Crop vaccination without side effects: optimising the cost-benefit balance of chemically induced immune priming in greenhouse vegetables.

Lead Research Organisation: University of Sheffield
Department Name: Animal and Plant Sciences

Abstract

It is possible that over half of the crops cultivated each year are lost to pests and diseases. The use of chemical pesticides helps to reduce these losses, but many pesticides are becoming less efficient as diseases develop resistance, and others are being removed from the market because of fears that they might affect the environment or human health. The objective of our project is to develop a different approach to protecting crops by boosting their own immune system. This strategy, known as chemically induced immune priming, has been used to protect some crops against commercially important diseases, including grey mould and downy mildew. However, the commercialisation of these resistance-inducing chemicals has not been successful, because they often also repress plant growth. A recent breakthrough by our research team showed that it is possible to separate the resistance-inducing effects of the chemical priming agent beta-aminobutyric acid (BABA) from its growth repressing effects. Although BABA protects plants against an exceptionally wide range of commercially relevant diseases, it also stunts plant growth and affects crop yield when applied at higher doses. Recently, we discovered that the receptor for BABA controls disease resistance and the accompanying plant stress response via separate signalling pathways (Luna et al. 2014; Nature Chemical Biology 10: 450 - 456). The current project aims to translate these research findings from the model system (Arabidopsis) to vegetable crops (tomato and lettuce). The international plant breeding company Enza Zaden, one of the world's largest suppliers of vegetable seeds to farmers, are so excited about the opportunity to enhance the benefits of immune priming that they have offered to make their research materials available to us and to co-fund our work. Together, we have designed an entirely novel project to optimize chemically-induced immune priming in vegetable crops, using an integrated approach of genetic and chemical strategies to reduce input of pesticides in the vegetable industry. If successful, our approach can be used effectively to protect vegetable crops in a more sustainable manner from damaging diseases.

Technical Summary

Immune priming provides plants with an enhanced defensive capacity against a broad spectrum of diseases. Beta-amino butyric acid (BABA) has emerged as a powerful priming agent in different plant species. However, a disadvantage of BABA is that over-stimulation reduces plant growth. This undesirable side effect has hampered commercialization of BABA as a crop defence activator. Previous work by the Ton lab has identified a key regulatory gene of BABA-induced priming in Arabidopsis, called IMPAIRED IN BABA-INDUCED IMMUNITY1 (IBI1). This gene encodes an aspartyl tRNA synthetase (AspRS), which binds in planta to the active R-enantiomer of BABA. This BABA-IBI1 interaction blocks default AspRS activity of IBI1, leading to a plant stress response that is caused by accumulation of uncharged tRNA and repression of gene translation. Independently of this stress response, BABA-IBI1 binding primes the IBI1 protein for an alternative defence function that becomes active after pathogen attack. Hence, IBI1 controls BABA-induced stress and BABA-induced resistance via separate pathways. Further evidence revealed that the trade-off between resistance and growth repression by BABA can be optimised by targeting IBI1 gene expression and chemical modification of the (R)-BABA molecule. This outcome forms the basis of this project, which follows a combination of genetic and chemical strategies to optimise IBI1-dependent resistance in tomato and lettuce. The project involves genetic alteration of the IBI1 gene, chemical synthesis and characterization of a new BABA analogue, quantification of chemical residues in crop products, and disease phenotyping to identify optimal crop-chemical combinations. The project will be conducted in partnership with ENZA Zaden, who are assisting with the identification of crop orthologs of IBI1. This project will allow Enza to generate cis-genic/non-GM mutant crop varieties that develop optimal levels of immune priming without affecting growth and yield.

Planned Impact

Fungal and oomycete diseases pose a serious threat to the production of vegetable crops in greenhouses. At present, these diseases are mostly controlled by repeated pesticide applications. There is growing concern about pesticide resistance and their impacts on health and environment, which is reflected by recent changes in EU legislation to limit pesticide usage (e.g. The European Union Directive 2009/128/EC). The objective of this project is exploit the plant's immune system to protect two vegetable crops (tomato and lettuce) against commercially relevant diseases without compromising crop growth and yield. The project will be conducted in close partnership with Enza Zaden, a world-wide supplier of vegetable seeds, and will follow a combination of genetic and chemical strategies. Supported by solid preliminary evidence, this integrated approach ensures high levels of efficiency and successful deliverables for the industrial partner. Consequently, we are confident that this project will make a significant contribution to a more effective and sustainable strategy of disease protection in greenhouse-cultivated vegetables, thereby reducing the need for repeated applications of pesticides.

Publications

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Description Despite delays in the final stages of the project due to the Covid19 pandemic, we have made substantial progress from both a fundamental and translational research perspective. This progress is illustrated by recent publications supported by this grant, including Buswell et al. 2018 (New Phytologist, 218: 1205-216) and Schwarzenbacher et al. 2020 (Molecular Plant, 13:1455-1469).

The project followed three complementary strategies to optimize the cost-benefit balance of chemically induced disease resistance in the vegetable crops tomato and lettuce:

• Objective 1: manipulation of IBI1 gene expression and IBI1 gene sequence to maximize chemically induced resistance and/or basal resistance in vegetable crops.

• Objective 2: to identify the mode of action and metabolic degradation pathway of a novel chemical priming agent acid (R-beta-homoserine) that induces disease resistance without repressing plant growth. This objective also involves the production of an economically viable synthesis route of R-beta-homoserine.

• Objective 3: to determine optimal combinations between crop genotype and chemical priming agents for maximum levels of induced resistance with minimal reductions in plant growth and yield.

Each of these objectives have been addressed in the correspondingly numbered project parts outlined below:

Project part 1: We have carried out functional complementation experiments with crop gene orthologs of the IBI1 gene, which encodes the receptor of beta-aminobutyric acid (BABA). These experiments have confirmed functional complementation and receptor activity of IBI1 gene orthologs from lettuce and tomato. ENZA Zaden has adopted this knowledge and is currently constructing transgenic crop varieties over-expressing these IBI1 orthologs. Based on our previous finding that IBI1 over-expression in Arabidopsis enhances basal resistance, increases the responsiveness to lower doses of BABA, and improves stress tolerance to BABA, these lines will be tested for (BABA-induced) resistance and yield under commercial greenhouse conditions (see Project Part 3 for more details). In addition, we have generated new results about the BABA-induced signalling events acting immediately downstream of the IBI1 receptor, using a combination of untargeted transcriptome and protein interaction analyses of the model interaction between Arabidopsis and downy mildew (Hyaloperonospora arabidopsidis; Hpa). Genome-wide transcriptome analysis revealed that IBI1-dependent expression of BABA-induced resistance (BABA-IR) against Hpa is associated with suppression of ABA-inducible abiotic stress genes. Protein-protein interaction studies revealed that IBI1 interacts with the VOZ1 and VOZ2 transcription factors (TFs), which are transcriptionally induced by ABA, but repress (pathogen-induced) expression of ABA-dependent genes regulating abiotic stress tolerance. We have demonstrated that the two VOZ TFs require nuclear localization for their contribution to BABA-IR, resulting in augmented expression of callose-related penetration resistance after pathogen attack. Hence, the IBI1-VOZ signalling module channels pathogen-induced ABA signalling towards cell wall defence, while simultaneously suppressing abiotic stress-responsive genes. A manuscript describing these results has provisionally been accepted for publication by Molecular Plant in their special issue on Plant-Microbe Interactions (Schwarzenbacher et al. 2020). Due to the Covid19 pandemic, the publication of this special issue has been delayed until October/November 2020. The requested revisions are relatively minor, mostly involving changes in the statistical analysis and a request for additional bio-assay results to clarify the role of role of ABA in IBI1/VOZ2-dependent penetration resistance. Despite the Covid19-related closure of the University of Sheffield, we have been able to address most experimental revisions under a restricted lab access arrangement, and are ready to submit our revised manuscript by the end of this month (30th June 2020; as agreed with the handling editor). In addition to VOZ1/2, our yeast-2-hyrbid screens identified various other protein candidates, including the ER-localised fatty acid hydroxylase FAH2. Bimolecular fluorescence complementation analysis (BiFC) confirmed the in planta interaction between IBI1 and FAH2. Moreover, we discovered that BABA induces low-level ER stress in an IBI1-dependent manner and that mutations in both FAH2 and the IRE1a/b-dependent unfolded protein response (UPR) affect BABA-induced penetration resistance against Hpa. The latter results establish a clear regulatory function of FAH2 and the ER in priming of callose-related penetration resistance, and from the basis of the BBSRC responsive mode grant proposal submitted on 23rd June 2012.

Project part 2: This project part has focused on the chemical component of BABA-IR and resulted in the discovery of R-beta-homoserine (RBH) as a resistance-inducing structural analogue of BABA. This beta-amino acid induces resistance without major non-target effects on plant growth and metabolism in both Arabidopsis and tomato. We have published this work in New Phytologist (Buswell et al. 2018 New Phytologist, 218: 1205-216). Unexpectedly, despite the structural similarity to R-BABA, this study also revealed that RBH acts through partially different defence signalling pathways and does not require the IBI1 receptor. Subsequent research under this project part has focused on two objectives: 1) the optimisation the chemical synthesis pipelines for R-BABA and RBH (as described in the original proposal) and 2) an additional mutant screen to identify novel regulatory genes of RBH-induced resistance (RBH-IR) against Hpa. Firstly, the chemical synthesis pipelines of R-BABA and RBH have been upscaled and streamlined. Initially, we used a route that relies on cyanation of D-alanine, which was labour-intensive and involved work with highly toxic KCN. Using this method, we obtained ~10 grams of pure R-BABA and synthesised a small amount (~100 mg) of 13C-labelled R-BABA. To eliminate the requirement for KCN and increase the throughput of the RBH synthesis pipeline, we adopted an alternative (KCN-independent) approach, using D-aspartic acid as the source material. This method, which relies on an acid-independent procedure to remove the Boc protection group from the NH group (heating in water, instead of prolonged incubation in 6 M HCl), proved successful and generated ~50g of enantiomer-specific RBH. The purified (unlabelled) RBH and R-BABA have been shared with ENZA Zaden to carry out greenhouse trials under project part 3. Secondly, based our discovery that RBH-IR is controlled by a different signalling pathway than BABA-IR (Buswell et al. 2018), we conducted a screen for homozygous Arabidopsis T-DNA mutants impaired in RBH-induced resistance (iri). In addition to a large number of iri mutants that are partially impaired in RBH-IR, this screen yielded four iri mutants that are fully impaired in RBH-IR against Hpa. Of these, the iri1 mutant was found to be impaired in a gene that encodes the broad-spectrum amino acid transporter LHT1 and was selected for further characterisation. Quantification of RBH uptake by the iri1/lht1 mutant, wild-type and IRI1/LHT1-overexpressing lines revealed that IRI1/LHT1 is critical for the uptake of RBH by the roots. This conclusion was further strengthened by our subsequent findings that IRI1/LHT1 regulates plant tolerance of RBH and that co-application of canonical amino acid substrates of IRI1/LHT1 interferes with RBH growth responses. A manuscript describing these results is ready for submission to Plant Physiology but awaits a final set of experiments to quantify RBH uptake/competition in transgenic yeast cells over-expressing Arabidopsis IRI1/LHT1. These latter experiments have been put on hold due to the Covid19-related lock down of the University of Sheffield. The other IRI genes identified in the screen, perform critical as-yet-unknown roles in RBH-IR. Together, the IRI genes could present alternative targets for genetic enhancement of RBH-primed immune responses and are currently investigated for IP potential.

Project part 3: The site-directed mutagenesis approach under project part 1 has revealed an critical role for the L-Asp-binding pocked of IBI1 in the perception of R-BABA (Buswell et al. 2018), but did not identify mutations that increase the resistance-inducing activity of the IBI1 protein in the absence of BABA. However, our evidence that over-expression of IBI1 not only increases the tolerance to BABA-induced stress (Buswell et al. 2018; Luna et al. 2014 Nat Chem Biol 10: 450-456), but also increases levels of basal resistance, BABA-IR and RBH-IR, illustrates that plants with elevated levels of IBI1 gene expression require lower concentrations of BABA/RBH to obtain full disease resistance. The translational potential of this discovery has been recognised by the R&D unit of ENZA Zaden, who are now focusing on GM and non-GM strategies to select vegetable varieties with increased IBI1 gene expression. The identified IBI1 orthologs (tomato, lettuce and cucumber) from project part 1 have been handed over to ENZA to generate transgenic vegetable lines over-expressing IBI1 orthologs. Due to the Covid19 lock-down, this work has been delayed, but will be continued by ENZA beyond the duration of the grant. In addition, ENZA has carried out greenhouse-based trials for BABA- and RBH-IR against commercially relevant diseases, using their own proprietary diversity panels of lettuce, tomato, cucumber and pepper. Based on these screens, the interaction between lettuce and the downy mildew pathogen Bremia lactucae has emerged as the most promising commercial target, including lettuce varieties that express high levels of stress tolerance to BABA/RBH in combination with long-lasting protection against virulent Bremia isolates (at >2 weeks after chemical treatment). Future R&D by ENZA will focus on the development of a marker-assisted breeding approach to select non-GM lettuce varieties that respond optimally to BABA and RBH in terms of stress tolerance and IR. This approach benefits from the on-going interaction with the Ton lab and the identification of new regulatory genes controlling chemically induced resistance in the Arabidopsis-Hpa model system.

In summary, this BBSRC-IPA grant has been of great mutual benefit to both the Ton lab and the industrial partner, ENZA Zaden. The grant has generated exciting new avenues of research and collaboration to advance the exploitation of chemical priming agents in vegetable crop protection schemes. Based on the long-lasting protective effects of BABA-induced priming, ENZA Zaden is willing to support a new BBSRC-IPA application (BB/W015250/1; 'Exploring and Exploiting Epigenetic Plant Immunity'), which focuses on gaining a better understanding of the epigenetic mechanisms driving immune priming against downy mildew diseases, and exploiting this knowledge through the implementation of new epigenetic breeding approaches in lettuce.
Exploitation Route As outlined above, our industry partner (Enza) is taking some of our discoveries forward towards the integration of immune priming chemicals and associated crop genes in IPM of greenhouse-cultivated vegetables. This is further evidenced by the fact that they are supporting a new BBSRC-IPA application to explore and exploit the epigenetic basis of immune priming in Arabidopsis and lettuce against downy mildew diseases (BB/W015250/1).
Sectors Agriculture, Food and Drink,Chemicals

 
Description The project entails a close collaboration with the R&D Phytopathology unit of Enza Seeds and aims to combine chemical and genetic strategies to optimize immune priming in vegetable crops (e.g. lettuce and tomato) in terms of maximizing effectiveness and reducing non-target effects. The company has begun trialling different priming chemicals discovered as part of of this BBSRC project (as published in Buswell et al. 2018, New Phytologist). In addition, based on our discovery that over-expression of the BABA receptor gene IBI1 increases the efficiency of both basal resistance and induced resistance by BABA and RBH, ENZA is now exploring on GM and non-GM strategies to select vegetable varieties with increased IBI1 gene expression. The identified IBI1 orthologs (tomato, lettuce and cucumber) from part 1 of this project have been handed over to ENZA to generate transgenic vegetable lines over-expressing IBI1 orthologs. Due to the Covid19 lock-down, this work has been delayed, but will be continued by ENZA beyond the duration of the grant. Furthermore, ENZA has carried out greenhouse-based trials for BABA- and RBH-IR against commercially relevant diseases, using their own proprietary diversity panels of lettuce, tomato, cucumber and pepper. Based on these screens, the selected crop-pathogen interactions have emerged as the most promising commercial targets, including crop varieties that express high levels of stress tolerance to BABA/RBH in combination with long-lasting protection against virulent pathogen strains (at >2 weeks after chemical treatment). Future R&D by ENZA will focus on the development of a marker-assisted breeding approach to select non-GM crop varieties that respond optimally to BABA and RBH in terms of stress tolerance and IR. Moreover, based on the highly effective and long-lasting nature of BABA-IR against downy mildew in lettuce (pat 3 of the project), ENZA has decided to support a new BBSRC-IPA application (BB/W015250/1) by PI Jurriaan Ton, which focuses on epigenetic mechanisms underpinning immune priming against downy mildew diseases.
First Year Of Impact 2021
Sector Agriculture, Food and Drink,Chemicals
Impact Types Economic