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International audience; Potato wart disease is considered one of the most important quarantine pests for cultivated potato and is caused by the obligate biotrophic chytrid fungus Synchytrium endobioticum. This review integrates observations from early potato wart research and recent molecular, genetic, and genomic studies of the pathogen and its host potato. Taxonomy, epidemiology, pathology, and formation of new pathotypes are discussed, and a model for molecular S. endobioticum-potato interaction is proposed. Taxonomy Currently classified as kingdom: Fungi, phylum: Chytridiomycota, class: Chytridiomycetes, order: Chytridiales, family: Synchytriaceae, genus: Synchytrium, species: Synchytrium endobioticum, there is strong molecular support for Synchytriaceae to be transferred to the order Synchytriales. Hosts and disease symptoms Solanum tuberosum is the main host for S. endobioticum but other solanaceous species have been reported as alternative hosts. It is not known if these alternative hosts play a role in the survival of the pathogen in (borders of) infested fields. Disease symptoms on potato tubers are characterized by the warty cauliflower-like malformations that are the result of cell enlargement and cell multiplication induced by the pathogen. Meristematic tissue on tubers, stolons, eyes, sprouts, and inflorescences can be infected while the potato root system seems to be immune. Pathotypes For S. endobioticum over 40 pathotypes, which are defined as groups of isolates with a similar response to a set of differential potato varieties, are described. Pathotypes 1(D1), 2(G1), 6(O1), and 18(T1) are currently regarded to be most widespread. However, with the current differential set other pathogen diversity largely remains undetected. Pathogen-host interaction A single effector has been described for S. endobioticum (AvrSen1), which is recognized by the potato Sen1 resistance gene product. This is also the first effector that has been described in Chytridiomycota, showing that in this fungal division resistance also fits the gene-for-gene concept. Although significant progress was made in the last decade in mapping wart disease resistance loci, not all resistances present in potato breeding germplasm could be identified. The use of resistant varieties plays an essential role in disease managemen

Key messageA Genome-Wide Association Study using 330 commercial potato varieties identified haplotype specific SNP markers associated with pathotype 1(D1) wart disease resistance.AbstractSynchytrium endobioticumis a soilborne obligate biotrophic fungus responsible for wart disease. Growing resistant varieties is the most effective way to manage the disease. This paper addresses the challenge to apply molecular markers in potato breeding. Although markers linked toSen1were published before, the identification of haplotype-specific single-nucleotide polymorphisms may result in marker assays with high diagnostic value. To identify hs-SNP markers, we performed a genome-wide association study (GWAS) in a panel of 330 potato varieties representative of the commercial potato gene pool. SNP markers significantly associated with pathotype 1 resistance were identified on chromosome11, at the position of the previously identifiedSen1locus. Haplotype specificity of the SNP markers was examined through the analysis of false positives and false negatives and validated in two independent full-sib populations. This paper illustrates why it is not always feasible to design markers without false positives and false negatives for marker-assisted selection. In the case ofSen1, founders could not be traced because of a lack of identity by descent and because of the decay of linkage disequilibrium betweenSen1and flanking SNP markers.Sen1appeared to be the main source of pathotype 1 resistance in potato varieties, but it does not explain all the resistance observed. Recombination and introgression breeding may have introduced new, albeit rare haplotypes involved in pathotype 1 resistance. The GWAS approach, in such case, is instrumental to identify SNPs with the best possible diagnostic value for marker-assisted breeding.

The obligate biotrophic chytrid species Synchytrium endobioticum is the causal agent of potato wart disease. Currently, 39 pathotypes have been described based on their interaction with a differential set of potato varieties. Wart resistance and pathotyping is performed using bioassays in which etiolated tuber sprouts are inoculated. Here, we describe an alternative method in which aboveground plant parts are inoculated. Susceptible plants produced typical wart symptoms in developing but not in fully expanded aboveground organs. Colonization of the host by S. endobioticum was verified by screening for resting spores by microscopy and by molecular techniques using TaqMan polymerase chain reaction and RNAseq analysis. When applied to resistant plants, none of these symptoms were detectable. Recognition of S. endobioticum pathotypes by differentially resistant potato varieties was identical in axillary buds and the tuber-based bioassays. This suggests that S. endobioticum resistance genes are expressed in both etiolated “belowground” sprouts and green aboveground organs. RNAseq analysis demonstrated that the symptomatic aboveground materials contain less contaminants compared with resting spores extracted from tuber-based assays. This reduced microbial contamination in the aboveground bioassay could be an important advantage to study this obligate biotrophic plant–pathogen interaction. Because wart resistance is active in both below- and aboveground organs, the aboveground bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs because it omits the requirement for tuber formation. In addition, possibilities arise to express S. endobioticum effectors in potato leaves through agroinfiltration, thereby providing additional phenotyping tools for research and breeding. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Phytophthora infestans, the causal agent of the Irish Potato Famine in the 1840s, is one of the most destructive crop pathogens that threaten global food security. Host resistance (R) genes may help to control the disease, but recognition by through the gene products can be evaded by newly emerging isolates. Such isolates are dangerous as they may cause disease outbreaks under favorable conditions. However, our lack of knowledge about adaptation in these isolates jeopardizes an apt response to resistance breakdown. Here we performed genome and transcriptome sequencing of HB1501 and HN1602, two field isolates from distinct Chinese geographic regions. We found extensive polymorphisms in these isolates, including gene copy number variations, nucleotide polymorphisms, and gene expression changes. Effector encoding genes, which contribute to virulence, show distinct expression landscapes in P. infestans isolates HB1501 and HN1602. In particular, polymorphisms at multiple effectors required for recognition (Avr loci) enabled these isolates to overcome corresponding R gene based resistance. Although the isolates evolved multiple strategies to evade recognition, we experimentally verified that several R genes such as R8, RB, and Rpi-vnt1.1 remain effective against these isolates and are valuable to potato breeding in the future. In summary, rapid characterization of the adaptation in emerging field isolates through genomic tools inform rational agricultural management to prevent potential future epidemics.

Key message Two novel major effect loci (Sen4 and Sen5) and several minor effect QTLs for potato wart disease resistance have been mapped. The importance of minor effect loci to bring full resistance to wart disease was investigated. Using the newly identified and known wart disease resistances, a panel of potato breeding germplasm and Solanum wild species was screened. This provided a state-of-the-art “hitch-hikers-guide” of complementary wart disease resistance sources. Abstract Potato wart disease, caused by the obligate biotrophic soil-born fungus Synchytrium endobioticum, is the most important quarantine disease of potato. Because of its huge impact on yield, the lack of chemical control and the formation of resting spores with long viability, breeding for resistant varieties combined with strict quarantine measures are the only way to efficiently and durably manage the disease. In this study, we set out to make an inventory of the different resistance sources. Using a Genome-Wide Association Study (GWAS) in the potato breeding genepool, we identified Sen4, associated with pathotypes 2, 6 and 18 resistance. Associated SNPs mapped to the south arm of chromosome 12 and were validated to be linked to resistance in one full-sib population. Also, a bulked segregant analysis combined with a Comparative Subsequence Sets Analysis (CoSSA) resulted in the identification of Sen5, associated with pathotypes 2, 6 and 18 resistance, on the south arm of chromosome 5. In addition to these two major effect loci, the GWAS and CoSSA allowed the identification of several quantitative trait loci necessary to bring full resistance to certain pathotypes. Panels of varieties and Solanum accessions were screened for the presence of Sen1, Sen2, Sen3, Sen4 and Sen5. Combined with pedigree analysis, we could trace back some of these genes to the ancestral resistance donors. This analysis revealed complementary resistance sources and allows elimination of redundancy in wart resistance breeding programs.

In order to better understand the Phytophthora infestans population structure in South Korea, 172 isolates were collected between 2009 and 2016 from four major potato cultivation areas. Fungicide (metalaxyl and dimethomorph) response, mating type, and microsatellite (SSR) genetic fingerprints were analysed to characterize these isolates. Ten isolates collected in Gyeongnam Province, which specializes in protected winter cultivation in polytunnels, were A2 mating type. All other isolates were A1 mating type. Overall, 42% of the isolates were resistant to metalaxyl, and 43% were sensitive. All isolates were sensitive to dimethomorph. From the SSR fingerprints, 45 distinct genotypes were identified, which could be clustered into four clonal lineages: KR_1_A1, KR_2_A2, SIB-1, and US-11. KR_1_A1 was the predominant P. infestans genotype in South Korea. KR_2_A2 was only found in Gyeongnam Province; all isolates were A2 mating type and resistant to metalaxyl. SIB-1 was dominant until 2013 but its frequency has gradually decreased in more recent years. US-11 was first found in 2014, after which its frequency has increased to become codominant with KR_1_A1. The calculated standardized index of association (IA) suggests that the South Korean P. infestans population is undergoing clonal reproduction. When compared with populations of P. infestans from the Netherlands, it has less genetic diversity and the dominant Netherlands P. infestans genotype, EU_13_A2 (Blue_13), was not found in South Korea. Such monitoring of the pathogen population contributes to a more efficient integrated pest management-based control strategy for potato late blight control in South Korea.

Synchytrium endobioticum is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum-specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response (HR), which cosegregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.

Late blight is an important disease in potato that is caused by the oomycete Phytophthora infestans. In the past, Solanum demissum late blight resistance (R) genes were introgressed into cultivated potato (Solanum tuberosum). Eleven of these resistant plants were selected to characterize the virulence spectrum of individual P. infestans isolates and to monitor the dynamics of virulence in P. infestans populations. These plants are referred to as the Mastenbroek and Black differential sets. It has long been assumed that each differential plant contained one single R gene. In the current study and previous studies, however, most Mastenbroek differential plants were shown to harbor multiple R gene(s), which blurs virulence typing of late blight isolates. In order to acquire more accurate virulence profiles, we extended the Mastenbroek differential set with Solanum spp. plants harboring reduced R gene complexity and with plants containing recently identified R genes from related but different Solanum species. In addition, a differential set of ten Genetically Modified (GM) plants harboring single late blight R genes in the same genetic background (Desiree). By analyzing the virulence spectra of recently collected isolates using both newly described differential sets, we found that the GM Desiree differential set was more accurate for isolate virulence typing than the conventional (extended) differential set. Besides, the GM Desiree differential set was shown to be useful as trap plants to isolate novel P. infestans strains and to monitor virulence towards particular R genes in P. infestans populations `on site´. Legislative restrictions are, however, limiting the use of the GM Desiree differential set.

Key message Ph-3 is the first cloned tomato gene for resistance to late blight and encodes a CC-NBS-LRR protein. Abstract Late blight, caused by Phytophthora infestans, is one of the most destructive diseases in tomato. The resistance (R) gene Ph-3, derived from Solanum pimpinellifolium L3708, provides resistance to multiple P. infestans isolates and has been widely used in tomato breeding programmes. In our previous study, Ph-3 was mapped into a region harbouring R gene analogues (RGA) at the distal part of long arm of chromosome 9. To further narrow down the Ph-3 interval, more recombinants were identified using the flanking markers G2-4 and M8-2, which defined the Ph-3 gene to a 26 kb region according to the Heinz1706 reference genome. To clone the Ph-3 gene, a bacterial artificial chromosome (BAC) library was constructed using L3708 and one BAC clone B25E21 containing the Ph-3 region was identified. The sequence of the BAC clone B25E21 showed that only one RGA was present in the target region. A subsequent complementation analysis demonstrated that this RGA, encoding a CC-NBS-LRR protein, was able to complement the susceptible phenotype in cultivar Moneymaker. Thus this RGA was considered the Ph-3 gene. The predicted Ph-3 protein shares high amino acid identity with the chromosome-9-derived potato resistance proteins against P. infestans (Rpi proteins). Electronic supplementary material The online version of this article (doi:10.1007/s00122-014-2303-1) contains supplementary material, which is available to authorized users.

Insect–plant interactions may be unintentionally affected when introducing genetically modified (GM) crops into an agro-ecosystem. Our aim was to test the non-target effects of a late blight-resistant GM potato on Myzus persicae in greenhouse and climate room experiments and understand how position and number of R gene insertions can affect non-targets in GM events. We also aimed to compare results to baseline differences among three conventional potato varieties varying in resistance to late blight. Aphid development and survival were affected by some GM events in the first generation, though effects disappeared in the second generation. Effects were not dependent on the presence of a marker gene or the insertion of a second resistance gene. Positional effects of gene insertion influenced aphid performance on certain GM events. However, aphid fitness varied considerably more between conventional potato varieties than between Désirée and the GM events. Comparing different GM events to the non-transformed variety is relevant, since unintended effects of insertion can occur. Our protocols can be recommended for in planta risk assessments with aphids. Ecological perspective is gained by selecting several measured endpoints and by comparing the results with a baseline of conventional cultivars.

Genetic transformation with resistance (R) genes is expected to enhance resistance durability against pathogens, especially for potato, a vegetatively propagated crop with tetrasomic inheritance and a long-term breeding program. In this study, 128 potato transformants were analysed for the presence of vector T-DNA genes, borders and backbone sequences. They were harvested after transformation using a construct containing neomycin phosphotransferase II (nptII) and three R genes against potato late blight (Phytophthora infestans). Our analysis revealed that 45 % of the R gene-containing transformants possessed a low T-DNA copy number, without the integration of vector backbone and borders. The integration of vector backbone sequences was characterized using eight genes, and backbone gene tetA was selected for the early prediction of plants with backbone sequence integration. Three transformants, two plants harbouring one T-DNA copy and one plant harbouring three T-DNA copies, were crossed with susceptible cv. Katahdin. Based on our results, we conclude that all four T-DNA genes were inherited as one cluster and segregated in a Mendelian fashion. The three T-DNA inserts from the transformant harbouring three T-DNA copies were statistically proven to be un-linked and inherited into the offspring plants independently. All of the R genes were functionally expressed in the offspring plants as in their parental transformants. This functional gene stacking has important implications towards achieving more durable resistance against potato late blight.

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 CDP(Sw5)4 and CDP(Sw5)5 flanked the R8 gene at the distal end (1 cM). CDP(Tm2)1-1, CDP(Tm2)1-2 and CDP(Tm2)2 flanked the R8 gene on the proximal side (2 cM). An additional co-segregating marker (CDP(Hero)3) was found, which will be useful for marker assisted breeding and map based cloning of R8.

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.

Esophageal gland secretions from nematodes are believed to include effectors that play important roles in plant parasitism. We have identified a novel gene family encoding secreted proteins specifically expressed in the dorsal esophageal gland of Globodera rostochiensis early in the parasitic cycle, and which contain the B30.2/SPRY domain. The secondary structure of these proteins, named the secreted SPRY domain-containing proteins (SPRYSEC), includes highly conserved regions folding into β-strands interspersed with loops varying in sequence and in length. Mapping sequence diversity onto a three-dimensional structure model of the SPRYSEC indicated that most of the variability is in the extended loops that shape the so-called surface A in the SPRY domains. Seven of nine amino acid sites subjected to diversifying selection in the SPRYSEC are also at this surface. In both yeast-two-hybrid screening using a library from a susceptible tomato and in an in vitro pull-down assay, one of the SPRYSEC interacted with the leucine-rich repeat (LRR) region of a novel coiled-coil nucleotide-binding LRR protein, which is highly similar to members of the SW5 resistance gene cluster. Given that the tomato cultivar used is susceptible to nematode infection, this SPRYSEC could be an evolutionary intermediate that binds to a classical immune receptor but does not yet, or no longer, triggers a resistance response. Alternatively, this SPRYSEC may bind to the immune receptor to downregulate its activity.

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thalianaS-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed. Electronic supplementary material The online version of this article (doi:10.1007/s11248-016-9964-2) contains supplementary material, which is available to authorized users.

To identify genes required for the hypersensitive response (HR), we performed expression profiling of tomato plants mounting a synchronized HR, followed by functional analysis of differentially expressed genes. By cDNA-AFLP analysis, the expression profile of tomato plants containing both the Cf-4 resistance gene against Cladosporium fulvum and the matching Avr4 avirulence gene of this fungus was compared with that of control plants. About 1% of the transcript-derived fragments (442 out of 50,000) were derived from a differentially expressed gene. Based on their sequence and expression, 192 fragments, referred to as Avr4responsive tomato (ART) fragments, were selected for VIGS (virus-induced gene silencing) in Cf-4-transgenic Nicotiana benthamiana. Inoculated plants were analyzed for compromised HR by agroinfiltration of either the C. fulvum Avr4 gene or the Inf1 gene of Phytophthora infestans, which invokes a HR in wild-type N. benthamiana. VIGS using 15 of the ART fragments resulted in a compromised HR, whereas VIGS with fragments of ART genes encoding HSP90, a nuclear GTPase, an L19 ribosomal protein, and most interestingly, a nucleotide binding-leucine rich repeat (NB-LRR)-type protein severely suppressed the HR induced both by Avr4 and Inf1. Requirement of an NB-LRR protein (designated NRC1, for NB-LRR protein required for HR-associated cell death 1) for Cf resistance protein function as well as Inf1-mediated HR suggests a convergence of signaling pathways and supports the recent observation that NB-LRR proteins play a role in signal transduction cascades downstream of resistance proteins. Additional keywords: CC-NB-LRR, defense.

Late blight, caused by Phytophthora infestans, remains the most devastating disease in potato resulting in economic costs that sum up 5.2 billion euros, globally. The use of resistant varieties is a powerful, viable and environmentally friendly alternative or supplement for the current, commonly deployed chemical control strategies. In this study, we set out to improve the susceptible potato variety Desiree by transformation with single or multiple late blight (R) resistance genes. Rpi-sto1, Rpi-vnt1.1 and a stack of Rpi-sto1:Rpi-vnt1.1:Rpi-blb3, were transformed and eight, eight, and ten independent transformants (events) respectively, were selected because of absence of vector backbone, low T-DNA copy number, responsiveness to the cognate Avr effectors, P. infestans resistance in detached leaf assays (DLAs) and preliminary field experiments. The performance of the selected events was studied under field conditions in The Netherlands and Belgium, after P. infestans inoculation and/or to natural late blight infection during two consecutive growing seasons. All selected events were more resistant than the non-transformed susceptible reference clone. The different individual R genes, however, contributed to different levels of resistance. The selected events were also compared to conventionally bred late blight resistant varieties with (partially) known R gene content. Generally, it was found that plants with single R genes showed a lower level of resistance than plants with R gene stacks. Only the events harbouring three late blight R genes remained unaffected until the end of the growing season, in both locations and in both growing seasons.

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated upon pathogen attack. We have previously shown that the fungal elicitor xylanase rapidly induces nitric oxide (NO), which is required for PI-PLCs activity and downstream defense responses in tomato cell suspensions. Here, we show that all six SlPLC genes are expressed in tomato cell suspensions. Treatment of the cells with xylanase induces an early increase in SlPLC5 transcript levels, followed by a raise of the amount of SlPLC2 transcripts. The production of NO is required to augment SlPLC5 transcript levels in xylanase-treated tomato cells. Xylanase also induces SlPLC2 and SlPLC5 transcript levels in planta. We knocked-down the expression of SlPLC2 and SlPLC5 by virus-induced gene silencing. We found that SlPLC2 is required for xylanase-induced expression of the defense-related genes PR1 and HSR203J. Fil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Ramírez, Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Abd El Haliem, Ahmed. Wageningen University; Países Bajos Fil: Vossen, Jack H.. Wageningen University; Países Bajos Fil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Joosten, Matthieu H. A. J.. Wageningen University; Países Bajos Fil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

Two of the domains most widely shared among R genes are the nucleotide binding site (NBS) and protein kinase (PK) domains. The present study describes and maps a number of new oat resistance gene analogues (RGAs) with two purposes in mind: (1) to identify genetic regions that contain R genes and (2) to determine whether RGAs can be used as molecular markers for qualitative loci and for QTLs affording resistance to Puccinia coronata. Such genes have been mapped in the diploid A. strigosa × A. wiestii (Asw map) and the hexaploid MN841801-1 × Noble-2 (MN map). Genomic and cDNA NBS-RGA probes from oat, barley and wheat were used to produce RFLPs and to obtain markers by motif-directed profiling based on the NBS (NBS profiling) and PK (PK profiling) domains. The efficiency of primers used in NBS/PK profiling to amplify RGA fragments was assessed by sequencing individual marker bands derived from genomic and cDNA fragments. The positions of 184 markers were identified in the Asw map, while those for 99 were identified in the MN map. Large numbers of NBS and PK profiling markers were found in clusters across different linkage groups, with the PK profiling markers more evenly distributed. The location of markers throughout the genetic maps and the composition of marker clusters indicate that NBS- and PK-based markers cover partly complementary regions of oat genomes. Markers of the different classes obtained were found associated with the two resistance loci, PcA and R-284B-2, mapped on Asw, and with five out of eight QTLs for partial resistance in the MN map. 53 RGA-RFLPs and 187 NBS/PK profiling markers were also mapped on the hexaploid map A. byzantina cv. Kanota × A. sativa cv. Ogle. Significant co-localization was seen between the RGA markers in the KO map and other markers closely linked to resistance loci, such as those for P. coronata and barley yellow dwarf virus (Bydv) that were previously mapped in other segregating populations.

Functional stacking of broad spectrum resistance (R) genes could potentially be an effective strategy for more durable disease resistance, for example, to potato late blight caused by Phytophthora infestans (Pi). For this reason, three broad spectrum potato R genes (Rpi), Rpi-sto1 (Solanum stoloniferum), Rpi-vnt1.1 (S. venturii) and Rpi-blb3 (S. bulbocastanum) were selected, combined into a single binary vector pBINPLUS and transformed into the susceptible cultivar Desiree. Among the 550 kanamycin resistant regenerants, 28 were further investigated by gene specific PCRs. All regenerants were positive for the nptII gene and 23 of them contained the three Rpi genes, referred to as triple Rpi gene transformants. Detached leaf assay and agro-infiltration of avirulence (Avr) genes showed that the 23 triple Rpi gene transformants were resistant to the selected isolates and showed HR with the three Avr effectors indicating functional stacking of all the three Rpi genes. It is concluded that Avr genes, corresponding to the R genes to be stacked, must be available in order to assay for functionality of each stack component. No indications were found for silencing or any other negative effects affecting the function of the inserted Rpi genes. The resistance spectrum of these 23 triple Rpi gene transformants was, as expected, a sum of the spectra from the three individual Rpi genes. This is the first example of a one-step approach for the simultaneous domestication of three natural R genes against a single disease by genetic transformation. Electronic supplementary material The online version of this article (doi:10.1007/s11248-011-9510-1) contains supplementary material, which is available to authorized users.

Potato defends against Phytophthora infestans infection by resistance (R)-gene-based qualitative resistance as well as a quantitative field resistance. R genes are renowned to be rapidly overcome by this oomycete, and potato cultivars with a decent and durable resistance to current P. infestans populations are hardly available. However, potato cultivar Sarpo Mira has retained resistance in the field over several years. We dissected the resistance of ‘Sarpo Mira’ in a segregating population by matching the responses to P. infestans RXLR effectors with race-specific resistance to differential strains. The resistance is based on the combination of four pyramided qualitative R genes and a quantitative R gene that was associated with field resistance. The qualitative R genes include R3a, R3b, R4, and the newly identified Rpi-Smira1. The qualitative resistances matched responses to avirulence (AVR)3a, AVR3b, AVR4, and AVRSmira1 RXLR effectors and were overcome by particular P. infestans strains. The quantitative resistance was determined to be conferred by a novel gene, Rpi-Smira2. It was only detected under field conditions and was associated with responses to the RXLR effector AvrSmira2. We foresee that effector-based resistance breeding will facilitate selecting and combining qualitative and quantitative resistances that may lead to a more durable resistance to late blight.

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.

Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato. Electronic supplementary material The online version of this article (doi:10.1007/s00122-011-1602-z) contains supplementary material, which is available to authorized users.

From 2006 through 2015, a research project on Durable Resistance in potato against Phytophthora (DuRPh) was carried out at Wageningen University and Research Centre. Its objective was to develop a proof of principle for durable resistance against late blight by cisgenesis. This public-funded project aimed at stimulating research on genetic modification and public debate on innovative genetic techniques. It was decided to clone and transfer late blight resistance (R) genes of crossable wild potato species (cisgenes) by Agrobacterium tumefaciens-mediated transformation without non-potato genes. A stack of multiple R genes were planned to be inserted into established varieties, thereby creating a dynamic variety in which the composition of the stacks may vary over space and time. Cisgenic plants were selected based on the expression of all inserted R genes and trueness-to-type. Within the project, 13 R genes from wild potato species were genetically mapped and three of them were cloned. Four varieties were transformed with one to three R genes. This was initially done using kanamycin resistance provided by a selectable marker gene of synthetic origin in order to quickly test the performance and stability of the introduced R genes and stacked R gene combinations. Once the functioning thereof was confirmed, marker-free transformations were conducted; thus, true cisgenic events were selected. The results about the different R genes, their chromosomal location, their specificity, the background dependence, the maximum size of a stack, its regeneration time and associated somaclonal variation frequency and its stability were studied. After selection and characterisation in the laboratory, the best cisgenic events were assessed in field trials for late blight resistance. This showed that inserted R genes were capable of turning a susceptible variety into a resistant one. Maximising longevity of the resistance was assured through resistance management research. It was shown that stacking of multiple R genes and monitoring how to deploy these stacks spatially and temporally could reduce fungicide use by over 80%. Communications through media and field demonstrations were manifold to allow public and policymakers to decide if cisgenesis is an acceptable tool to make potato farming more sustainable. Future deployment of the DuRPh strategy will depend largely on its status as a genetically modified crop or its exemption thereof. Worldwide near eradication of late blight would increase global annual potato production by close to 80 million tons, thereby contributing considerably to the needed additional global future food supply.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00122-011-1757-7) contains supplementary material, which is available to authorized users.

Late blight of potato (Solanum tuberosum), caused by Phytophthora infestans, can effectively be managed by genetic resistance. The MaR9 differential plant provides durable resistance to a broad spectrum of late blight strains. This resistance is brought about by at least seven genes derived from S. demissum including R1, Rpiabpt1, R3a, R3b, R4, R8 and, so far uncharacterized resistance gene(s). Here we set out to genetically characterize this additional resistance in MaR9. Three BC1 populations derived from MaR9 were identified that segregated for IPO-C resistance but that lacked R8. One BC1 population IX PCR marker, 184-81, fully co-segregated with R9a. The map position of R9a on the distal end of the lower arm of chromosome IX was confirmed using PCR markers GP101 and Stm1021. Successively, cluster-directed profiling (CDP) was carried out, revealing six closely linked markers. CDPSw58, CDPSw59 and CDPSw510 flanked the R9a gene at the distal end (5.8 cM) and, as expected, were highly homologous to Sw-5. CDPTm22 flanked R9a on the proximal side (2.9 cM). CDPTm26 and CDPTm27 fully cosegregated with resistance and had high homology to Tm-22, showing that R9a resides in a cluster of NBS–LRR genes with homology to Tm-22. Besides R9a, additional resistance of quantitative nature is found in MaR9, which remains to be genetically characterized. showed a continuous scale of resistance phenotypes, suggesting that multiple quantitative resistance genes were segregating. In two other BC1 populations esistance and susceptibility were segregating in a 1:1 ratio, suggesting a single qualitative resistance gene (R9a). A chromosome IX PCR marker, 184-81, fully co-segregated with R9a. The map position of R9a on the distal end of the lower arm of chromosome IX was confirmed using PCR markers GP101 and Stm1021. Successively, cluster-directed profiling (CDP) was carried out, revealing six closely linked markers. CDPSw58, CDPSw59 and CDPSw510 flanked the R9a gene at the distal end (5.8 cM) and, as expected, were highly homologous to Sw-5. CDPTm22 flanked R9a on the proximal side (2.9 cM). CDPTm26 and CDPTm27 fully cosegregated with resistance and had high homology to Tm-22, showing that R9a resides in a cluster of NBS–LRR genes with homology to Tm-22. Besides R9a, additional resistance of quantitative nature is found in MaR9, which remains to be genetically characterized.

High levels of resistance to Phytophthora infestans in Solanum are predominantly based on the gene-for-gene interaction. Identification of hitherto unknown R genes is essential for future pyramiding approaches. This could be achieved either through classic introgression breeding or through cisgenesis and could lead to sustainable control of late blight. Here, we report on the mapping of Rpi-cap1 and Rpi-qum1, two late blight R genes identified in the wild species Solanum capsicibaccatum and Solanum circaeifolium ssp. quimense, respectively, to very similar positions on the long arm of chromosome 11. Despite the difficulties encountered for marker development, a high-resolution genetic map with cleaved amplified polymorphic sequence markers was constructed. Furthermore, an R gene cluster-directed profiling approach led to the development of markers that closely linked to or co-segregated with the Rpi-cap1 gene. Both R genes are hypothesized to be homologous to the N gene, a toll-interleukin1 receptor–nucleotide-binding site–leucine-rich repeat domain type of R gene to tobacco mosaic virus from tobacco. To confirm this hypothesis, cloning of Rpi-cap1 and Rpi-qum1 should be pursued. Cloning would also be instrumental to facilitate the introduction of these valuable R genes into potato crops using cisgenic- and marker-assisted breeding approaches.