Unveiling and deploying durability of late blight resistance in potato : from natural stacking to cisgenic stacking

The potato, which receives an increased attention as a food crop, has long been in threats from the oomycete Phytophthora infestans, the causal agent of late blight. This disease still remains the most important constraint in potato producing regions of the world. It might cause the complete destruction of the foliage and tubers of potato if meteorological conditions are conducive to the onset and spread of late blight epidemics. Although fungicides applications provide sufficient levels of late blight control, they impose high input costs to the farmer, are detrimental to human and environment and increase the capacity of the pathogen to develop resistance to the active ingredients of fungicides applied. The increased genetic diversity in P. infestanspopulations due to sexual recombination between two mating types in many parts of the world and the emergence of fungicide resistant strains poses the necessity to develop potatoes that possess high levels of durable resistance as an alternative to the use of fungicides. Clones MaR8 and MaR9 from the Mastenbroek differential set, used to assess virulence towards Rgenes, have been known for their strong resistance to P. infestans. This also holds for cultivar Sarpo Mira which has retained resistance in the field over several years without fungicide applications. Uncovering genetic basis of such, partly naturally-formed, late blight resistance is the prerequisite for the implementation of durable resistance in a breeding scheme. In this study, MaR8, MaR9and cv. Sarpo Mira were used as plant materials for unveiling durability of late blight resistance in potato. First, F1 mapping populations from crosses between these resistant materials with susceptible parents were assessed for late blight resistance in field trials and in detached leaf assays (DLA) after inoculation with an incompatible P. infestans isolate IPO-C. A 1:1 segregation of resistance and susceptibility was observed in the MaR8derived-F1 population in field trials, but not in detached leaf assays. NBS profiling and Rgene cluster directed profiling (CDP), followed by marker landing in the newly sequenced potato genome, referred to as “anchored scaffold approach”, led to the mapping of R8at a new locus on chromosome IX rather than on chromosome XI, the previously suggested chromosomal position (Chapter 2). The Rgene mediated resistance reaction in potato is a consequence of an (in)direct interaction between the pathogen Avrand host Rgene product that leads to a hypersensitive cell death (HR). We screened a wide collection of RXLR effectors of P. infestansfor eliciting cell death in the differential potato MaR8 by agroinfiltration (Chapter 3). R8-specific cell death to one effector PITG_07558, termed AVR8, co-segregated with the R8-mediated resistance to P. infestansisolate IPO-C in a F1 population. From the notion that Avr8is identical to effector AvrSmira2that was previously found to associate with field resistance in cultivar Sarpo Mira, we performed genetic mapping studies in a Sarpo Mira-based F1 population and indeed Rpi-Smira2localized in the R8locus. To investigate the geographical and phylogenetic origin of R8in the Solanumgene pool, we conducted functional screens for AVR8 responsiveness in 98 wild genotypes (72 accessions of 40 species) of Solanumsection Petota. We identified twelve AVR8 responding Solanum accessions originating both from Central and South America. Interestingly, our study involving late blight resistance from the differential plant MaR9described that it is near the R8 locus on chromosome IX (Chapter 4). An integrated approach combining 1. a Rgene ”de-stacking” approach using Rgene specific marker analysis and effector responses, 2. the whole plant climate cell assay, and 3. CDP profiling enabled a clear picture for the presence of two closely linked genes, termed R9aand R9b. It was shown that R9alocates in a Tm-22 cluster of NB-LRR genes and, most likely will be a member of the Tm-22 Rgene family (Chapter 4). The identified fully co-segregating Tm-2 likeCDP markers were used to select the R9agene-containing BAC clone, demonstrating the possibility of BAC landing by marker saturation in the targeted chromosomal regions (Chapter 5). For cloning R9agene, a bacterial artificial chromosome (BAC) library derived from the differential plant MaR9, was screened with co-segregating Rgene CDP markers whereby two overlapping BAC clones carrying CDP markers were obtained. Sequence annotation of the complete insert of these BAC clones identified the presence of two complete Rgene analogs (RGA9.1 andRGA9.2) of the NB-LRR class in one BAC clone. Two RGAs, including their natural regulatory transcriptional elements, were subcloned by long-range PCR into a binary vector for plant transformation. After transformation, it was found that RGA9.1was able to complement the susceptible phenotype in cultivar Desiree. RGA9.1, now designated R9a,encodes a CC-NB-LRR protein of the Tm2 family, where the LRR consensus is only loosely fitted. Agroinfiltration-based effector screens for identifying the Avrgenes matching the R9agene was performed, leading to the discovery of Avrblb2 homologs which trigger R9amediated hypersensitivity in Nicotiana benthamiana (Chapter 5).Resistance profiling with 54 P. infestans isolates showed that MaR9 and S.xedinense accessions had similar resistance spectra as the Rpi-blb2containing cultivar Bionica. Transformation of potato with resistance genes and antibiotic resistance markers encounters consumers’ criticism. These criticisms are considerably less if only resistance genes from crossable species, and no antibiotic resistance selection marker is used. Genes deriving from crossable species are referred to as cisgenes. For the production of cisgenic potatoes with a broader resistance spectrum and potential durability, Agrobacterium-mediated marker free transformation and PCR selection of transformants was performed. This way four potato cultivars (Atlantic, Bintje, Potae9 and Doip1) were successfully transformed with a construct containing two cisgenic Rpigenes (Rpi-vnt1from Solanum venturiiand Rpi-sto1from Solanum stoloniferum) (Chapter 6). Resistance assays in untransformed varieties with five P. infestansisolates showed that cvs. Potae9 and Doip1 were already resistant to certain isolates. Single Rpigene containing transgenic plants for all 4 varieties were obtained and used as references. Marker free transformation with a construct containing two Rpi genes (cisgenesis) was compared to kanamycin assisted selection (transgenesis) in terms of regeneration and transformation frequency, vector backbone integration, and T-DNA copy number. In addition, the different time tracks to harvest regenerated shoots for the selection of PCR positive regenerants for one or both Rpi-genes were studied. Through further analyses involving phenotypic evaluations in the greenhouse, agroinfiltration of avirulence (Avr) genes and detached leaf assays, totally eight cisgenic plants were selected. Two cisgenic plants of cv. Altantic and four of cv. Bintje, were selected that showed broad spectrum late blight resistance due to the activity of both Rpigenes. Based on characterization of two cisgenic transformants of cv. Potae9, it was demonstrated that the existing late blight resistance spectrum has been broadened by adding the two Rpigenes. Finally, results from this study are discussed in terms of genetic and molecular mechanism of durability and cisgenic deployment to address the challenges of the durable resistant potato variety development (Chapter 7). We pursue possible options for durability in the nature of the Rgenes or their cognate Avrgenes. The comparative analysis of several features of available R-AVR pairs shows that major components for producing durability are the copy number variation in the P. infestansgenome and abundance of the Avrgene in different isolates. As a counterpart of such an Avrgene, potato Rgenes that display broad spectrum resistance and often have abundant functional homologs among various wild Solanumspecies could be optional for Rgene combinations providing durability. Multiple years’ on-site-monitoring of resistance spectrum in natural Rgene stacks demonstrates that stacking of several broad spectrum Rpigenes or even “defeated” Rgenes could sum up to high levels of resistance potentially capable to provide durability to commercial potato cultivars. Our data about acquirement of complementary resistance spectrum by cisgenic introduction of two broad spectrum resistance genes into cultivars support a first step into that direction. </p

Mapping of the S. demissum late blight resistance gene R8 to a new locus on chromosome IX

The use of resistant varieties is an important tool in the management of late blight, which threatens potato production worldwide. Clone MaR8 from the Mastenbroek differential set has strong resistance to Phytophthora infestans, the causal agent of late blight. The F1 progeny of a cross between the susceptible cultivar Concurrent and MaR8 were assessed for late blight resistance in field trials inoculated with an incompatible P. infestans isolate. A 1:1 segregation of resistance and susceptibility was observed, indicating that the resistance gene referred to as R8, is present in simplex in the tetraploid MaR8 clone. NBS profiling and successive marker sequence comparison to the potato and tomato genome draft sequences, suggested that the R8 gene is located on the long arm of chromosome IX and not on the short arm of chromosome XI as was suggested previously. Analysis of SSR, CAPS and SCAR markers confirmed that R8 was on the distal end of the long arm of chromosome IX. R gene cluster directed profiling markers CDPSw54 and CDPSw55 flanked the R8 gene at the distal end (1 cM). CDPTm21-1, CDPTm21-2 and CDPTm22 flanked the R8 gene on the proximal side (2 cM). An additional co-segregating marker (CDPHero3) was found, which will be useful for marker assisted breeding and map based cloning of R8

Broad spectrum late blight resistance in potato differential set plants MaR8 and MaR9 is conferred by multiple stacked R genes

Phytophthora infestans is the causal agent of late blight in potato. The Mexican species Solanum demissum is well known as a good resistance source. Among the 11 R gene differentials, which were introgressed from S. demissum, especially R8 and R9 differentials showed broad spectrum resistance both under laboratory and under field conditions. In order to gather more information about the resistance of the R8 and R9 differentials, F1 and BC1 populations were made by crossing Mastenbroek (Ma) R8 and R9 clones to susceptible plants. Parents and offspring plants were examined for their pathogen recognition specificities using agroinfiltration with known Avr genes, detached leaf assays (DLA) with selected isolates, and gene-specific markers. An important observation was the discrepancy between DLA and field trial results for Pi isolate IPO-C in all F1 and BC1 populations, so therefore also field trial results were included in our characterization. It was shown that in MaR8 and MaR9, respectively, at least four (R3a, R3b, R4, and R8) and seven (R1, Rpi-abpt1, R3a, R3b, R4, R8, R9) R genes were present. Analysis of MaR8 and MaR9 offspring plants, that contained different combinations of multiple resistance genes, showed that R gene stacking contributed to the Pi recognition spectrum. Also, using a Pi virulence monitoring system in the field, it was shown that stacking of multiple R genes strongly delayed the onset of late blight symptoms. The contribution of R8 to this delay was remarkable since a plant that contained only the R8 resistance gene still conferred a delay similar to plants with multiple resistance genes, like, e.g., cv Sarpo Mira. Using this “de-stacking” approach, many R gene combinations can be made and tested in order to select broad spectrum R gene stacks that potentially provide enhanced durability for future application in new late blight resistant varieties

New Phytophthora resistance gene

The invention relates to a new gene that produces a protein which is capable of inferring oomycete resistance, more preferably resistance to Phytophthora infestans when expressed in a plant, wherein said nucleotide sequence encodes a protein that is encoded by the protein produced by the nucleotide sequence of FIG. 10 or the protein depicted in FIG. 11 or a nucleotide sequence that codes for a protein that has an identity of at least 95% with said protein produced by the nucleotide sequence of FIG. 10 or the protein depicted in FIG. 11. The invention also relates to a method for providing at least partial resistance or increasing resistance in a plant against an oomycete infection comprising providing a plant or a part thereof with a nucleotide sequence as indicated above or a functional fragment thereof, preferably wherein said plant is a plant from the Solanaceae family, more preferably Solanum tuberosum

New Phytophthora resistance gene

The invention relates to a new gene that produces a protein which is capable of inferring oomycete resistance, more preferably resistance to Phytophthora infestans when expressed in a plant, wherein said nucleotide sequence encodes a protein that is encoded by the protein produced by the nucleotide sequence of FIG. 10 or the protein depicted in FIG. 11 or a nucleotide sequence that codes for a protein that has an identity of at least 95% with said protein produced by the nucleotide sequence of FIG. 10 or the protein depicted in FIG. 11. The invention also relates to a method for providing at least partial resistance or increasing resistance in a plant against an oomycete infection comprising providing a plant or a part thereof with a nucleotide sequence as indicated above or a functional fragment thereof, preferably wherein said plant is a plant from the Solanaceae family, more preferably Solanum tuberosum

Mining the Genus Solanum for Increasing Disease Resistance

Plant Breeding is the art of selecting and discarding genetic material to achieve crop improvement. Favourable alleles resulting in quality improvement or disease resistance must be added, while unfavourable alleles must be removed. The source for novel alleles can be other varieties, landraces or crop wild relatives. The identification of allelic variation is referred to as allele mining. Before allelic variation can be used for breeding purposes several steps need to be taken. First of all an inventory is needed of the available genetic resources. Phenotypic screens are needed to uncover potential expected and even unanticipated alleles. Next, using genetic and molecular tools, the alleles responsible for the identified traits must be traced and distinguished in order to be introgressed into new varieties. In this review we focus on the identification of novel disease resistance traits in the agronomically important genus Solanum. The fact that R genes are present in multigene clusters within the genome, which often include many paralogs necessitates thorough discussion on the distinction between alleles and paralogs. Often such a distinction cannot easily be made. An overview is given of how natural resources can be tapped, e.g. how germplasm can be most efficiently screened. Techniques are presented by which alleles and paralogs can be distinguished in functional and/or genetic screens, including also a specific tagging of alleles and paralogs. Several examples are given in which allele and paralog mining was successfully applied. Also examples are presented as to how allele mining supported our understanding about the evolution of R gene clusters. Finally an outlook is provided how the research field of allele mining might develop in the near future

The Solanum demissumR8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties

<p> The potato late blight resistance geneR8has been cloned.R8is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato.Abstract: The broad spectrum late blight resistance gene R8 from Solanum demissum was cloned based on a previously published coarse map position on the lower arm of chromosome IX. Fine mapping in a recombinant population and bacterial artificial chromosome (BAC) library screening resulted in a BAC contig spanning 170 kb of the R8 haplotype. Sequencing revealed a cluster of at least ten R gene analogues (RGAs). The seven RGAs in the genetic window were subcloned for complementation analysis. Only one RGA provided late blight resistance and caused recognition of Avr8. From these results, it was concluded that the newly cloned resistance gene was indeed R8. R8 encodes a typical intracellular immune receptor with an N-terminal coiled coil, a central nucleotide binding site and 13 C-terminal leucine rich repeats. Phylogenetic analysis of a set of representative Solanaceae R proteins shows that R8 resides in a clearly distinct clade together with the Sw-5 tospovirus R protein from tomato. It was found that the R8 gene is present in late blight resistant potato varieties from Europe (Sarpo Mira), USA (Jacqueline Lee, Missaukee) and China (PB-06, S-60). Indeed, when tested under field conditions, R8 transgenic potato plants showed broad spectrum resistance to the current late blight population in the Netherlands, similar to Sarpo Mira.</p

Towards Sustainable Potato Late Blight Resistance by Cisgenic R Gene Pyramiding

This chapter provides an overview of the possibilities of genetic modification (GM) potato breeding in general and specifically to combat the most important disease, late blight. Potato plants are vulnerable to a number of pests and diseases. An enigmatic question concerns whether individual Rpi genes can confer sufficient broad-spectrum resistance to impart durability. The presence of Rpi genes or transcripts as determined using molecular markers does not warrant their functional expression. Late blight resistance resources from crossable species can be deployed for intragenic or cisgenic breeding strategies. The chapter presents and discusses a pipeline for cisgenic late blight resistance breeding that was developed at Wageningen UR Plant Breeding. If only two Rpi genes are present, often isolates can be identified that overcome recognition mediated by one Rpi gene but not by the other. The late blight-resistant GM potato which was probably closest to commercialization was “Fortuna”