Synchytrium endobioticum, the potato wart disease pathogen.

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 management.

Allelic variants of the NLR protein Rpi-chc1 differentially recognize members of the Phytophthora infestans PexRD12/31 effector superfamily through the leucine-rich repeat domain.

Phytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that trigger effective defense responses upon the recognition of cognate RXLR avirulence (Avr) effector proteins. To deploy these R genes in a durable fashion in agriculture, we need to understand the mechanism of effector recognition and the way the pathogen evades recognition. In this study, we cloned 16 allelic variants of the Rpi-chc1 gene from Solanum chacoense and other Solanum species, and identified the cognate P. infestans RXLR effectors. These tools were used to study effector recognition and co-evolution. Functional and non-functional alleles of Rpi-chc1 encode coiled-coil nucleotide-binding leucine-rich repeat (CNL) proteins, being the first described representatives of the CNL16 family. These alleles have distinct patterns of RXLR effector recognition. While Rpi-chc1.1 recognized multiple PexRD12 (Avrchc1.1) proteins, Rpi-chc1.2 recognized multiple PexRD31 (Avrchc1.2) proteins, both belonging to the PexRD12/31 effector superfamily. Domain swaps between Rpi-chc1.1 and Rpi-chc1.2 revealed that overlapping subdomains in the leucine-rich repeat (LRR) domain are responsible for the difference in effector recognition. This study showed that Rpi-chc1.1 and Rpi-chc1.2 evolved to recognize distinct members of the same PexRD12/31 effector family via the LRR domain. The biased distribution of polymorphisms suggests that exchange of LRRs during host-pathogen co-evolution can lead to novel recognition specificities. These insights will guide future strategies to breed durable resistant varieties.

Genome Analysis of Two Newly Emerged Potato Late Blight Isolates Sheds Light on Pathogen Adaptation and Provides Tools for Disease Management.

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.

A Hitchhiker's guide to the potato wart disease resistance galaxy.

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. 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.

Distribution of P1(D1) wart disease resistance in potato germplasm and GWAS identification of haplotype-specific SNP markers.

KEY MESSAGE: A Genome-Wide Association Study using 330 commercial potato varieties identified haplotype specific SNP markers associated with pathotype 1(D1) wart disease resistance. Synchytrium endobioticum is 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 to Sen1 were 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 chromosome 11, at the position of the previously identified Sen1 locus. 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 of Sen1, founders could not be traced because of a lack of identity by descent and because of the decay of linkage disequilibrium between Sen1 and flanking SNP markers. Sen1 appeared 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 Synchytrium endobioticum AvrSen1 Triggers a Hypersensitive Response in Sen1 Potatoes While Natural Variants Evade Detection.

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.

Potato late blight field resistance from QTL dPI09c is conferred by the NB-LRR gene R8.

Following the often short-lived protection that major nucleotide binding, leucine-rich-repeat (NB-LRR) resistance genes offer against the potato pathogen Phytophthora infestans, field resistance was thought to provide a more durable alternative to prevent late blight disease. We previously identified the QTL dPI09c on potato chromosome 9 as a more durable field resistance source against late blight. Here, the resistance QTL was fine-mapped to a 186 kb region. The interval corresponds to a larger, 389 kb, genomic region in the potato reference genome of Solanum tuberosum Group Phureja doubled monoploid clone DM1-3 (DM) and from which functional NB-LRRs R8, R9a, Rpi-moc1, and Rpi_vnt1 have arisen independently in wild species. dRenSeq analysis of parental clones alongside resistant and susceptible bulks of the segregating population B3C1HP showed full sequence representation of R8. This was independently validated using long-range PCR and screening of a bespoke bacterial artificial chromosome library. The latter enabled a comparative analysis of the sequence variation in this locus in diverse Solanaceae. We reveal for the first time that broad spectrum and durable field resistance against P. infestans is conferred by the NB-LRR gene R8, which is thought to provide narrow spectrum race-specific resistance.

The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties.

KEY MESSAGE: The potato late blight resistance gene R8 has been cloned. R8 is 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. 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.

Silencing of six susceptibility genes results in potato late blight resistance.

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. thaliana S-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.

Inoculation of Transgenic Resistant Potato by Phytophthora infestans Affects Host Plant Choice of a Generalist Moth.

Pathogen attack and the plant's response to this attack affect herbivore oviposition preference and larval performance. Introduction of major resistance genes against Phytophthora infestans (Rpi-genes), the cause of the devastating late blight disease, from wild Solanum species into potato changes the plant-pathogen interaction dynamics completely, but little is known about the effects on non-target organisms. Thus, we examined the effect of P. infestans itself and introduction of an Rpi-gene into the crop on host plant preference of the generalist insect herbivore, Spodoptera littoralis (Lepidoptera: Noctuidae). In two choice bioassays, S. littoralis preferred to oviposit on P. infestans-inoculated plants of both the susceptible potato (cv. Desiree) and an isogenic resistant clone (A01-22: cv. Desiree transformed with Rpi-blb1), when compared to uninoculated plants of the same genotype. Both cv. Desiree and clone A01-22 were equally preferred for oviposition by S. littoralis when uninoculated plants were used, while cv. Desiree received more eggs compared to the resistant clone when both were inoculated with the pathogen. No significant difference in larval and pupal weight was found between S. littoralis larvae reared on leaves of the susceptible potato plants inoculated or uninoculated with P. infestans. Thus, the herbivore's host plant preference in this system was not directly associated with larval performance. The results indicate that the Rpi-blb1 based resistance in itself does not influence insect behavior, but that herbivore oviposition preference is affected by a change in the plant-microbe interaction.

Characterisation of the late blight resistance in potato differential MaR9 reveals a qualitative resistance gene, R9a, residing in a cluster of Tm-2 (2) homologs on chromosome IX.

KEY MESSAGE: The durable late blight resistance in potato plant Ma R9 is genetically characterized. A novel R -gene is mapped. The monogenic nature and map positions of R9 are negated and rectified. 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, Rpi-abpt1, 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 showed a continuous scale of resistance phenotypes, suggesting that multiple quantitative resistance genes were segregating. In two other BC1 populations resistance 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. CDP(Sw)58, CDP(Sw)59 and CDP(Sw5)10 flanked the R9a gene at the distal end (5.8 cM) and, as expected, were highly homologous to Sw-5. CDP(Tm2)2 flanked R9a on the proximal side (2.9 cM). CDP(Tm2)6 and CDP(Tm2)7 fully co-segregated with resistance and had high homology to Tm-2 (2) , showing that R9a resides in a cluster of NBS-LRR genes with homology to Tm-2 (2) . Besides R9a, additional resistance of quantitative nature is found in MaR9, which remains to be genetically characterized.

Development of late blight resistant potatoes by cisgene stacking.

BACKGROUND: Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. RESULTS: We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. CONCLUSIONS: We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.

Vector integration in triple R gene transformants and the clustered inheritance of resistance against potato late blight.

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.

Qualitative and quantitative late blight resistance in the potato cultivar Sarpo Mira is determined by the perception of five distinct RXLR effectors.

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.

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.

Functional stacking of three resistance genes against Phytophthora infestans in potato.

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.

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 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.

Understanding and exploiting late blight resistance in the age of effectors.

Potato (Solanum tuberosum) is the world's third-largest food crop. It severely suffers from late blight, a devastating disease caused by Phytophthora infestans. This oomycete pathogen secretes host-translocated RXLR effectors that include avirulence (AVR) proteins, which are targeted by resistance (R) proteins from wild Solanum species. Most Solanum R genes appear to have coevolved with P. infestans at its center of origin in central Mexico. Various R and Avr genes were recently cloned, and here we catalog characterized R-AVR pairs. We describe the mechanisms that P. infestans employs for evading R protein recognition and discuss partial resistance and partial virulence phenotypes in the context of our knowledge of effector diversity and activity. Genome-wide catalogs of P. infestans effectors are available, enabling effectoromics approaches that accelerate R gene cloning and specificity profiling. Engineering R genes with expanded pathogen recognition has also become possible. Importantly, monitoring effector allelic diversity in pathogen populations can assist in R gene deployment in agriculture.

Cloning and characterization of r3b; members of the r3 superfamily of late blight resistance genes show sequence and functional divergence.

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.

Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance.

The perception of pathogen-derived elicitors by plants has been suggested to involve phosphatidylinositol-specific phospholipase-C (PI-PLC) signalling. Here we show that PLC isoforms are required for the hypersensitive response (HR) and disease resistance. We characterised the tomato [Solanum lycopersicum (Sl)] PLC gene family. Six Sl PLC-encoding cDNAs were isolated and their expression in response to infection with the pathogenic fungus Cladosporium fulvum was studied. We found significant regulation at the transcriptional level of the various SlPLCs, and SlPLC4 and SlPLC6 showed distinct expression patterns in C. fulvum-resistant Cf-4 tomato. We produced the encoded proteins in Escherichia coli and found that both genes encode catalytically active PI-PLCs. To test the requirement of these Sl PLCs for full Cf-4-mediated recognition of the effector Avr4, we knocked down the expression of the encoding genes by virus-induced gene silencing. Silencing of SlPLC4 impaired the Avr4/Cf-4-induced HR and resulted in increased colonisation of Cf-4 plants by C. fulvum expressing Avr4. Furthermore, expression of the gene in Nicotiana benthamiana enhanced the Avr4/Cf-4-induced HR. Silencing of SlPLC6 did not affect HR, whereas it caused increased colonisation of Cf-4 plants by the fungus. Interestingly, Sl PLC6, but not Sl PLC4, was also required for resistance to Verticillium dahliae, mediated by the transmembrane Ve1 resistance protein, and to Pseudomonas syringae, mediated by the intracellular Pto/Prf resistance protein couple. We conclude that there is a differential requirement of PLC isoforms for the plant immune response and that Sl PLC4 is specifically required for Cf-4 function, while Sl PLC6 may be a more general component of resistance protein signalling.