Multivariate equivalence testing for food safety assessment.

Products for food and feed derived from genetically modified (GM) crops are only allowed on the market when they are deemed to be safe for human health and the environment. The European Food Safety Authority (EFSA) performs safety assessment including a comparative approach: the compositional characteristics of a GM genotype are compared to those of reference genotypes that have a history of safe use. Statistical equivalence tests are used to carry out such a comparative assessment. These tests are univariate and therefore only consider one measured variable at a time. Phenotypic data, however, often comprise measurements on multiple variables that must be integrated to arrive at a single decision on acceptance in the regulatory process. The surge of modern molecular phenotyping platforms further challenges this integration, due to the large number of characteristics measured on the plants. This paper presents a new multivariate equivalence test that naturally extends a recently proposed univariate equivalence test and allows to assess equivalence across all variables simultaneously. The proposed test is illustrated on plant compositional data from a field study on maize grain and on untargeted metabolomic data of potato tubers, while its performance is assessed on simulated data.

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.

Improved One-Class Modeling of High-Dimensional Metabolomics Data via Eigenvalue-Shrinkage.

One-class modelling is a useful approach in metabolomics for the untargeted detection of abnormal metabolite profiles, when information from a set of reference observations is available to model "normal" or baseline metabolite profiles. Such outlying profiles are typically identified by comparing the distance between an observation and the reference class to a critical limit. Often, multivariate distance measures such as the Mahalanobis distance (MD) or principal component-based measures are used. These approaches, however, are either not applicable to untargeted metabolomics data, or their results are unreliable. In this paper, five distance measures for one-class modeling in untargeted metabolites are proposed. They are based on a combination of the MD and five so-called eigenvalue-shrinkage estimators of the covariance matrix of the reference class. A simple cross-validation procedure is proposed to set the critical limit for outlier detection. Simulation studies are used to identify which distance measure provides the best performance for one-class modeling, in terms of type I error and power to identify abnormal metabolite profiles. Empirical evidence demonstrates that this method has better type I error (false positive rate) and improved outlier detection power than the standard (principal component-based) one-class models. The method is illustrated by its application to liquid chromatography coupled to mass spectrometry (LC-MS) and nuclear magnetic response spectroscopy (NMR) untargeted metabolomics data from two studies on food safety assessment and diagnosis of rare diseases, respectively.

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.

Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process.

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.

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.

Comparative Subsequence Sets Analysis (CoSSA) is a robust approach to identify haplotype specific SNPs; mapping and pedigree analysis of a potato wart disease resistance gene Sen3.

BACKGROUND: Standard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data. RESULTS: As a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties. CONCLUSIONS: CoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers.

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.

NLR network mediates immunity to diverse plant pathogens.

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that "sensor" NLR proteins are paired with "helper" NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.

Effects of a genetically modified potato on a non-target aphid are outweighed by cultivar differences.

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.

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.

Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity.

Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

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.

The tomato phosphatidylinositol-phospholipase C2 (SlPLC2) is required for defense gene induction by the fungal elicitor xylanase.

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.