Natural Diversity as a Rich Resource for Basic and Applied Wheat Genetics
The study of natural diversity at the molecular level in the form of molecular allele mining does not only allow for the identification of new functional disease resistance alleles, but also for the characterization of the molecular basis of resistance gene function and specificity. By screening about 2,000 landraces from different geographical regions we were able to increase the number of known functional Pm3 alleles from seven to 16 (Bhullar et al. 2009, 2010).
Based on the available large dataset of functional and non-functional Pm3 alleles, we could derive a hypothesis on functionally important amino acids in this protein. In particular, we focused on the nucleotide-binding-site domain (NBS) of the PM3 protein. Alleles with a broad and a narrow resistance spectrum have been described. We found that a broad Pm3-spectrum range correlates with a fast and intense hypersensitive response (HR) in a Nicotiana benthamiana transient-expression system. This activity can be attributed to two particular amino acids in the ARC2 subdomain of the NBS. The combined substitution of these two amino acids in narrow-spectrum Pm3 alleles enhances their capacity to induce a HR in Nicotiana and, very interestingly, we found that the same substitutions also broaden the resistance spectrum of the Pm3f allele in wheat, resulting in an improved version of this gene. These results demonstrate the possibility for improvement of the NBS-“molecular switch” acting in the conversion of initial pathogen perception by the LRR into resistance-protein activation. Thus, we have found a way to enhance the resistance spectrum of an existing gene via minimal targeted modifications in the NBS domain. Ultimately, this might also allow the design of synthetic genes with new specificities and, ideally, to develop a more durable type of resistance based on major genes. The application of such findings could be made with transgenic lines, but given that only very small changes are needed in the protein sequence, new technologies such as transcription activator-like effector nucleases (TALEN) and related approaches might be used for gene editing in wheat.
Natural diversity also reveals mechanism of resistance gene evolution. We have found that the Pm8 gene derived from rye and present on the frequently used 1RS/1BL translocation in wheat is an ortholog of the wheat Pm3 resistance gene (Hurni et al. 2013). The finding that orthologous genes have maintained their function against the mildew pathogen over an estimated seven million years revealed a surprising evolutionary stability of powdery mildew resistance gene activity. This is even more surprising given the fact that after the introduction of Pm8 in cultivated wheat lines around 70 years ago, this resistance was rapidly overcome by the pathogen. Thus, we propose that the evolutionary events might have been quite different in the natural grassland ecosystems before agriculture compared to the modern agricultural environments.
Transgenic Strategies for a More Durable Use of Major Resistance Genes
We have recently explored the transgenic use of the Pm3 resistance alleles in wheat resistance breeding. We have isolated the Pm3a–g alleles from different wheat lines and transformed them into the wheat genotype Bobwhite S26 under control of the maize ubiquitin promoter. A large field trial performed in Switzerland in the years 2008–2010 has shown that most of these alleles confer improved resistance to powdery mildew when overexpressed (Brunner et al. 2011). In particular, it is noteworthy that some transgenic lines had an improved resistance compared to the donor line with the same gene, indicating that overexpression can improve resistance activity.
Moreover, resistance was improved when transgenic lines with different functional Pm3 alleles were mixed in the field (Brunner et al. 2012). This so-called multiline approach has been classically used in agro-ecosystems. However, the lines were never completely isogenic as the different resistance genes were introduced into the same genotypes by backcross breeding, resulting in relatively large chromosomal segments from the donors. Using a transgenic approach, we established true isogenic lines in the same genotype for a number of different Pm3 alleles, with different race spectrum of resistance to wheat powdery mildew. We found a clear improvement of resistance in mixed stands containing lines carrying two different Pm3 alleles, demonstrating the effectiveness of this approach.
The availability of many Pm3 alleles in transgenic form also allowed us to pyramid two alleles in the same genotype. Whereas in classical breeding alleles can only be combined temporarily in F1 hybrids, the transgenes have inserted at different locations in the genome and can therefore be combined in a stable, homozygous form after crosses and selection for the presence of two or more alleles in the same plant. We are currently studying the different double homozygous lines for several allelic combinations. We find situations of the expected additivity of gene function, but also interference. The molecular analysis of these lines is ongoing and promises to give fundamental new insight into resistance gene function.
Although the three approaches (overexpression, multilines, gene pyramidization) described above for the transgenic use of major R genes are still in an early phase in relation to applied wheat breeding, they show the potential of such new strategies. In particular it should be considered that wild relatives of crop plants hold great gene/allelic diversity for resistance. It is often difficult and very timeconsuming to transfer genes from wild species into crops by classical breeding: it can take decades to derive agronomically useful material from such introgressions. Transgenic technology, together with a more rapid identification of the relevant genes in the wild germplasm, provide a promising way to use the natural diversity more efficiently and to rapidly develop pre-breeding material for further use.