Award details

Isolation and characterisation of pathogenicity genes from the downy mildew Peronospora parasitica using phage display and phage antibody approaches.

Reference	BBS/E/C/00823332
Principal Investigator / Supervisor	Dr Paul Bowyer
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	77,015
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

The fungi and pseudo-fungi (Oomycetes) are probably the most damaging agents of plant disease worldwide causing crop losses of ield100s of millions in the UK alone. Despite the significant advances which have been made in our understanding of fungal pathogenic processes in plants over the past years there has been little progress in the study of the common and economically important obligate biotrophs, due to problems in culture, transformation and handling of these difficult" organisms. Obligate biotrophs must undergo a series of distinct developmental steps during the pathogenic process. Each developmental step requires the formation of specific cell types or infection structures eg. germtubes, appressoria, penetration hyphae, infection hyphae and haustoria. Much attention has been focussed on understanding early developmental stages, such as germination and appressorium formation, as it is expected that interference with such early developmental events (eg. by fungicides) will provide effective control of the disease. Appressoria enable penetration of the plant surface through production of directed turgor pressure or degradative enzymes and are essential for disease. Chemicals that interfere with factors involved in appressorium development would thus be a good candidates for new fungicides. There have been no detailed studies on the cell biology of appressorium differentiation in the commercially important downy mildews. We propose to identify factors involved in the formation of appressoria of the downy mildew Peronospora parasitica (Pp), which arise as apical swellings from germ tubes and are formed above anticlinal cell walls. This plant- pathogen system has the advantage that large numbers of spores can be produced rapidly and synchronously on rapid-cycling brassica. Recent work at LARS has shown that appressoria can be synchronously produced from spores in large numbers on scored hydrophobic surfaces such as polythene or polycarbonate, thus allowing immediate protein (or RNA) isolation. Attempts to identify appressorium specific factors are usually focussed on raising antibodies against infection structures isolated from infected plants. This approach has been successful but has the drawbacks that: 1) production of monoclonal antibodies is very time consuming, 2) the infection structures are ex planta for 12 - 48 hrs before purification is complete, raising questions as to whether their protein profiles reflect the situation in planta, and 3) the ethical consideration that antibodies are raised in animals that are subsequently sacrificed. We propose to use newly available phage antibody technology, in which antibodies are displayed on the surface of E. coli bacteriophage M13, to rapidly (1-2 weeks in total) raise multiple monoclonal phage antibodies against appressorial proteins (and potentially other structural molecules) from Peronospora parasitica. Phage antibodies have several advantages over conventional monoclonals: they are rapid to produce, they can be raised against many proteins and compounds against which no antibodies could previously be raised, they suffer from no problems of immunodominance, and they by-pass animal immunization. Phage antibodies will be used to examine spatial and temporal distribution of antigens during appressorial development. Although RNA differential screening processes are problematic in cases where developmental structures form in plants, they have been shown to work in cases where structures can be induced to form ex planta. We will attempt to use RNA from Pp appressoria formed ex planta from germinating spores and RNA from germinating Pp spores to differentially screen for genes expressed specifically during appressorial development. Objectives 1996 Not supplied. 1997 Establish in vitro appressorium production systems, raise phage antibodies against appressorial proteins and screen to identify appressorium specific antigens."

Summary

unavailable

Committee	Closed Committee - Agri-food (AF)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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