Engineering disease resistance gene networks: synthetic helpers for expanded sensors

Project Information

BoostR

Grant agreement ID: 708899

DOI

10.3030/708899

Project closed

EC signature date 11 March 2016

Start date 1 July 2016

End date 30 June 2018

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 195 454,80

EU contribution

€ 195 454,80

Coordinated by

THE SAINSBURY LABORATORY
United Kingdom

Periodic Reporting for period 1 - BoostR (Engineering disease resistance gene networks: synthetic helpers for expanded sensors)

Reporting period: 2016-07-01 to 2018-06-30

Summary of the context and overall objectives of the project

One of the biggest challenges facing agricultural research is to determine how to produce more food on less land. Plant diseases negatively impact agricultural productivity by reducing the quality and quantity of food produced. To generate long-lasting resistance to plant diseases, an improved understanding of how pathogens cause disease and how the plant responds to the invading pathogens is needed.
A class of specialized intracellular immune receptors known as NLRs (Nucleotide binding leucine rich repeat containing proteins) form one of the most important genetic components of the plant immune system. Often referred to as resistance genes, NLRs are useful targets for generating disease resistant crops, and have long been (un)knowingly selected by plant breeders for crop improvement. However, NLRs are frequently overcome by pathogen evolution. Conventional breeding is limited by the availability of NLRs with useful recognition specificities, one way to overcome this is to design synthetic NLRs that possess novel properties.
The functional principles of NLR mediated immunity is far more complex than previously thought. We know that some NLRs work in pairs, in which a sensor NLR, specialized to recognize the pathogen, is coupled with a helper NLR that is involved in initiating the defence signal. More recently, a group of NLRs in the Solanaceae plant family, which include potatoes and tomatoes, were shown to form an intricate signalling network. In this network, a small number of helper NLRs, named NRCs (NLRs-required for cell death; NRC2, NRC3, NRC4) are paired with a group of agronomically important sensor NLRs, with varying degrees of specificity, to confer resistance against diverse pathogens.
Advancing our understanding of mechanisms underlying plant immunity will provide crucial input for improving disease control measures to ameliorate agricultural production. The overall aim of my proposal is to exploit new knowledge of NLR sensor-helper interaction networks to generate synthetic NRC proteins that possess broad-spectrum disease resistance. My hypothesis is that altering NRC helper proteins will improve disease resistance against a number of pathogens that devastate Solanaceae crops.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Since many sensor NLRs in the NLR network rely on NRCs for immune activation in the Solanaceae, it was hypothesized that pathogens may secrete virulence proteins, known as effectors, to target NRC function. As part of my MSCA fellowship, BoostR, I screened collections of effector proteins from several agronomically important plant pathogens, including bacteria, aphids, nematodes and oomycetes. I identified two effectors that were able to suppress NRC2 and NRC3, but not NRC4 function. These are AVRcap1b from the late blight pathogen, Phytophthora infestans, and SPRYSEC from the potato cyst nematode pathogen, Globodera rostochiensis. While both these effectors suppress NRC2 and NRC3 function, they do so in different ways. SPRYSEC directly binds to NRC2 and NRC3 in planta, while AVRcap1b does not. This suggests that these pathogens use diverse mechanisms to target the NLR network. Further investigation revealed that SPRYSEC targets the NB-ARC domain of NRC2 and NRC3, a region involved in nucleotide binding, NLR activation and mediating conformational changes within the protein. Based on these findings, I co-authored a research perspective in the journal Science (doi: 10.1126/science.aat2623). The paper highlights fundamental concepts of immune receptor networks and the implications these have for breeding crops for disease resistance. To promote the paper, I was involved in creating a YouTube video titled “Plants have an immune system… and it’s complicated” and participated in an interview for the “Talking Biotech” podcast titled “Plant disease networks”. The video is complementary to the Science perspective and together with the podcast allowed us to reach a broad audience. Additionally, I also co-authored a paper that summarizes the current knowledge of the interplay between effectors, their host targets, and their matching immune receptors (doi: 10.1094/MPMI-08-17-0196-FI). Already these papers have impacted ongoing research in the plant-pathogen interaction community.
Finally, I generated and screened NRC chimeras for expanded sensor specificity and evasion of suppression by pathogen effectors. I identified two NRC chimeras that maintained signalling with their NLR sensor counterparts and that evaded suppression by AVRcap1b and SPRYSEC. For a proof of concept, I transformed these chimeras into the model Solanaceae plant, Nicotiana benthamiana. I also plan to transform them into tomato and potato plants to determine their value in breeding for disease resistance. Results from this part of my project was presented in the form of a research lecture at Imperial College London to students of the Masters in Applied Biosciences and Biotechnology course.
Moving forward, I will continue to advance findings from my MSCA project in my host lab. I am working to narrow down the region within the NB-ARC domain targeted by SPRYSEC and plan to confirm the region within SPRYSEC that governs binding to NRC2 and NRC3. I will also explore the potential target of AVRcap1b. Furthermore, I will screen the transformed potato and tomato plants for enhanced resistance to P. infestans and G. rostochiensis to determine their value in breeding programs. Upon completion, I aim to publish two research papers detailing my findings. Moreover, I co-supervise and mentor a PhD student that is studying the biochemical characteristics of AVRcap1b and SPRYSEC, including determining the mechanisms these effectors use to target NRCs.

In addition to the proposed work, I was also the lead author of two review articles: one which describes oomycete species that are current threats (doi:10.1098/rstb.2015.0459) and another which provides an overview on elicitins (doi: 10.1111/nph.14137). These provide a great overview of the molecular interactions between plants and oomycete pathogens, and fall within the broader umbrella of plant pathogen interaction studies.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Findings from my MSCA project, BoostR, have resulted in new knowledge in host-microbe interaction studies. Identification of pathogen effectors that target NRC proteins will enable us to improve our understanding of the molecular mechanisms underlying pathogen infection and host recognition. In addition, the fact that these pathogens are evolutionarily divergent but converged to target the same host pathway, highlights the importance of NRC helper proteins in mediating immunity against various Solanaceae infecting pathogens. The generation of chimeric NRCs that maintain signalling to their corresponding sensor NLRs, and that evade pathogen suppression, has allowed us to improve of understanding of NLR function and provides us with new strategies for breeding disease resistant crops. Overall, I believe results from my project will have far-reaching implications, as the NRC network provides resistance to a diversity of agronomically important pathogens.
Furthermore, by promoting my research via various media outlets, such as the aforementioned YouTube video and Podcast interview, I have been able to reach a broad audience, including members of the general public. This has provided me with a platform where I can promote my research and improve public awareness of research in the area of plant-microbe interactions.

Hypersensitive cell dealth and disease resistance