Omics-enabled detection and studies of phytophthora capsici disease in the field
Lead Research Organisation:
University of Dundee
Department Name: School of Life Sciences
Abstract
Phytophthora. capsici is one of the most important pathogens in pepper production. The early detection and diagnosis of disease forms a front line strategy in our efforts to limit damage caused by epidemics on crops. To help deliver on this strategy, we will chase the following milestones:
1. Assess the presence of a set of P. capsici specific genes (PSGs) across a diverse set of isolates (Year 1). Candidate PSGs will be amplified from DNA from a diverse set of available P. capsici isolates and sequenced. PSGs that are conserved between isolates will be taken forward towards Aim 2. PCR and sequencing is routine in the laboratory and we thus do not envision any problems.
2. Design P. capsici specific primer sets for a suitable PCR test (Years 1-2). We will design a test that is fully compatible with Enza's automated genotyping facility. We will use primers defined in aim 1 to devise a quick and reliable PCR based test. For this we will generate infected material in the lab, including other Phytophthora species as negative controls. The test will be validated by using field samples from 2 EZ breeding sites (Asia). These will be harvested on site and sent to ENZA for processing (DNA and RNA isolation). With the vast experience in genotyping, automated sample processing and molecular biology expertise in the Huitema Lab, we do not envision any problems with this aspect of this research. The student will work at ENZA for 3 months on this assay to ensure full implementation within the facility.
3. Sequence the transcriptomes and characterize the proteomes of P. capsici infected plants (Years 2-3). We will use diseased plants (identified in 2) for further characterization by means of RNASEQ and proteomics analyses. We will extract RNA and protein from diseased plants and analyse biotrophic as well as necrotrophic tissues. This should lead to the identification of the most abundant PSG encoded proteins in natural infections. These proteins will be used towards aim 4. In addition to work towards aim 4, we will also identify the (effector) genes that are expressed in the field. These are important targets for (R-gene based) control measures and should help prioritize effector genes for detailed studies in the laboratory. This will provide a unique view of the genes that are expressed during field epidemics. Some P. capsici transcripts will have low abundance. If required we will try to solve this problem with the use of exon capture, a technology that we are considering to employ in the lab.
4. Generate P. capsici-specific antibodies, suited for detection in the field (Years 3-4). Given that generally, the shipment and processing of diseased materials presents limitations, work towards this aim will help design a (Elisa based) test, suited for on-site diagnostics of P. capsici infection. This test is complementary to diagnostics developed in aim 2 and can be used as cross-validation. We will extract proteins from infected plant tissue and submit these for LC-MSMS. Peptide spectra will be searched against P. capsici and pepper proteomes to identify species specific and abundant P. capsici proteins in field samples. 5 Candidate proteins will be expressed and used to raise specific antibodies against P. capsici. These antibodies will then be used for validation against a diverse set of P. capsici strains and other Phytophthora samples (negative controls). The student will work for at least another 3 months at ENZA towards this aim.
1. Assess the presence of a set of P. capsici specific genes (PSGs) across a diverse set of isolates (Year 1). Candidate PSGs will be amplified from DNA from a diverse set of available P. capsici isolates and sequenced. PSGs that are conserved between isolates will be taken forward towards Aim 2. PCR and sequencing is routine in the laboratory and we thus do not envision any problems.
2. Design P. capsici specific primer sets for a suitable PCR test (Years 1-2). We will design a test that is fully compatible with Enza's automated genotyping facility. We will use primers defined in aim 1 to devise a quick and reliable PCR based test. For this we will generate infected material in the lab, including other Phytophthora species as negative controls. The test will be validated by using field samples from 2 EZ breeding sites (Asia). These will be harvested on site and sent to ENZA for processing (DNA and RNA isolation). With the vast experience in genotyping, automated sample processing and molecular biology expertise in the Huitema Lab, we do not envision any problems with this aspect of this research. The student will work at ENZA for 3 months on this assay to ensure full implementation within the facility.
3. Sequence the transcriptomes and characterize the proteomes of P. capsici infected plants (Years 2-3). We will use diseased plants (identified in 2) for further characterization by means of RNASEQ and proteomics analyses. We will extract RNA and protein from diseased plants and analyse biotrophic as well as necrotrophic tissues. This should lead to the identification of the most abundant PSG encoded proteins in natural infections. These proteins will be used towards aim 4. In addition to work towards aim 4, we will also identify the (effector) genes that are expressed in the field. These are important targets for (R-gene based) control measures and should help prioritize effector genes for detailed studies in the laboratory. This will provide a unique view of the genes that are expressed during field epidemics. Some P. capsici transcripts will have low abundance. If required we will try to solve this problem with the use of exon capture, a technology that we are considering to employ in the lab.
4. Generate P. capsici-specific antibodies, suited for detection in the field (Years 3-4). Given that generally, the shipment and processing of diseased materials presents limitations, work towards this aim will help design a (Elisa based) test, suited for on-site diagnostics of P. capsici infection. This test is complementary to diagnostics developed in aim 2 and can be used as cross-validation. We will extract proteins from infected plant tissue and submit these for LC-MSMS. Peptide spectra will be searched against P. capsici and pepper proteomes to identify species specific and abundant P. capsici proteins in field samples. 5 Candidate proteins will be expressed and used to raise specific antibodies against P. capsici. These antibodies will then be used for validation against a diverse set of P. capsici strains and other Phytophthora samples (negative controls). The student will work for at least another 3 months at ENZA towards this aim.
People |
ORCID iD |
| Edgar Huitema (Primary Supervisor) | |
| Rory McLeod (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M016676/1 | 01/10/2015 | 30/09/2019 | |||
| 1644375 | Studentship | BB/M016676/1 | 01/10/2015 | 30/09/2019 | Rory McLeod |
| Description | iCASE PhD studentship scheme |
| Amount | £95,042 (GBP) |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 10/2015 |
| End | 09/2019 |
| Description | James Hutton Institute collaboration on project |
| Organisation | James Hutton Institute |
| Department | Cell and Molecular Sciences |
| Country | United Kingdom |
| Sector | Public |
| PI Contribution | We have held regular meetings to discuss projects on our understanding of Phytophthora gene expression during infection of plants. we have generated a combined P. infestans and P. capsici Y1H library for our collaborators and screened motifs (present in both organisms) for interactors (transcription factors). in addition, we have tried to over-express P. infestans TFs in P. capsici to get to grips with possible roles in infection. |
| Collaborator Contribution | Complementary efforts toward understanding these processes allowed us to test the possible roles of regulators in two distinct Phytophthora species. these efforts include expression profiling of P. infestans and P. capsici candidate transcriptional regulators and comparative analyses. |
| Impact | Comparative gene expression analyses and datasets for candidate TFs. Candidate DNA motifs present in either P. capsici, P. infestans or both |
| Start Year | 2013 |
| Description | iCASE studentship |
| Organisation | Enza Zaden |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Phytophthora capsici is a devastating pathogen capable of infecting important crops worldwide. Disease in the field can lead to eradication of crops and large financial losses if left untreated. Current diagnosis of the pathogen from the field is time consuming, difficult and requires highly trained specialists to handle and process samples. A more efficient diagnosis method is needed to ensure farmers can effectively maintain and manage their crops. Working with recently isolated P. capsici strains from the field is beneficial for both phytopathologists and plant breeders to identify the mechanisms used by the pathogen to cause infection and to develop resistant, commercial crops. To bridge the gap between laboratory and field knowledge, successful diagnosis and isolation of the pathogen is necessary. A genome of a single isolate of P. capsici has been sequenced and is publicly available. I have used this genome - as well as sequencing three other field isolates using three different sequencing technology - to use an omics approach to address the diagnostic issues that are currently faced. Here, I have employed two bioinformatic pipelines to aid the diagnosis effort to diagnose P. capsici from the field. The first (OEDs), designs diagnostic primers that are species-specific; and the second (PDP), designs primers that can discriminate within the species, resulting in isolate-specific primers. We conclude that both pipelines can design discriminatory diagnostic primers, but more sequence data and validation is required. These pipelines show great promise for diagnosing eukaryote plant pathogens found in the field. |
| Collaborator Contribution | Enza Zaden were our formal collaborators and partners in this project. As part of the iCASE agreement, they hosted the PhD student, Rory McLeod, in their research department where he looked at virulence of P. capsici on elite pepper breeding lines. In addition, they have made the following contributions: Supervision and management of student, including regular site visits make available greenhouse facilities and breeding materials for experimentation Sequencing (MiSeq) of 4 P. capsici isolates (in-house) Provide access to breeding locations in Indonesia (through Ewindo) |
| Impact | Grant proposal (IPA). Failed. |
| Start Year | 2015 |
| Title | Improved Pipeline for the design of species and isolate specific primers |
| Description | Modification of existing pipelines to handle sequence data from eukaryote microbes. OEDS: This pipeline takes (publically) available genome sequences for a range of pathogens (within the Phytophthora genus) and identifies regions of genomes that are unique to a given pathogen. By using sequences from multiple isolates within a species, the pipeline can automatically design species-specific primers for validation and deployment in the lab and field respectively. PDP: Primer Diagnostic Pipeline (PDP) is a modular pipeline derived from the Find Differential Primers pipeline. The published pipeline has seven steps - validate input config file, concatenate sequences, identify features, predict primer locations, cross amplification, BLAST screen and classify. PDP has the ability to produce diagnostic primer candidate-specific at species, sub-species and isolate level (depending on the dataset). The sub-species-specific primers can group particular isolates together whilst distinguishing them from other isolates. |
| Type Of Technology | Software |
| Year Produced | 2019 |
| Impact | We have deployed and conducted some limited tests on the rapid design and evaluation of primers that are species-specific and/or can discriminate between isolates of a given pathogen. |
| Description | Research talk & Visit to Enza Zaden |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Industry/Business |
| Results and Impact | Research talk and exploration of future collaboration with EZ staff. Discussion has led to one successful iCASE application (see portfolio) and one BBSRC-IPA grant application (not funded). |
| Year(s) Of Engagement Activity | 2014 |
| Description | Scientific Talk at Enza Zaden |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Industry/Business |
| Results and Impact | Presentation of work towards understanding P. capsici biology to a breeder audience (Enza Zaden Company) in the Netherlands |
| Year(s) Of Engagement Activity | 2018 |