Defining and deploying Rpi gene diversity in S. americanum to control late blight in potato
Lead Research Organisation:
University of Dundee
Department Name: School of Life Sciences
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
Technical Summary
Wild potato and tomato relatives can show heritable variation in late blight (LB) resistance due to Resistance to P. infestans (Rpi) genes.
AIM1 is to clone all the Rpi-amr genes in our Solanum americanum stocks. We developed a new method for fast Rpi gene cloning- sequence capture with long PacBio reads- and used this to clone Rpi-amr3. In unpublished work, we thus cloned at least 3 more. We aim to clone the remaining 3-6 Rpi genes in our S. americanum populations, and provide at least 8 genes that could be deployed in stacks.
AIM2 is to test functionality of each Rpi-amr gene in transgenic potato in field environments, and to verify their non-interference in each other's function.
AIM3 is to discover P. infestans effectors (AvrAmrs) detected by our cloned Rpi-amr genes. Rpi genes enable plants to sense P. infestans effectors, and upon recognition, activate plant defence. All P. infestans Avr secreted effectors carry an RxLR motif. It is possible to identify all RxLR effectors in a reference genome. Libraries of such effectors have been constructed in DNA vectors that enable them to be transiently expressed in plant cells; co-expression of Rpi gene and recognized effector causes a hypersensitive cell death response; we will screen existing libraries for such Rpi/effector combinations. With the Hein/Birch labs, we will also use sequence capture ("Pathseq") to refine our knowledge of the full RxLR effector repertoire of Pi. New RxLRs revealed by PathSeq will be added to the list of RxLRs tested to discover AvrAmr effectors. In addition, a refined bait library for sequence capture (PathSeq2.0) will be used to screen world-wide Pi diversity.
AIM4 of the project is to introduce the Rpi- genes into their orthologous chromosomal positions in potato. These "New Breeding Technologies", taking advantage of CrispR/Cas9 editing methods, may enable less controversial control of LB using genetics.
AIM1 is to clone all the Rpi-amr genes in our Solanum americanum stocks. We developed a new method for fast Rpi gene cloning- sequence capture with long PacBio reads- and used this to clone Rpi-amr3. In unpublished work, we thus cloned at least 3 more. We aim to clone the remaining 3-6 Rpi genes in our S. americanum populations, and provide at least 8 genes that could be deployed in stacks.
AIM2 is to test functionality of each Rpi-amr gene in transgenic potato in field environments, and to verify their non-interference in each other's function.
AIM3 is to discover P. infestans effectors (AvrAmrs) detected by our cloned Rpi-amr genes. Rpi genes enable plants to sense P. infestans effectors, and upon recognition, activate plant defence. All P. infestans Avr secreted effectors carry an RxLR motif. It is possible to identify all RxLR effectors in a reference genome. Libraries of such effectors have been constructed in DNA vectors that enable them to be transiently expressed in plant cells; co-expression of Rpi gene and recognized effector causes a hypersensitive cell death response; we will screen existing libraries for such Rpi/effector combinations. With the Hein/Birch labs, we will also use sequence capture ("Pathseq") to refine our knowledge of the full RxLR effector repertoire of Pi. New RxLRs revealed by PathSeq will be added to the list of RxLRs tested to discover AvrAmr effectors. In addition, a refined bait library for sequence capture (PathSeq2.0) will be used to screen world-wide Pi diversity.
AIM4 of the project is to introduce the Rpi- genes into their orthologous chromosomal positions in potato. These "New Breeding Technologies", taking advantage of CrispR/Cas9 editing methods, may enable less controversial control of LB using genetics.
Planned Impact
Phytophthora infestans causes late blight disease and is the most significant threat to potato production, the world's third largest food crop. Late blight is thus a considerable threat to global food security.
Building on established research excellence and jointly developed tools (RenSeq and PathSeq), this proposal will identify and functionally characterise NB-LRR gene resistances from the wild species Solanum americanum (Rpi_amrs). To predict the durability of these Rpi genes, corresponding pathogen avirulence (Avr) genes must be identified. PathSeq is an unpublished tool designed to facilitate the enrichment of pathogen effectors prior to sequencing. A key component of the proposed research is the utilisation of PathSeq to refine the pan-RXLRome of P. infestans effectors and to identify cognate Avr genes for Rpi-amrs. All known avirulence genes belong to this class of effectors, which contain a signal peptide followed by a canonical RXLR motif.
A pilot study of PathSeq in six P. infestans isolates including the reference strain T30-4, provided evidence that the currently described RXLRome is under-representative. By using PathSeq in combination with long-read sequencing platforms such as PacBio and further mining existing, non-enriched PacBio sequences from T30-4, 3928A (13_A2), 2010_7822B (6_A1) and 110059 (US-23) and further mining existing, non-enriched PacBio sequences from T30-4, we will refine the RXLR set of P. infestans and develop a new bait library that included all identified RXLRs (PathSeq2.0). This information, alongside the development of PathSeq, will be made available to the research community through open access publications. Similar to the NB-LRR gene re-annotation of the sequenced potato clone DM, which is accessible through a bespoke potato gbrowser hosted at the James Hutton Institute, the refined effector repertoire of P. infestans will be made easily accessible through a customized P. infestans genome browser that will includes a specific, downloadable track for effectors. There will be no identification restrictions for this Internet resource. Based on the refined RXLR analysis, novel core effectors will be synthesised and cloned into a suitable expression system for transient delivery in Solanum species to help elucidating Avr-amrs. We will share this resource with international groups such as Wageningen University (WU) and INRA Avignon who utilize Effectoromics to characterise host and non-host resistances.
The pan-RXLRome of P. infestans will be established by analysing the genomes of 24 diverse isolates that represent the most diverse genotypes from America, Europe and Asia. PathSeq reads will be obtained from gDNA and cDNA to identify presence/absence, expression and sequence polymorphisms. The nature of the Avr-amrs and the observed sequence/expression polymorphism for Avr-amrs alongside other, known Avr genes will be shared with the commercial partner, Simplot, and the scientific community to assess the potential durability of Rpi-amrs resistances in different geographic locations.
PathSeq will be developed to enable the future use of the technology for rapid and cost-effective analyses of RXLR effectors from diverse P. infestans isolates collected, for example, from the field. As such, PathSeq will complement existing P. infestans genotyping platforms such as currently used SSR markers and will enable a detailed insight into the molecular diversity of infective isolates. This information can be utilised as a diagnostic to chart future changes in P. infestans populations, allowing growers to deploy appropriate resistances to sustain durable disease resistance in the face of such changes. In addition, PathSeq could be established, in the future, as a component of precision farming and inform farmers about the necessity to apply chemical controls agents if deployed resistances could be threatened by emerging P. infestans isolates that can evade recognition by Rpi genes.
Building on established research excellence and jointly developed tools (RenSeq and PathSeq), this proposal will identify and functionally characterise NB-LRR gene resistances from the wild species Solanum americanum (Rpi_amrs). To predict the durability of these Rpi genes, corresponding pathogen avirulence (Avr) genes must be identified. PathSeq is an unpublished tool designed to facilitate the enrichment of pathogen effectors prior to sequencing. A key component of the proposed research is the utilisation of PathSeq to refine the pan-RXLRome of P. infestans effectors and to identify cognate Avr genes for Rpi-amrs. All known avirulence genes belong to this class of effectors, which contain a signal peptide followed by a canonical RXLR motif.
A pilot study of PathSeq in six P. infestans isolates including the reference strain T30-4, provided evidence that the currently described RXLRome is under-representative. By using PathSeq in combination with long-read sequencing platforms such as PacBio and further mining existing, non-enriched PacBio sequences from T30-4, 3928A (13_A2), 2010_7822B (6_A1) and 110059 (US-23) and further mining existing, non-enriched PacBio sequences from T30-4, we will refine the RXLR set of P. infestans and develop a new bait library that included all identified RXLRs (PathSeq2.0). This information, alongside the development of PathSeq, will be made available to the research community through open access publications. Similar to the NB-LRR gene re-annotation of the sequenced potato clone DM, which is accessible through a bespoke potato gbrowser hosted at the James Hutton Institute, the refined effector repertoire of P. infestans will be made easily accessible through a customized P. infestans genome browser that will includes a specific, downloadable track for effectors. There will be no identification restrictions for this Internet resource. Based on the refined RXLR analysis, novel core effectors will be synthesised and cloned into a suitable expression system for transient delivery in Solanum species to help elucidating Avr-amrs. We will share this resource with international groups such as Wageningen University (WU) and INRA Avignon who utilize Effectoromics to characterise host and non-host resistances.
The pan-RXLRome of P. infestans will be established by analysing the genomes of 24 diverse isolates that represent the most diverse genotypes from America, Europe and Asia. PathSeq reads will be obtained from gDNA and cDNA to identify presence/absence, expression and sequence polymorphisms. The nature of the Avr-amrs and the observed sequence/expression polymorphism for Avr-amrs alongside other, known Avr genes will be shared with the commercial partner, Simplot, and the scientific community to assess the potential durability of Rpi-amrs resistances in different geographic locations.
PathSeq will be developed to enable the future use of the technology for rapid and cost-effective analyses of RXLR effectors from diverse P. infestans isolates collected, for example, from the field. As such, PathSeq will complement existing P. infestans genotyping platforms such as currently used SSR markers and will enable a detailed insight into the molecular diversity of infective isolates. This information can be utilised as a diagnostic to chart future changes in P. infestans populations, allowing growers to deploy appropriate resistances to sustain durable disease resistance in the face of such changes. In addition, PathSeq could be established, in the future, as a component of precision farming and inform farmers about the necessity to apply chemical controls agents if deployed resistances could be threatened by emerging P. infestans isolates that can evade recognition by Rpi genes.
Organisations
People |
ORCID iD |
| Paul Birch (Principal Investigator) |
Publications
He Q
(2018)
Plant pathogen effector utilizes host susceptibility factor NRL1 to degrade the immune regulator SWAP70.
in Proceedings of the National Academy of Sciences of the United States of America
McLellan H
(2022)
Exploiting breakdown in nonhost effector-target interactions to boost host disease resistance.
in Proceedings of the National Academy of Sciences of the United States of America
Thilliez GJA
(2019)
Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes.
in The New phytologist
Wang H
(2021)
Evolutionarily distinct resistance proteins detect a pathogen effector through its association with different host targets.
in The New phytologist
| Description | We have redefined and identified new RXLR effectors in Phytophthora infestans We have identified new resistance genes that can be utilised by the IPA partner Simplot for developing resistant potatoes We have developed new markers for breeding late blight resistance in potato which are being used in the UK and internationally We have identified new methods by which Resistacne proteins identify Avirulence effectors We have revealed that different R proteins can detect AVR proteins from the pathogen via their actions on independent effector targets in the host |
| Exploitation Route | We have identified new resistance genes that can be utilised by the IPA partner Simplot for developing resistant potatoes We have developed new markers for breeding late blight resistance in potato which are being used in the UK and internationally |
| Sectors | Agriculture, Food and Drink |
| Description | The Americanum resistance gene Rpi-Am3 has been taken up by the company JR Simplot in transgenic potato lines for deployment. New methods for gene enrichment for effectors and for receptor like kinases have been developed |
| First Year Of Impact | 2019 |
| Sector | Agriculture, Food and Drink |
| Impact Types | Societal,Economic |
| Title | Potato Y2H library |
| Description | This potato yeast-2-hybrid library has been well characterised and has been demonstrated to have excellent coverage in terms of appropriate genes and in terms of the gene sizes covered. |
| Type Of Material | Biological samples |
| Year Produced | 2010 |
| Provided To Others? | Yes |
| Impact | It has facilitated identification of protein-protein interactions in potato and has identified the potato targets of effectors from the late blight pathogen Phytophthora infestans |
| Description | Plant Power day |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | The public engagement aims of the School of Life Sciences are to: 1. Build on our creative partnerships to deliver a high quality, innovative engagement programme. 2. Engage a range of people with our research. 3. Collaborate with our local communities to meet their needs and widen our reach. 4. Promote and support a culture of active participation in public engagement within our life sciences community. This includes students, research and non-research staff. In the Division of Plant Sciences, research includes topics such as genetic modification, food security and sustainability. "Plant Power" is an annual event that takes place in collaboration with and at the University of Dundee Botanical Gardens where various different groups and organisations participate with plant related activities/shows. A science strand was delivered by researchers from the Division of Plant Sciences at the University of Dundee and the James Hutton Institute. The aim of the science strand is to engage the general public, mainly family groups, to learn about the plant research taking place locally and why this research is important. Topics such as genetic modification, food security and sustainability are covered. As a division wide event, it addresses our School strategy aim of promoting and supporting a culture of active participation in public engagement within our life sciences community. This includes students, research and non-research staff. The researchers presented different (12 in total) interactive hands-on outreach activities related to their respective groups' research to the visitors. These activities are either brand new or have been developed by researchers over a number of years at various events. Various modes were used to communicate the research as shown by the diversity of activities undertaken such as the use of simple games (Jumping Genes & 1,000 year Old Farm); craft activities (flower felting & Mendelian Genetics); and accessible science experiments (DNA extraction from raspberries & detection of microbes on the roots of plants). Over 1000 people came to the Botanical Gardens for the event. They are generally family groups with young children (below 10 years of age). We estimate that around 200-250 people visited our science strand area. Feedback from the public indicated that they enjoyed all the activities and learned lots of new plant related facts. Feedback comments included: - Learnt about multicoloured corn - I did not know it existed. - I did not know corn could be multicoloured. - I've learnt where bananas and pineapples come from. - I learnt about potatoes - Learnt about microbes on roots Legacy: Follow on plans are for the activities developed for Plant Power to become formal educational borrow boxes for teachers to utilise for an in-class learning resource. An overall Plant Sciences box aligned with the Curriculum for Excellence for Scottish schools and investigating formally sharing activities via publications would be a subsequent step. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Plant Power day 2019 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | Plant Power is an annual event that takes place at the University of Dundee Botanic Garden where various different groups and organisations participate with plant related activities/shows. A science strand is delivered by researchers from the Division of Plant Sciences at the University of Dundee and the James Hutton Institute. We presented different interactive hands-on activities related to their respective groups research to the visitors. These activities are either brand new or have been developed over a number of years at various events. The aim is to allow the public to learn about the research taking place locally and why this research is important. Various modes were used to communicate the research as shown by the diversity of activities e.g. use of games (pin the plant & botany trail); craft activities (chromosome modelling & lino printing); science experiments (raspberry DNA extraction); art (animating science). My research was represented in this program of work by Hazel McLellan, Qin He, Petra Boevink, Haixia Wang and Dionne Turnbull. Approximately 970 people came to the Botanic Garden for the event. They are generally family groups with young children (below 10 years of age). We estimate that around 200-250 people visited our activities. Feedback from the public indicated that they enjoyed all the activities. Researchers fed back the questions and statements from the public asked while interacting with them. Examples include people not realising that DNA is everywhere in a plant (DNA extraction activity); asking questions about how experiments are conducted with plants and the differences between plant and animal cells (chromosome modelling). |
| Year(s) Of Engagement Activity | 2016,2017,2018,2019 |
| Description | Plant Science Gatsby Lectures 2019 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Speakers from the division of Plant Sciences delivered two lectures to secondary pupils and teachers on the topics of climate change and pharming. Lectures lasted roughly 40 minutes and were followed by hands-on activities and the chance for pupils to speak to scientists and postgraduate students about the topics. Students were very positive about the experience, the hands-on activities in particular, and shared that they had learned new information that was pertinent to the curriculum. Following the lectures two of the schools expressed interest in working with Life Sciences on further projects, and a collaborative project around sustainability and lab research will begin at the end of February 2020 with them. |
| Year(s) Of Engagement Activity | 2017,2018,2019 |
| Description | organisation of the IS-Molecular Plant Microbe Interactions congress in Glasgow |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | I organised the IS-MPMI meeting in Glasgow. This was the largest ever such meeting, with 1450 attendants. The attendants spent at least a week in Scotland, primarily in Glasgow, which impacted the local economy. The meeting was feateured on Radio BBC Scotland |
| Year(s) Of Engagement Activity | 2019 |
| URL | https://www.ismpmi.org/Congress/2019/Pages/default.aspx |