Temporal Co-regulation of Pathogenesis in Phytophthora

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

How do plant pathogens, such as the potato late blight pathogen, Phytophthora infestans, regulate the timing of their different infection stages, and which genes are required at specific stages of plant infection? Despite the enormous cost and impact of Phytophthora diseases, we know little about how this group of pathogens regulate and coordinate specific stages of plant infection that culminate in disease development.
Late blight, caused by P. infestans, is the most devastating disease of potato, the third most important food crop globally. The very broad host range pathogen P. capsici is a major threat to vegetables, against which (durable) resistance is not available in most crops. Crop plant diseases caused by Phytophthora pathogens are thus a threat to global food security. The situation in Europe is compounded by legislation banning or restricting some chemicals that farmers rely on to prevent Phytophthora diseases. Changes in pathogen populations, coupled with the need to produce more food with a diminished environmental footprint, means that new avenues of disease control must be sought. In addition to P. infestans and P. capsici, more than 120 species of Phytophthora have been characterized, which collectively cause significant disease on almost all dicot crops. Some are limited in host range, and the resources for host genetics and genomics provide novel opportunities to identify and harness natural disease resistance. However, others, such as P. ramorum and P. kernoviae, are emerging as threats to natural ecosystems, infecting a broad range of tree and shrub species with which they have not co-evolved. To combat these, breeding for resistance is not a viable strategy. A deep understanding of Phytophthora infection biology is required to provide novel, next generation targets for highly specific and environmentally benign chemical control, and to identify new avenues that lead to disease resistance in plant hosts.
In order for it to be a successful pathogen, Phytophthora must grow within living plant tissue and then spread to new plants by producing spores. This requires the formation of different pathogen infection structures, which involves the action of many different genes, many of which are only active at these specific stages of infection. The DNA sequences of P. infestans and P. capsici have revealed hundreds (over 500) of candidate virulence factors that are transferred into plant cells to promote disease. These pathogens also have many other potential virulence proteins about which little is known. By identifying which of these candidate virulence genes are most active during specific infection of plants, this project will allow us, for example, to identify how Phytophthora coordinates its gene expression to form specialised infection structures, and what nutrients it obtains from its host plants.
However, the main focus of this project is to identify the 'switches' that initiate and regulate expression of the large numbers of genes required for infection. We will search for those regulatory switches that are common to P. infestans and P. capsici, as essential and conserved are likely to be more promising for later development of broadly applicable disease control strategies. As these are likely to be the central controls of Phytophthora disease development, it is likely that disruption of their function will also severely compromise the ability of Phytophthora to cause plant disease.
Gene expression underlying specific stages of disease development could be exploited through identification of crop plant traits that interfere with, or otherwise reduce, production of Phytophthora virulence factors. Alternatively, as we are seeking the regulatory components that are common to both narrow and broad host range Phytophthora species, these may be attractive targets for development of new chemical control agents that may also be active against other oomycete plant pathogens.

Technical Summary

Driven by the availability of the Phytophthora genome sequences, recent years have seen the identification of vast collections of secreted effector proteins which suppress PAMP-triggered immunity. Much research focuses on the RXLR class of effectors, as these are delivered inside plant cells to directly manipulate host defences.
Many Phytophthora pathogenicity proteins and RXLR effectors show highly coordinated temporal changes in expression. Although the importance of both pathogen protein classes to epidemics is undisputed, we know little about how such tight regulation of expression is achieved. This project aims to combine next generation sequencing of Phytophthora-host interaction transcriptomes, bioinformatic and functional gene promoter analyses, DNA-protein interaction assays, and silencing and overexpression of transcription factors to identify and validate the protein 'switches' that specify Phytophthora gene expression changes during disease establishment.
Transcriptome sequencing will reveal the conserved groupings of genes that exhibit differential expression during infection, giving insight into the mechanisms of infection establishment. Identification of the transcription factors, conserved in narrow (P. infestans) and broad host range (P. capsici) Phytophthora species, that initiate expression of large groups of essential pathogenicity factors will provide next generation targets for the control of Phytophthora disease. That is, wide-ranging disruption of effector and other infection-related gene regulation in Phytophthora, though targeting of their regulators, forms an attractive strategy for disease control. This may be realised through development of novel chemical agents designed to generally inhibit infection processes conserved across Phytophthora species, or through traits present in crop plant germplasm. As such, this project will provide the basis for control solutions for new threats to crops and natural ecosystems.

Planned Impact

More than 120 species of Phytophthora have been described, all of which cause diseases of dicot plants. Some, such as P. infestans, which is the major constraint to global potato production, are limited in host range, and the resources for host genetics and genomics provide novel opportunities to identify and harness natural disease resistance. However, others, such as P. capsici, infect a broad range of economically important crop hosts and strong resistance traits are often lacking. Furthermore, species such as P. ramorum and P. kernoviae are emerging as threats to natural ecosystems, infecting a broad range of tree and shrub species with which they have not co-evolved. To combat these, breeding for resistance is not a viable strategy. A deep understanding of Phytophthora infection biology is required to provide novel, next generation targets for highly specific and environmentally benign chemical control, and to identify new targets for disease resistance in crop plant hosts.

To date, all characterized host resistances to oomycetes have been found to detect RXLR effectors, which are delivered to the inside of plant cells, and exhibit elevated levels of transcript accumulation during infection. All other factors from Phytophthora found to be essential for infection also exhibit elevated expression during infection. This suggests that effective targets for control of Phytophthora disease may be identified from the interaction transcriptome. Although not the immediate focus of this project, it will deliver potential conserved pathogen targets that may encompass previously uncharacterized secreted effectors for detection by host R genes, conserved metabolic proteins to be targeted for chemical control, or the regulators of infection-specific gene expression themselves. An additional outcome that is outside the scope of this project will be the identification of plant genes that exhibit modified expression in the presence or absence of specific groups of pathogen effectors, and which may be involved in plant defence against disease. Each of these outcomes can potentially lead to next-generation targets for precise and oomycete-specific disease control.

Earlier Phytophthora gene expression studies have either lacked sensitivity (microarray) or systematic sampling (qRT-PCR) of early time points of infection. For example, 48 hours after infection has been the earliest infection time assessed by microarray, although much transcriptional activity has already occurred before this time. By combining sequencing of the interaction transcriptomes at a series of time points, combined with protein/DNA interaction assays, promoter functional assays in the pathogens, and silencing of transcription factors, this project will provide a fundamental understanding of coordinated gene expression and components of regulation during Phytophthora infection.

Exploitation of plant germplasm in breeding programmes of solanaceous crops are part of JHI's (potato) and Syngenta's (tomato, pepper) business. As we are working with a common solanaceous host plant in this project, findings will be directly translatable to the three major crop plants of interest to the project partners. With Syngenta as a partner in this project, there is scope for immediate dialogue with end users of the data produced from this project. Added value is also derived from knowledge transfer from the academic partners at UoD and JHI to Syngenta regarding action and targets of effectors, and essential pathogenicity factors in Phytophthora. This direct connection with industry will facilitate the conversion of academic knowledge to commercial outcome, with more rapid benefits to industry and agriculture.

Publications

10 25 50
 
Description So far, we have

1. Identified motifs that may underpin gene regulation in P. capsici (and possibly P. infestans). we are about to start screening for the TFs that may bind these elements

2. We have analysed a large RNAseq experiment to further refine motif predictions. we are able to quantify P. capsici gene expression at the very early stages, a capability that we will seek to exploit for further motif identification.

3. We have identified a candidate TF in P. capsici that when over expressed, gives a phenotype during infection. We are currently characterising this protein further (Co-IP, Y2H) and have completed microarray analyses on over expression lines to identify candidate target genes. we are currently preparing an MS that describe these results.

4. We have identified TFs that appear to be differentially expressed in P. capsici. We are currently studying their function in vivo by means of OE and by attempting knock-down/outs. We have demonstrated that PcNMRAL1 may regulate the transition from biotrophy to necrotrophy in P. capsici. This work has now been published (Pham et al., 2018)
Exploitation Route We may be able to target NMRA like proteins in P. capsici (chemically) to disable pathogenesis.
Sectors Agriculture, Food and Drink,Chemicals,Creative Economy

URL https://www.ncbi.nlm.nih.gov/pubmed/?term=29419371
 
Description We have communicated our results to Syngenta, our industrial partner. We will send the Y1H materials to this company as well as the sequences and expression data of all genes differentially expressed during infection. Since this is an industrial collaboration, the intent on data use has not been disclosed to me. I therefore cannot fully comment on future use by our partners.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Creative Economy,Other
Impact Types Economic

 
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 Syngenta (Industrial Partner) 
Organisation Syngenta International AG
Country Global 
Sector Public 
PI Contribution We have contributed gene expression data, expertise and our latest results of our research to Syngenta in the form of reports and meetings.
Collaborator Contribution Syngenta made a £35,000 contribution as they are part of this IPA award.
Impact Gene expression information on P. capsici-host interactions. Annotation and characterisation of candidate transcriptional regulators. Site visits and exchange of ideas, expertise and information (knowledge exchange).
Start Year 2013
 
Description Cafe Science Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Cafe Science to the public about my research. included research funded by BBSRC
Year(s) Of Engagement Activity 2015
 
Description Collaborator event Syngenta 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation paper presentation
Geographic Reach National
Primary Audience Industry/Business
Results and Impact This was an annual event that is organised at Syngenta to present work on projects that are sponsored by Syngenta. Dr. Jasmine Pham presented the work of the project (in collaboration with project lead Dr. Steve Whisson) to a mostly academic audience.

None known to us
Year(s) Of Engagement Activity 2014
 
Description Invited speaker LFC workshop in Hohhot (Inner Mongolia, China) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation to the LFC workshop focussed on Molecular plant-microbe interactions
Year(s) Of Engagement Activity 2017
 
Description Invited speaker Nanjing Agricultural University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Presentation to the faculty of NAU, dept of plant pathology.
Year(s) Of Engagement Activity 2017
 
Description Phytophthora capsici¬†effectors target the host nucleus to promote virulence 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Scientific talk at COST meeting: Workshop on cellular dynamics of effector action and recognition Presentation and Poster

no actual impacts realised to date
Year(s) Of Engagement Activity 2014
 
Description Presentation at the Oomycete Genomics Network Meeting, Asilomar (CA, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation given by Dr. Jasmine Pham (PDRA employed on grant) at a scientific meeting.
Year(s) Of Engagement Activity 2015
 
Description Research Collaboration Event, Syngenta 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Jasmine Pham, PDRA employed on this grant, attended a Research Collaboration Event at Syngenta, on the 2nd and 3rd of Sept 2013. At this event, a poster was presented on her most recent work on this project. None Poster

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
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
 
Description Visit to Syngenta 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Visit to Syngenta where a research talk was given. presentation was broadcasted to Syngenta research sites world wide.
Year(s) Of Engagement Activity 2015