An evolutionary approach to develop durable disease resistance to bacterial canker of cherry

The resurgence of cherry production in the UK from 400 tonnes in the year 2000 to 3500 tonnes in 2014, achieved through the adoption of high-density plantings, has led to bacterial canker, which is caused by Pseudomonas syringae, becoming the main disease of cherry, for which there is no effective control. Our recent work has shown that bacterial canker is not caused by a single bacterial population but by three distinct groups of Pseudomonas, each having independently acquired the ability to cause disease on cherry and each manipulating the host in subtly different ways in order to subvert plant defences and survive in long term associations with the tree. This phenomenon is termed convergent evolution and is an interesting finding, as from it several fundamental scientific questions arise. In this proposal we seek to answer four questions, based upon recent research into this commercially important, yet understudied, pathogen.

First, what is the basis of niche survival and persistence of P. syringae on cherry? We wish to understand if different complements of toxins, effectors (a special class of proteins secreted by the pathogen that are involved in suppressing plant immunity and promoting pathogen growth) and other gene clusters, implicated in manipulation of host defences, determine survivability in particular niches (in woody tissues for example) or at particular times of year, and whether these are different in our three Pseudomonas clades?

Second, how is host specificity determined by effector content? Do conserved effectors, over-represented in convergently evolved Pseudomonas groups when compared to closely related non-cherry or plum pathogens, play an essential role in adaptation onto these hosts? This evolutionary approach, integrating information from many different strains of Pseudomonas is a novel way of utilising patterns of molecular evolution to provide insights into which pathogen genes are important targets for further study.

Addressing these two questions will provide fundamental insights into how pathogens evolve onto their hosts and may provide new avenues to pursue when considering how to control these pathogens in the field.

Third, which effectors control known resistance responses in cherry? This is a crucial question. Part of the plant immune system is controlled by specific classes of genes (broadly termed resistance or R genes) that have evolved to encode proteins that recognise pathogen attack, by monitoring for the presence or activity of effectors and then rapidly activating plant defence responses. Effectors are often referred to as the pathogen's Achilles heel, as they are both required for pathogenicity but leave the pathogen vulnerable to detection. Understanding which effectors are recognised in cherry material is important to predict the likely usefulness of particular plant resistances. For example, recognition of a rare effector at low frequencies in a pathogen population is less useful than an R gene that recognises a highly conserved effector, essential for pathogenicity. Our approach seeks to identify resistance genes that target effectors that are common to all strains of cherry infecting Pseudomonas, as well as other previously identified, but uncharacterised, resistances.

Fourthly, what is the genetic architecture of resistance to P. syringae in cherry? By identifying the regions of the cherry genome that control resistance, using a technique called genetic mapping, molecular markers tagging R genes can be developed and used by the UK industry (plant breeders) in order to breed cherry cultivars resistant to all three groups of pathogenic Pseudomonas.

Answering these questions provides plant breeders with the information that they require to develop resistant cultivars, improving yield, quality and the profitability of the industry and reducing waste in the supply chain.

Within this proposal we plan to carry out the following experiments:

1) Dissection of effector and toxin complements in Pseudomonas
Using the DeNoGAP orthology database and analysis toolkit developed by Prof David Guttman, we will include pilot and resequenced Prunus Ps strains, providing over 500 whole genome Ps pathovars for analysis. Carrying out a co-occurrence analysis, taking into account core genome phylogeny and phylogenetic autocorrelation, we will identify Prunus-essential effectors or effector/toxin groups. We will then create polymutants in pv syringae (Pss) and subsequently in the effector rich Ps morsprunorm strains to validate our theoretical predictions.

2) Unbiased detection of Ps genes regulating colonisation and persistence in woody tissue
Creation of a transposon-insertion saturated pool for Pss strains will enable parallel competition experiments to determine the basis of niche adaptation. Using a modified Inseq method, a plasmid expressing a transposon, creating multiple mutations in all possible TA sites in the genome will be expressed in different phenotyping experiments to uncover transposon insertion frequency, which can be related back to fitness effects controlled by single loci.

3) Screening cherry germplasm for resistance
Phenotype the germplasm and populations, using leaf infection and luciferase growth assay techniques using first genomic library screening and later effector null mutants to establish the link between specific effector presence/absence and resistance responses. This information will be utilised in order to use specific effectors as a tool for phenotyping segregating host populations for resistance loci.

4) Mapping of resistance
Generate genotypic data for cherry populations and germplasm and carry out QTL mapping for canker resistance, using data from WP3 to maximize QTL resolution, and provide molecular markers for disease resistance.

This grant will have a global impact, both on the research field internationally and on the international industry, especially the UK industry, as bacterial canker of Prunus is present in all regions of the globe. The importance of this pathogen cannot be underestimated, with P. s. pv. aesculi exemplifying an epidemic strain rapidly spreading and devastating Horse Chestnut populations in northern Europe; and P. s. pv. actinidiae causing huge economic loss of the major plant export crop of kiwi, worldwide. Through full engagement with industry stakeholders, maximum translation of this research will be ensured, driving forward the UK plant breeding industry in a globally competitive market.

Direct beneficiaries:

1. Commercial private sector
The UK and international plant breeding sector will benefit enormously from this endeavour and will allow these industries to first develop markers for QTL and later move from marker level associations to candidate gene associations. This is important for next-generation genome editing approaches and functional validation of candidate genes. This moves the industry very quickly to a point where pedigree-based selection and genome-wide selection are affordable and tractable options for crop improvement. Placing this in the hands of the UK partners will give the UK business a significant competitive edge (Benefit within 7-10 years)..

2. Fruit growing sector in the UK
UK industry will benefit as it will be able to access a resource that is beyond its means to create. Longer term it is anticipated that the UK partners will make significant use of this resource and knowledge generated from this pre-competitive work. This may lead to further competitive work funded by other research bodies (e.g. innovate UK or AHDB). Advancing genomic resources in horticultural crops and their pathogens is a key aim of the AHDB-Horticulture and evidenced by its support in this proposal. Ultimately, if patterns in effector gain and loss could be understood, prediction of a pathogen's host range and specificity may one day be possible from sequence data alone.
(Benefit within 5-10 years).

3. Public and retail sector-
Several UK retailers aim to double sales of UK-produced fruit by 2020; this project will assist that aim and improve UK productivity and competitiveness. Downstream science conducted utilising the resources generated in this project will lead to more reliable production methods and potentially reduce wastage in the supply chain (through reduced inputs and better variety development) (Benefit within 7-10 years).

Indirect beneficiaries
The wider cherry growing industry (UK and beyond)
As a result of resistance markers to bacterial canker, the rate of change of varietal development will increase, leading to greater benefits to downstream growers, packers and producers. (Benefit within 10-12 years)

Government, public and policy benefits
The public will benefit, not only from the improved position of UK agribusiness (and access of breeders to novel technologies), but also through the long term improvement in supply chain resilience through improved cultivar development. In the longer term the public will benefit through increased food security and sustainability, as a result of scientific improvements on horticultural crops. This feeds into many UK Government and EU policy agendas including: health (improving produce quality, pesticides (reducing residues through improved resistance), water (ability to grow nearer water courses), climate (growing crops perennially will improve carbon sequestration) and environment (reduced carbon and pesticides) (Benefit within 5-10 years).