The Structure and Workflow in BIOEXPLOIT

Subproject 1: To identify targets for durable resistance by analysing fungal effector molecules.

Plants have developed an advanced and highly specific surveillance system to detect invading pathogens. Pathogens betray their presence by passively or actively releasing effector molecules at the interface with the host. These effector molecules collectively facilitate growth and reproduction of the fungus on a host plant. Co-evolution between pathogens and plants has resulted in numerous resistance genes capable of recognising a wide range of effector molecules (avirulence genes). Only a small number of these resistance genes is known and currently used for pest control in agriculture. Some of these resistance genes have a prolonged life span in agro-ecosystems, because they recognise fungal effector molecules that are essential for the fitness of the pathogen. In Subproject 1 we will undertake a large-scale search to identify and to characterise pathogen effector molecules from Phytophthora infestans, Septoria tritici, Blumeria spp., and Puccinia spp. The output of Subproject 1 will be a range of pathogen genes encoding effector molecules of which some are recognised by cognate R proteins studied in Subproject 2. Laboratory and field experiments will be undertaken to study the cost of losing avirulence effector activity for the pathogen as predictor of durable resistance. The evolution of avirulence genes in response to selection in agriculture will be studied, to predict resistance deployment strategies that maximise the durability of resistance. The fungal effector molecules will be used in Subproject 2 to determine the recognition specificity of resistance genes. Subproject 3 will use Avr - R gene pairs to reveal the mechanisms underlying recognition specificity and activation of disease resistance signalling in resistance responses. For Subproject 4 the fungal effector molecules will be captured in expression libraries to facilitate a high throughput screening of the allelic variance in customised core collections on resistance loci using agroinfiltration assays. In Subproject 5 specific avirulence genes will be used as a molecular marker for marker-assisted breeding.

Subproject 2: To map, isolate and characterise genes responsible for qualitative and quantitative resistance in potato and wheat.

In Subproject 2 we will identify, clone, and characterise the genomic organisation and molecular structure of novel disease resistance loci (R genes and QTLs) to the major fungal pathogens of wheat and potato i.e. Septoria tritici, Blumeria spp., Puccinia spp., Fusarium spp., and Phytophthora infestans. To facilitate genetic and physical mapping existing segregating populations will be used from external studies and current breeding programmes of the SMEs. In addition, germplasm with novel disease resistances identified in genebanks in Subproject 4 will serve as major input for Subproject 2. This resistant germplasm will be converted into immortalised segregating mapping populations. Using marker technologies developed in Subproject 5 molecular markers will be used that are closely linked and polymorphic to the resistance traits for genetic mapping of these populations. Physical maps and BAC libraries will subsequently be generated to clone the resistance gene or the QTL, and to study the genomic organisation of R gene clusters and major QTLs. This knowledge will provide insight in the evolution of R genes and QTLs, their genomic diversity, and the degree of synteny in chromosomal segments carrying these R genes and QTLs. The protein sequences of orthologous and paralogous resistance loci will be used to model surface topology of the protein structure and to identify amino acids involved in pathogen recognition specificity. The data of matching avirulence gene (SP1) and R gene (SP2) pair will collectively feed into Subproject 5 for marker-assisted breeding and Subproject 6 for genetic engineering these resistance traits. A principle deliverable of Subproject 2 are the R gene loci and QTLs that will be used for allele mining of the plant biodiversity in wild germplasm of related plant species in genebanks (SP4). This cyclic interaction between the Subprojects 2 and 4 plays an important role in this integrated project. Subproject 2 will also feed into Subproject 3 to unravel the mechanism underlying innate resistance.

Subproject 3: To unravel the molecular mechanisms underlying innate resistance.

The principle objective of Subproject 3 is to study the mechanism underlying recognition specificity and subsequent activation of the defence response in plants to avirulent plant pathogens. The activities in this Subproject will collectively establish a molecular inventory of the components in recognition complexes and disease resistance signalling pathways. The molecular inventory of innate disease resistance will be built by using proteomics (e.g. mass spectrometry of complex components) and (post)genomics tools (e.g. RNA profiling of activated defence responses and virus induced gene silencing). We also aim to  clarify the biophysical and biochemical principles in the R protein activation and disease resistance signal transduction components identified in the previous two workpackages. It will also provide an in planta validation of intra- and intermolecular protein-protein interactions identified earlier in the Subproject. Defence responses activated by different classes of R proteins recognising a variety of plant pathogens have shown to involve molecular interactors that are conserved throughout the plant kingdom. The idea is that Avr-induced recognition complexes in plants activate disease resistance signalling pathways that converge into a relatively small number of conserved interconnected molecular networks. The input of Subproject 3 will, therefore, initially come from external studies involving Avr-R gene pairs from other plant microbe interactions to serve as model for the pathogens under scrutiny of BIOEXPLOIT. At a later stage of the project the focus of this Subproject will shift to Avr-R gene pairs identified in Subprojects 1 and 2. The data generated in the Subproject 2 and 3 collectively will facilitate the engineering of novel recognition specificities in R proteins. The genetic loci of disease signalling components will be further studied in subproject 4 to reveal potential allelic variation.

Subproject 4: To explore biodiversity on loci associated with disease resistance in wheat and potato accessions in genebanks. 

The genetic variation in wild accessions of wheat and potato is still largely unexplored. Only a small fraction of the natural biodiversity in disease resistance is currently included in the genetic bases of commercial varieties. A major goal of this Subproject is to explore the genetic resources stored in genebanks for designing new varieties. To this purpose we will establish in Subproject 4 customised core collections containing the allelic variance at genetic loci associated with major R genes and QTLs for resistance. The genetic variation at disease resistance loci identified in Subproject 2 will be mined by extensive genotyping and subsequent phenotyping of wild germplasm. Existing breeding programmes from the SMEs will be integrated at this level to assess the allelic variation in breeding materials used in these programmes. Efficient molecular methods for targeted genotypic and phenotypic evaluation of wheat and potato genebanks will be developed to facilitate a high throughput-screening format. The outputs of Subproject 4 will feedback into Subproject 2 to initiate the cloning of the novel disease resistance alleles, and will feed forward into Subproject 5 to initiate marker-assisted breeding of the exotic resistance allele into an elite background. Phenotyping the allelic variance in the customised core collections will include the biodiversity in the centres of origin of the fungal and oomycete pathogens. Subproject 1 will feed into the phenotypic evaluations of allelic variation by providing pathogen-derived effector expression libraries for use as molecular markers. Furthermore, Subproject 4 will facilitate the phenotypic screening of all intermediate breeding phases from germplasm to the elite variety.

Subproject 5: To design durable disease resistance through marker-assisted breeding.

The principle of marker-assisted breeding is a realistic option for developing new varieties with multiple disease resistances. The development of high through-put technologies for selecting plants at the seedling stage may shorten the time between the first cross involving wild species and introduction on the market considerably, for some crops even up to 50%. SMEs play a prominent role in the plant breeding industry in wheat and potato, but often lack the investment capital to develop and validate marker-assisted breeding in their commercial breeding programmes. It is expected that the application of marker-assisted breeding will probably increase exponentially when a high throughput format is combined with low cost molecular markers. The overall objective of Subproject 5 is to develop and to validate high throughput molecular marker technologies for implementation in commercial breeding programmes. Subproject 2 and existing breeding programmes will feed polymorphic molecular markers linked to disease resistance loci into subproject 5. These markers will be converted to reliable, robust and cheap PCR based markers. HTP protocols for these markers will be developed and validated in existing breeding programmes of participating SMEs. Two recent developments in multiplex HTP marker technologies, the AFLP and/or SNPWave and Tagged Microarray Marker technology, will be investigated for their potential in commercial breeding programmes. The technological innovations in this Subproject will be directly applied to the introgression breeding of R genes and QTLs identified in Subproject 2 and in existing commercial breeding programmes. A novelty in these disease resistance breeding programmes will be the application of Avr genes as molecular markers in phenotypic resistance screening. The activities comprise stacking or pyramiding of novel identified or known resistance sources, validation of the effectiveness of sources or marker-assisted backcross of sources into elite germplasm. The outputs of Subproject 5 are validated breeding lines, molecular markers, and HTP protocols that will either feedback into the commercial breeding programme of the participating SME, or will be made available to the plant breeding industry in general through the Technology Transfer Platform.

Subproject 6. To design durable disease resistance through genetic engineering (Objective 6).

Genetic engineering allows the rapid introduction of new genetic variation derived from unrelated species into crop species and it enables designing disease resistance by modifying natural resistance genes. Our goal is to obtain durable broad-spectrum resistance by introducing synthetic expression cassettes combining R genes and/or genes involved in disease signalling and to create novel recognition specificities by the manipulation of R genes using site-directed mutagenesis and domain swaps. A second objective is to mediate broad resistance in wheat by expressing mutated host transcription factors (defence response [DR] genes ) regulating disease resistance or susceptibility mechanisms. Subproject 2 and Subproject 4 will feed major R genes and genes controlling QTLs for resistance into subproject 6. Subproject 3 will feed defence response genes into subproject 6. The aim is to build resistance cassettes of natural and modified genes in a standard elite background. The resistance cassettes may include components of the R protein containing recognition complexes identified in subproject 3. The output of Subproject 6 will be GM wheat and potato harbouring one or more natural or modified R genes, DR genes, or QTLs in an elite European background or in the background of an elite variety from a breeding programme of participating SMEs.

Subproject 8: To disseminate the results and to transfer technology to industry

Because the impact of genomic research on classical breeding is relatively new, there are no traditional strong links between breeders and genomics-orientated researchers. BIOEXPLOIT will provide a new platform for communication among researchers from different disciplines. Within this project we will also establish a Technology Transfer Platform to exploit the knowledge generated within this project. To increase the awareness among consumers with regard to marker-assisted breeding and GM techniques, we will organise workshops and symposia for a broad audience. The attitude of the consumers now and in future will be of utmost importance for private companies to decide which route should be taken to develop varieties with durable resistance.