesicle transport in late blight disease development

Fungi and oomycetes cause the most devastating crop diseases and thus significantly threaten global food security. Critical components of their virulence arsenal are proteins called cytoplasmic effectors that are delivered inside plant cells to suppress immunity. A key scientific challenge in the plant-microbe interaction field is to understand how effectors are secreted and translocated into host cells in order to better develop new approaches to prevent disease.

An exciting breakthrough in our laboratory revealed that cytoplasmic effectors from the potato late blight pathogen Phytophthora infestans may accumulate in extracellular vesicles (EVs), implicating this as a delivery route. In this proposal, supported by industrial partner Syngenta, we will investigate the protein cargo of EVs, how EVs are synthesised and secreted, the means by which cytoplasmic effectors are delivered into plant cells to reach their destinations, and whether these processes can be inhibited.

To investigate how cytoplasmic effectors are delivered into plant cells by means of EVs we envisage 3 inter-related objectives:

Firstly we will use proteomic approaches to define the Phytophthora secretome, and especially the contents of EVs. Inhibitors that promote or prevent EV production in other systems, along with inhibitors from Syngenta that alter vesicle biology, will be employed to define the EV secretome. Transformation of Phytophthora and cell biology will be used to confirm whether specific effector proteins are associated with EVs and whether they are secreted at the haustorial interface between pathogen and host cells.

Secondly, we will investigate the molecular cell biology of EV biogenesis to define the routes of effector secretion from the pathogen. The subcellular localisation in Phytophthora of effectors that are secreted to act either inside or outside of host cells will be determined, identifying the specific endomembrane compartments associated with their secretion. The functions of key genes identified in proteomic studies will be investigated by gene silencing; are they required for EV biosynthesis?

Thirdly we will use molecular and cell biology approaches to observe and to perturb entry routes into host cells in order to define the pathway travelled by cytoplasmic effectors to reach their subcellular destination. In particular, gene silencing and biochemical inhibitors will be used to attenuate endocytosis to investigate the impact of this upon EV, and thus effector, uptake.

The proposal aims to provide an understanding of the mechanisms underlying translocation of filamentous pathogen effectors into plant cells and particularly the potential involvement of EVs in this. It will provide the basis to explore the potential that the EV secretory route provides novel targets for disease control. The involvement of EVs in molecular transfer between host and microbe cells is a rapidly emerging and exciting area in medical and agricultural research fields. This proposal will provide a deeper understanding of the roles of EVs in the delivery of effectors from filamentous pathogens into plant cells.

The major crop diseases are caused by fungi and oomycetes. These pathogens translocate so-called cytoplasmic effectors into plant cells to manipulate immunity and facilitate disease. How these effectors are delivered into host cells is a fiercely debated topic of central importance to understanding disease development. Recently, we have discovered that a cytoplasmic effector we can visualise being delivered from Phytophthora infestans into host cells is associated with extracellular vesicles (EVs) when secreted from the pathogen. In contrast, apoplastic effector EPIC1 is conventionally ER-to-Golgi secreted and is not associated with EVs. This timely breakthrough prompts a detailed investigation into potential roles of EVs in effector delivery, which is of importance and impact to both agricultural and medical research.
We will investigate the hypothesis that cytoplasmic effectors are secreted from pathogen cells and translocated into host cells via EVs. We will employ proteomics under a range of growth conditions, and in the presence of various inhibitors, to determine the EV-associated secretome. Transformation of P. infestans with fluorescently-tagged effectors will be used to confirm whether they are secreted in association with EVs. Mutation of the RXLR motif in cytoplasmic effectors will be used to investigate the role of this peptide sequence in endocytic sorting. A recently developed endocytic compartment-specific fluorescent protein 'trapping' approach will be used to investigate whether effectors are secreted via either Golgi or multivesicular bodies. Constitutive or conditional silencing in P. infestans of genes emerging from proteomic studies will be used to investigate their involvement in EV biogenesis or secretion. Finally, we will use molecular and cell biology to observe and to perturb routes of endocytic entry into host cells to determine whether known endocytic pathways are travelled by cytoplasmic effectors to reach their subcellular destination.

Who will benefit from this research?

Pests and diseases are a major constraint to achieving food security. Up to 50% of crop losses in developing nations are due to pests and diseases. A deep understanding of how major oomycete pathogens deliver effector proteins into host plant cells will provide new routes to prevent infection, offering a long-term opportunity to make a significant impact on food security across the world. The proposed research is expected to benefit:
1. The agro-chemical industry, and especially our industrial partner Syngenta, who are looking for new targets for control of oomycete diseases;
2. The host-microbe interactions research communities (plant and animal) as novel, and potentially generic, insight is provided into the delivery of effector proteins into host cells;
3. Societies worldwide as new approaches can be developed to control disease, leading to greater food security;
4. The public, with whom regular engagement will be sought each year to communicate our science and the underlying reasons for it;
5. Staff working on the project, who will receive a broad experimental training and experience communicating their research to the public.

How will they benefit from this research?

The agro-chemical industry, and especially Syngenta, will directly benefit from this research as it will provide to them: 1) An understanding of the importance of distinct secretory pathways, and especially of vesicle formation for effector delivery, leading to infection and disease; 2) Information on the impact of inhibitors, including their own chemicals, on the secretion and delivery of effector proteins; 3) The identification of potential new proteins involved in vesicle-mediated delivery that act as targets for control chemicals; 4) Transgenic Phytophthora infestans reporter lines for secretory pathways that can be used for phenotypic screens in agro-chemical discovery; 5) New cell biological phenotypic methods that can be adopted for chemical screens; 6) New tools and approaches to manipulate oomycetes such as P. infestans for in-house discovery programmes. In the long-term it is hoped that findings from this research would provide new ways to enhance food security through targeted, environmentally benign chemical control. This would benefit societies in both the developed and developing world.

All microbial pathogens, including filamentous fungal and oomycete pathogens, deliver effector proteins into host (plant or animal) cells. Insight into the cell and molecular processes required for effector delivery will thus have potentially wide and generic relevance. The breakthroughs leading to this proposal, including publication of the first visual demonstration of effector translocation in oomycetes (Wang et al 2017), and the demonstration that this effector is associated with extracellular vesicles (EVs), along with the timely observations of EV involvement in cell-to-cell delivery of molecules in medical and plant research, means that this proposal is timely to make a very broad international impact on a number of research fields. The proposal will provide evidence for how EVs are formed, what proteins are associated with EVs, whether they are delivered into host cells, and the means by which EVs may be taken up by host cells to deliver effectors to their destinations.

The breadth of molecular and cell biological techniques and their application to strategic and applied outcomes, will benefit the career development of PDRAs employed on the project.

Finally, we will interact with the public to describe and discuss the issues underlying food security, environmental change, and chemical control of crop diseases.