The contribution of pathogen effectors to host range and non-host resistance

Lead Research Organisation: University of York
Department Name: Biology

Abstract

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Planned Impact

Solanaceous crops form an essential component of the world's food supply with potato ranking as the most important global non-cereal food crop. 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. New, durable and sustainable means of combating crop disease therefore offer an opportunity to make a significant impact on food security across the world. The proposed research is expected to benefit a) growers worldwide, especially those in developing countries, b) consumers, c) biotechnology and industry, d) researchers investigating crop diseases and disease resistance and e) the environment by reducing the amount of chemical sprays required for crop protection.
Historically, growers in Europe have relied on pesticides to produce most of their crops in the face of pressure from pests and diseases. Recent EU directives however, have prohibited or restricted the use of many active ingredients. Thus, growers now face diseases which are difficult to control. Breeding offers an environmentally benign method of controlling diseases by intogression of resistance genes but is inefficient to deal with rapidly changing virulent pathogens. Furthermore, resistances are often not readily deployable into cultivars.
Non-host resistance underlies the inability of a pathogen to cause disease in all plants outside its host range and is thus, by definition, durable. This proposal aims to understand the molecular constraints governing host ranges of distantly related pathogens/pests of Solanaceae crops, and thus the basis of non-host resistance between closely-related species in the Solanaceae. Understanding the molecular basis of non-host resistance offers the prospect of durable resistance against damaging pathogens and thereby a reduction of chemical inputs (including fungicides) and economic losses to growers. This allows sustainable use and management of ecosystem resources; a key aims of the LWEC programme. In addition to addressing food security, resistance transferred from non-host plants offers the prospect of sustainable pathogen management and crop production. This will be of great benefit to consumers, especially in the developing world where food is scarce. Importantly, this proposal aims to translate conceptual advances emerging from fundamental research on both Solanaceae models and Arabidopsis thaliana to important crops. The outcomes of this proposed research will impact the biotechnology industry. Critically, this is an IPA proposal, with the companies Simplot and Syngenta directly investing in the research as Industrial Partners. They aim to assess and exploit the potential of disease resistance-associated genes arising from this work to achieve durable resistance in Solanaceous crops, with Simplot focussing on potato late blight, and Syngenta focussing on Phytophthora diseases of tomato and pepper.
The work described in this proposal is timely as it will exploit genome information for 2 economically important oomycete pathogens, and the recently completed potato, tomato, pepper and N. benthamiana genome sequences. Outcomes of the project will have a high impact on the research community and raise the profile of research staff involved through peer-reviewed publications and invitations to conferences. This project will provide excellent training and career development opportunities for two PDRAs, and exposure directly to commercial exploitation of their research.

Publications

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Description This project commenced on the 1st of January 2014 and aims to generate a fundamental understanding of the contribution of Phytophthora virulence determinants (effectors), their plant targets, and host resistance proteins to non-host resistance (NHR).
The first Objective was to investigate whether effectors from each pathogen trigger non-host specific resistance responses. The first part of this objective was to clone priority effectors from P. infestans and P. capsici into plant expression vectors. Following microarray studies performed in-house for P. infestans, 90 RXLRs-containing virulence determinants were cloned into agro-binary vectors. These have been cloned into the plant expression vector pGRAB, and 77 have so-far been cloned into the viral vector pGR106 (PVX). Following homology-based MCL clustering and microarray studies for P. capsici, 50 RXLR-containing virulence determinants have been cloned or synthesised and are currently being transferred into plant expression vectors.
The second part of this objective was to identify recognition events of RXLR-containing virulence determinants in non-host plants. We determined that PVX was the best vector for RXLR expression screens in genotypes of potato, pepper, and tomato. However, agro-infiltration of expression constructs into Nicotiana sylvestris provides the most reproducible and facile screening procedure for potential NHR recognition. N. sylvestris emerged as a promising route to seek candidate NHR resistance genes - it is diploid, the genome has been sequenced, and transient expression is as facile as in N. benthamiana. Critically, as a common non-host, any follow-up work to seek R genes could involve cloning all R gene candidates from only this plant for subsequent functional assays in N. benthamiana with recognised effectors from both P. infestans and P. capsici.
As a result, all 90 P. infestans RXLRs have been expressed in N. sylvestris in replication and 12 P. infestans RXLRs are recognised in N. sylvestris, 5 of which are Avrblb2 family members. Of the remainder, 5 have not been associated to our knowledge with recognition in the host potato. Screens of P. capsici effectors on N. sylvestris have been performed to identify effector families that are recognised.

Objective 2 aimed at establishing whether effector activity determines pathogen host ranges within Solanaceae and/or Arabidopsis by identifying host protein targets of candidate effectors from P. infestans and P. capsici.
On a previous grant (BBSRC LOLA), >70 Pi effectors have been screened to considerable depth in a potato-P. infestans interaction Y2H library. This has revealed approx. 200 potato proteins as candidate targets of these effectors.
As our ultimate aims are to transfer Arabidopsis (At) orthologues of Pi/Pc effector targets into Solanceous crops (potato, tomato, pepper), we have focussed on Arabidopsis as a main priority. As part of the previous grant indicated above, 90 P. infestans RXLRs have been screened against the set of 12000 Arabidopsis genes cloned into yeast that has recently been reported for screening by effectors from G. orontii (Wessling et al 2014 Cell Host-Microbe 16:364-375). This has formed a basis to compare the non-host interactome of Pi-At with the host interactomes of At with H. arabidopsidis (Hpa), P. syringae tomato (Pst) and G. orontii. Analyses of the data are ongoing.
So far, more than 160 candidate At orthologues of the potato proteins interacting with P. infestans RXLRs have been cloned into yeast for Y2H analyses.
The At orthologues of potato interactors (following P. infestans RXLR screens) have themselves been screened against 90 P. infestans effectors and against all cloned effectors from Hpa. Potential breakdown of interactions for several Pi RXLRs with At orthologues of their interactors in potato has been observed. Interestingly, several of these are nevertheless apparently targeted by effectors from the host-adapted pathogen Hpa, suggesting they are 'common' targets.

The At orthologues have been categorised according to their interactions. 30 of them have been expressed in N. benthamiana to see whether they provide resistance to late blight. Two At orthologues have demonstrated that they provide this resistance. One of these has progressed to transgenic plants and has demonstrated that it does indeed provide resistance. This is of interest to the company Simplot who have transformed the At genes into potato to test whether they provide enhanced resistance. A major publication is being written to report the key findings of the RXLR-target screens, leading to transfer of an Arabidopsis protein into Solanaceae to provide late blight resistance
Exploitation Route Two At orthologues have demonstrated that they provide this resistance. One of these has progressed to transgenic plants and has demonstrated that it does indeed provide resistance. This is of interest to the company Simplot who have transformed the At genes into potato to test whether they provide enhanced resistance. A major publication is being written to report the key findings of the RXLR-target screens, leading to transfer of an Arabidopsis protein into Solanaceae to provide late blight resistance
Sectors Agriculture, Food and Drink

 
Description The scientific progress of this project has been reported to the scientific community at various national and international meetings including the OMGN meeting (Norwich 2014, Asilomar 2017), MPMI (Rhodes 2014) and COST FA 1208 (Zakopane, Poland); and at countless small international meetings and invitations to give seminars. A number of peer-reviewed publications have arisen thus far. The data have been presented to the Industrial partners at progress meetings and Simplot has taken forward some of the Arabidopsis orthologues genes that evade interactions with P. infestans effectors if they yield resistance in transient assays in N. benthamiana. We have discovered that a particular Ubiquitin E3 ligase targeted by a Phytophthora effector in potato is a positive regulator of immunity. The Phytophthoras RXLR effector cannot target the equivalent (orthologous) protein from a 'nonhost' plant, Arabidopsis. The Arabidopsis gene has thus been expressed in Solanaceae and it provides enhanced resistance to Phytophthora infestans. This demonstrates the principle of the project and has been taken in house for further studies by the industrial partner, Simplot. In addition, this work has led to further successful funding from BBSRC with the support of the same industrial partner, JR Simplot, in order to study host 'susceptibility factors' targeted by Phytophthora RXLRs. It has also led to additional funding from BBSRC responsive mode to study ubiquitin E3 ligases targeted by RXLR effectors, including the one referred to above.
First Year Of Impact 2017
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Description ISCF Agri-food Technology Catalyst Seeding Award
Amount £5,000 (GBP)
Funding ID BB/SCA/York/17 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 02/2018