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.

Publications

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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