The genomic basis of host specificity and niche adaptation of Pseudomonas syringae on Prunus
Lead Research Organisation:
University of the West of England
Department Name: Faculty of Health and Applied Sciences
Abstract
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Technical Summary
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.
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.
Planned Impact
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).
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).
Publications
Neale HC
(2020)
An improved conjugation method for Pseudomonas syringae.
in Journal of microbiological methods
Description | Work package 2 We created Tn mutant libraries of one strain for each of the major cherry canker pathogens Pss, Psm R1 and Psm R2 and screened the mutants on immature cherry fruit to look for changes in virulence. Mutants with reduced and also enhanced virulence recovered from the fruit assay were further characterised by in vitro growth parameters including biofilm formation and leaf and cut shoot pathogenicity tests. The mutated genes in a selection of mutants with altered pathogenicity were identified by sequencing and complementation experiments. Most interestingly a mutation of the effector gene, hopAU1, led to an increase in virulence in Psm R2 (Neale et al., 2021) potentially indicating this is a factor involved in quantitative resistance. Alongside this, a method was developed to improve plasmid conjugation frequency in order to achieve saturating transposon mutagenesis of the genome of strains of Pseudomonas syringae. Manipulation of the growth stage of donor and recipient cells allowed the required increase in frequency and facilitated conjugation of otherwise recalcitrant strains, in some cases increasing the conjugation frequency 1000-fold (Neale et al., 2019). This improved method allowed for the creation of saturating mutant libraries with mariner plasmid PKMW3 of the three strains of interest. |
Exploitation Route | Not clear yet |
Sectors | Agriculture Food and Drink |