Novel tools to breed for resistance against tuber diseases caused by obligate biotrophic pathogens
Research on potato disease resistance towards obligate biotrophic pathogens is impaired by inherently complex disease assays. The lack of pure pathogen isolates and variation in tuber quality cause irrepressible variability among years and test locations. In this project we will design and deploy novel tools for S. endobioticum resistance breeding, similar approaches could also be effective for other biotrophic potato pathogens.
Central in such approaches are effector responses which serve as a proxy for conventional phenotyping methods. Also, information on effector diversity is used to predict the resistance spectra of cognate resistance (R) genes. Recently, we identified the first S. endobioticum effector, Avrsen1, which is specifically recognised by the potato R gene product Sen1, that provides resistance to pathotype 1 isolates.
Although only five different pathotypes are presently described in western Europe, genetic diversity among isolates from the same pathotype is unexpectedly high. A single isolate can in fact contain multiple genotypes including mixtures of virulent and avirulent genotypes. This explains why resistance to higher pathotypes is sometimes incomplete. Stacking of multiple R genes in new varieties is mandatory to provide full resistance to the breadth of intra-isolate diversity.
In order to stack wart R genes, closely flanking and haplotype specific markers are needed. Furthermore, to limit the number genes in a stack, it is preferred to use R genes with a broad pathogen recognition spectrum.
In this project we will identify the cognate Avrs of at least three major R genes in breeding material. The availability of these Avrs allows to efficiently fine-map R genes and to distinguish resistance sources in breeding material. In addition, common features in the sequences of the Avrs will serve to identify effector signatures, which will allow us to select effectors that are conserved among and within isolates. The Solanum germplasm collection from WUR will be screened for hypersensitive responses triggered by these “orphan” effectors to identify their cognate broad spectrum wart R genes. Finally we expect that, having multiple effectors and their natural variation in over a hundred isolates, will help us to shed light on the function of these effectors in virulence and provide a better understanding on how S. endobioticum and pathogenic Chytridiomycota in general are manipulating their hosts.
Our effector based studies will provide important insights in pathogen population structures in relation to R genes deployed in varieties. Thereby, we provide unambiguous and more complete information for quarantine regulation in comparison to currently deployed assays with heterogeneous pathogens and a limited variety differential set.
The breeding tools that are developed in this project will facilitate the selection of resistant potato varieties with that ward-off the breadth of the pathogen diversity. Thereby, novel outbreaks are less likely to occur and moreover, a broader selection of varieties will be available for cultivation in the “protection zones” that are subject to quarantine oversight. Potato production will be better aligned with market demands. These two main results will together contribute to more efficient use of agricultural space.
Completion of Avr candidate set (2019):
Avrs matching R genes with differential isolate resistance spectra can be identified because of their variability in the genomes of the different isolates. 20 of such Avr candidates are available. In addition, we will identify effectors with expression variation. RNA from susceptible and partially resistant variety/pathotype interactions will be isolated and subjected to RNAseq. This comparative secretome analysis will reveal additional Avr candidates.
Validation of Avrs (2019-2020):
Avr candidates will be synthesised, cloned in a modular (signal peptides, epitope tags) golden gate system, and expressed in the leaves of a panel of resistant potato plants through agroinfiltration. The panel will consist of the EPPO differential set (5 different resistance sources) and two additional resistance sources which are not represented in the EPPO set. Differential HR responses will be identified and subsequently, we will verify that these effector genes are the cognate Avr of these QTLs using co-segregation of resistance with HR responses in segregating populations.
Identification of broad spectrum orphan effectors (2020):
Common features among effector that trigger HR (effector signatures) will be identified based on sequences homology/protein-structure/gene expression patterns. Next, genes with effector signatures will be selected(orphan effectors). The diversity of orphan effectors between and within isolates will be studied and a maximum of 10 broad spectrum effectors.
Germplasm screen (2020-2021):
Broad spectrum effectors will cloned and expressed in 100 Solanum accessions from the WUR in vitro collection. These accessions are selected for their sexual compatibility, agro-competence. Differentially responding accessions will be identified. Responsive accessions will be screened for wart resistance directly, using above-ground inoculation. Also, a selection of Solanum species with presumed wart resistance will be screened using the above ground inoculation method (Vossenberg et al 2019)
Validation and mapping of novel resistance (2020-2022):
Differentially responsive and resistant accessions will be crossed with non-responsive accessions to produce F1 populations. Populations in which wart resistance co-segregates with effector response will provide breeding material for broad spectrum wart resistance.