Agricultural Bio-Markers for Array Technology

Objective

A. Background

Micro-array technology refers to techniques that allow simultaneous analysis of many thousands of individual tests. The availability of this new technology opens up outstanding perspectives for the field of molecular diagnostics in its broadest sense. For the first time, it is now possible to devise diagnostic tests that cover very broad fields of application. For example, diagnosis of all agronomically important arthropod pests on one single micro-array chip with an area of less than 5 square centimetres may now be possible. Micro-array-based molecular diagnostics therefore is an extremely powerful tool that will significantly advance modern agricultural diagnosis.

The full potential of modern phytodiagnosis can only be exploited if the relevant technology is highly reliable, fast and economical. Furthermore, as one major limitation in the field of phytodiagnosis is the limited throughput of the currently available methods, the technology must also be amenable to mass-screening. Finally, because the field of phytodiagnosis covers so many different organisms, the technology should be highly flexible. Ideally, it should allow combining tests for hundreds of organisms in one single assay.

A variety of methods are currently used to address these problems. Antibody-based methods, such as ELISA, are still widely used to diagnose detectable levels of virus diseases, and culturing methods to phenotypically identify bacterial and fungal infections. New nucleic acid-based assays, mainly based on the polymerase chain reaction (PCR), are now available to identify many of the most important viruses and bacteria, as well as eukaryotic pests and diseases. The main problem with these methods is that they generally require a targeted approach. For example, PCR technology requires specific primers to amplify diagnostic DNA or RNA sequences before the specific identification reactions, such as enzymatic digestion (RFLP, restriction fragment length polymorphism) or sequencing, can be performed. These methods are technically demanding and require sophisticated laboratory skills. The many diverse methods and conditions that are needed for accurate identification make it difficult to cover the entire field of phytodiagnosis in a single laboratory. Even when these aspects are all mastered, within-species genetic diversity requires highly redundant analyses for precise identifications.

The advent of micro-arrays marks a quantum leap in genome research. They have already proved their great potential in many studies on gene regulation. Applied to genotyping, micro-arrays offer the possibility of determining alleles at hundreds of thousands of loci from hundreds of DNA samples. The feature of massively parallel analysis of hundreds of genetic markers in a short time and under identical conditions makes micro-arrays an ideal tool for molecular phytodiagnostics.

Micro-array technology is based on the same concept as Northern and Southern blots, i.e. hybridisation of target molecules to probes attached to a solid support. Miniaturisation of the probe spots enables us to place in an array format up to several 10,000 samples on a single microscope slide, often referred to as a "chip". To date, the probes generally represent oligonucleotides or short DNA fragments, and such micro-array chips have been used very successfully in assessing gene regulation for several years. Protein based arrays have only recently been used yet their potential is very promising. Despite their undisputed potential, micro-arrays have so far not been widely used for diagnostic purposes, and this is especially true for the field of plant science.

Need for a new COST Action

Micro-array technology offers several major advantages over currently existing diagnostic methods. For the first time, the technology will enable us to produce standardised and harmonised test methodology for a wide range of economically important pests and pathogens across all European countries. Furthermore, the widespread use of the technology, using the same micro-array chips will allow significant savings in overall costs of phytodiagnostics.

Among the main problems that has hampered the widespread use of micro-arrays in diagnostics is the vast amount of information needed to build the chips. For each organism to be identified, species-specific sequence information has to be elaborated and the most reliable probes have to be developed and tested. Considering the wealth of species important in phytodiagnosis, this task by far exceeds the potential of a single laboratory or even a single nation.

The success of introducing this new, multidisciplinary technology as a general tool in phytodiagnostics in Europe depends critically on broad acceptance, commercialisation, and cross-linking between different fields of application in the early stages of implementation. In addition, awareness of developments in the different disciplines working on array application is of great advantage especially in the process of evaluating the suitability of the various micro-array techniques.

A COST concerted action serving as a platform for interdisciplinary communication and as a distribution organisation for elaborated methods and progress on this new technology is ideally suited to address this demanding task. This COST Action will form a European network of laboratories with similar interests that will collaborate in the evaluation of the suitability of the developed array-products on a pan-European scale, and it will establish the awareness of its advantages even in those European countries that do not actively take part in developing array-technology based products.

B. OBJECTIVES AND BENEFITS

The main objective of the Action is to establish and support micro-array technology - in the form of nucleic acid- and protein-based arrays - as a new tool for breeding, diagnosis, and high throughput screening in the field of agriculture.

The need for a flexible system that allows molecular diagnostics of large sample sizes of many species has long been recognised. However, current technology generally requires focusing on narrow taxonomic groups. Micro-array technology, being based on highly parallel simultaneous analysis, has shown its potential for large-scale genome analysis. The technology would allow for simultaneous querying of hundreds of species specific markers and will thus enable the production of micro-array chips that can be used for larger taxonomic groups such as the entire bacteria or, in higher eukaryotes, for example the entire class of insecta.

The broad availability of this widely applicable technology will promote and protect development of agricultural products in Europe. The Action will provide a platform to introduce and support this important new technology to many European countries.

The benefits that can be expected from the Action can be summarised as follows:

1.Assessment of the suitability of different micro-array techniques to the field of phytodiagnostics and animal/plant breeding

2.Establishment of a database containing all relevant information on nucleic acid sequences and proteins suitable for micro-array - based phytodiagnosis

3.Co-ordination of micro-array chip composition and production, profiting from the combined knowledge of all participating countries

4.Co-ordination of implementation of specific micro-array technologies in participating countries, thereby establishing this important new technology in European phytodiagnosis

5.Significant improvement in harmonisation of European phytodiagnosis

6.Support of this new phytodiagnostic technique on a broad European level

C. SCIENTIFIC PROGRAMME

Micro-array technology today is realised by a number of different methods. Differences concern the type of probe that is attached to the solid surface, the surface substrate, the spot density of the array, and the marker system to detect probe-target interactions. The different systems combine various advantages and disadvantages that make a careful evaluation of their suitability for phytodiagnosis an essential first step in this Action. This evaluation is a highly multidisciplinary activity, covering aspects of choice of substrate, choice of probes and marker system, and aspects of detection technology and bio-informatics. Consequently, the process requires collaboration between specialists from many different fields including industrial R&D. To exploit the huge potential of this technology, it is further necessary to establish a data collection on suitable genetic markers for the intended purpose. The selection of probes for micro-array chip production will be based on this data collection. This requires intensive database searches and close collaboration between the

research laboratories involved in the Action. Many of these activities have to be addressed separately for nucleic acid based and protein based micro-arrays. To accommodate these tasks the following Working Groups (WG's) are proposed:

WG 1:Nucleic-Acid Based Micro-arrays
Subgroup 1:Viral Diagnostics
Subgroup 2:Prokaryotic Diagnostics
Subgroup 3:Eukaryotic Diagnostics

The objective of this WG is to collect all available information on suitable DNA and RNA markers (i.e. sequences) for phytodiagnostic purposes. As this task requires in-depth knowledge of different fields, such as virology and phytopathology, the WG is divided into three Subgroups. Each Subgroup focuses on one large field with its peculiarities. For example, many viruses are RNA based and thus require the corresponding markers, and this will be one of the main activities of Subgroup 1. Subgroup 2 will be mainly involved in bacterial diagnostics. Quite a number of bacterial genomes are now fully sequenced, which allows for database search of the most suitable genomic regions for species-level identification. One marker system, the 16S rDNA, is already widely and successfully used for species identification in bacteria and a corresponding chip already exists. Therefore, this marker sequence is a good candidate to be used in micro-array based prokaryotic identification. Consequently, one focus of this Subgroup will be to assess the suitability of such a chip on a broad scale in phytodiagnostics and to evaluate potential alternatives. The focus of Subgroup 3 will be the genomes of higher eukaryotes, mainly fungi, nematodes, and arthropods. The large size of each of these three taxa may require splitting this Subgroup further.

WG 2:Protein Based Micro-arrays

In principle, this WG will have the same tasks as WG 1, i.e. to collect all available information on suitable protein markers for phytodiagnostic purposes. As this technology is even more recent than the DNA-based micro-array technology, potential Subgroups will be established within the first year, if necessary.

WG 3:Bio-informatics and Information Dissemination
Subgroup 1:Internet Office

This WG will provide the expertise in bio-informatics required for completion of this Action, and it will provide a platform for co-ordination and dissemination of the accumulated information. The main tasks will be: 1) To search public databases, e.g. Genebank, for potential probes; 2) to establish a database with all potential probes, i.e those from the database search and those communicated by WG 1 and WG 2; 3) to adjust probe design to optimal reaction conditions; and 4) to perform micro-array data analysis (in collaboration with WG 4). These tasks may eventually be organised in several Subgroups, yet one specific Subgroup 1 will be formed at the onset of the Action, and its task will be to create an interactive internet homepage for information dissemination. This Subgroup will obtain reports, summaries and discussion contributions from all WG's to be placed on the web-page.

WG 4:Chip Production and Analysis

Micro-array production requires sophisticated equipment that to date is not available in many laboratories. Essentially, a robotic spotting device is needed that allows the probes to be spotted onto the surface of choice for the desired micro-array. Alternatively, methods exist for production of short oligonucleotides directly on the chip. These methods, however, are covered by patents and are not easily accessible. In addition to the spotter a micro-array reader is required. The members of WG 4 either have this equipment in their own laboratories or they have access to such devices. The main task of this WG will be: 1) micro-array spotting and distribution (in collaboration with WG 1 and 2), and 2) micro-array scanning and data analysis (in collaboration with WG 3). Close contacts to companies involved in micro-array technology will be established to ensure that optimal technology is used and to enable potential collaboration.

Selection of topics

The use of micro-arrays in the field of agronomy depends heavily on economic factors, i.e. they need to be inexpensive and there has to be a market. Only if these conditions are met, private companies are likely to join our efforts. Focus therefore mainly will be on pests and diseases of fruit and potatoes. There is a large international trade volume with these crops and there is strict European-wide regulation on testing, which provides a solid basis for later commercial use of the micro-arrays developed for these crops. More specifically, focus will be on viroids and virus diseases for potatoes and on virus, bacterial and phytoplasma diseases on fruit. A micro-array for eukaryotic pests such as nematodes and insects will also be developed for these crops. If other opportunities appear during the Action, they will be included in ongoing programmes.

Definition of the output

Several running COST Actions are using molecular diagnostic methods. However, these actions are not involved in the "de novo" development of new tools for molecular identification of pests and pathogens. These Actions will be contacted as soon as the first micro-arrays are available and to put these new tools on a broad evaluation basis and to further the acceptance of this technology in Europe.

Interactions with other COST Actions already running

A strong complementary interaction can be expected and will be sought for the following topics: Actions 831; 835 and 838, where differentiation and identification by serological and/or nucleic acid based methodology is an essential technique but not the predominant aim. It was already agreed during the 1st InterCOST Action in Lisbon 1998, that especially for these Actions a very close mutual interest can be expected without duplicating topics.

Connections with the two Previous Actions COST 88 and 823

Looking back to previous Actions 88 and 823 where the main emphasis was to promote the methodologies of "Early detection", the results of these Actions were the establishment of a network of laboratories and governmental institutions involved in phytodiagnosis. Ideas and instruments were developed to spread information between participating countries, not only among participating laboratories but also to legal institutions and organisations to support them. It was also the merit of these Actions that the monoclonal, recombinant antibodies, and finally PCR became widely accepted as plant pathogen detection methods.

Finally, as a replacement of the database project, the new technique offered by the world-wide-web was exploited and a method prepared which allows information distribution much easier and cheaper.

Now we are facing a new impetus of a technology called "Micro-Array-Technology" or DNA-chips. Participants of 823 have unanimously agreed that this technology will revolutionise diagnosis and lead to a completely new dimension in this field. It can be foreseen that completely new applications in many different fields like breeding, development of new protective substances and quarantine standardisation will develop.

Interactions with other activities including 5th FP.

Interactions with a project with the working title "Array-Technology as Analytical Tool for Detection and Identification of Plants and Plant Pathogens" submitted within the 5th European Framework Programme is envisaged. In addition, close links to another micro-array project submitted to the EU by the Central Science Laboratory, and to several nationally funded micro-array projects in other European countries will be established.

With respect to industrial participation: in the above mentioned Consortium are at least 4 industrial partners involved, which should be definitely integrated parts also of the new COST Action. An integration of the European Plant Protection Organisation (EPPO) as legal organisation is foreseen. EPPO is the platform for discussions on plant quarantine measures and diagnosis harmonisation in Europe and would be a well-suited body to distribute information from the COST Action into legal bodies of the signatory countries. A first request to explore their willingness was met with great interest.

D. ORGANISATION AND TIMETABLE

The structure of the New Action is shown in the organigram.

Each Working Group will have one co-ordinator. If it appears necessary from the topic of a WG to install additional co-ordinative power, this might be possible on a time-limited basis. During the Action, if need arises, it will also be possible to create sub-WG's which are temporarily active. Decisions on this will be made by the MC during its annual meetings.

The activities which can be foreseen already during the planning stage, as well as the regular activities that arise from organisatory and administrative needs can be summarised as follows:

1.One Management Meeting will be scheduled every year, preferably in January (also to discuss the annual reports).

2.Each WG is expected to hold one WG-Meeting per year. This will be the routine schedule for the years 2-4.

3.For the first year the first MCM will not be jointly with a WG. During this MC Meeting the Action will assemble to agree to and demonstrate the WWW activities. This meeting will enable also to contact and invite the Chair Persons of other COST Actions with related or mutual interests and discuss future co-operation possibilities.

4.In year 3 of the Action Plan an interactive COST meeting should be initiated, because it can be expected that after this periods of activity spin-offs should be available to be shared or distributed.
A duration of five years is considered necessary. The reason is that, working in a field with strong seasonality (the crop field seasons), there are restrictions on sampling. The Action will, try to optimally co-ordinate field season and corresponding sampling with laboratory work. However, there are a large number of organisms that need to be identified. Validation of the samples is very important and will most probably show that partial re-sampling in the next season is required, possibly from different collection sites. In this sense, validation of probes and thus robust identification will be an iterative process. Therefore, it is highly desirable to be able to collect during four field seasons, which will significantly increase the quality of results.

Milestones

The following milestones can be defined:

Year 1:

Establishment of a WWW-platform for communication and information dissemination.
Agreement on a European list of plant pathogens which are suitable for array-technology.
Establishment of a list of suitable targets from sequence data and proteins
Distribution of tasks described in the Scientific Programme between the signatory countries.

Year 2:

Collection of data (sequence and proteins) for organisms with no prior information on the target molecules

Year 3:

Evaluation of the chosen targets for suitability to co-exist on one chip (e.g. optimisation of hybridisation parameters).
Establishment of a database containing generally agreed diagnostics.

Year 4:

Reproducibility testing of chips (evaluation and validation)
Development of a pattern database suitable to serve as future aid for diagnosis.

Year 5:

Implementation of a common format for pattern recognition and evaluation for European arrays.

E. DISSEMINATION PLAN

Beyond its tasks outlined in the Scientific Programme section, Working Group 3 will provide a platform for co-ordination and dissemination of the accumulated information. This WG will establish one Subgroup (No. 1) whose main task will be to create an interactive internet homepage for information dissemination for the Action. This Subgroup will obtain reports, summaries and discussion contributions from all WG's to be placed on the web-page. In addition, this Subgroup will actively disseminate relevant information to all potential users by contributions to web-based discussion groups and to representatives of interested institutions.

F. ECONOMIC DIMENSION

20 countries will participate with at least 35 senior scientists, who will actively be involved in the new Action. It can further be safely estimated that the Action will involve 60 junior scientists and about 120 technicians. This will amount to a total of personnel costs of Euro 6.9 million/year. For scientific work in natural sciences about the same amount of money can be estimated for overheads, investments and consumables. The overall costs of the Action can thus roughly be estimated at Euro 60 million, provided the number of signatory countries becomes twenty.

Programme(s)

IC-COST - European cooperation in the field of scientific and technical research (COST), 1971-

Topic(s)

4 - Agriculture and Biotechnology

Call for proposal

Data not available

Funding Scheme

Data not available

Coordinator

N/A

EU contribution

No data

Address

Switzerland

Total cost