Molecular and cellular biology of resistance to Phytophthora infestans in Solanum species

Ever since the late blight epidemics of the mid-nineteenth century, man has endeavored to protect his potato crop. Every year, extensive chemical protection is applied, which is expensive and may be harmful to the natural environment. The use of resistant potato cultivars would provide an elegant alternative, however breeding for late blight resistance has not yet resulted in adequately resistant cultivars. In addition to the demand for a high level of resistance, the resistance should be durable. The causal agent of the late blight disease is Phytophthora infestans , a biotrophic oomycete pathogen. Oomycetes are often incorrectly referred to as fungi, but they evolved the ability to infect plants independently from fungi, and may therefore have distinct mechanisms for interacting with plants. Insight in the mechanisms of resistance to P. infestans may assist the breeders in their operation to achieve durable resistance.Genetic resistance can be determined at the subspecies or variety level (race-specific resistance), or at the species or genus level (nonhost resistance). Nonhost resistance is full resistance, and is present in most plant species. In addition, resistance may be a quantitative trait (partial resistance). A rich pool of diverse resistances to P. infestans has been found in wild Solanum species, with levels ranging from full resistance to various levels of partial resistance. Some old potato cultivars also exhibit partial resistance, which proved to be durable. To achieve a durable late blight resistance, a better understanding of the molecular basis of the various types of resistances is essential. To this end, we compiled a set of Solanum species with various types and levels of resistance to P. infestans , and studied the cellular and molecular aspects of the resistance mechanisms present in these plants.Laboratory studies at the cellular and molecular level require an experimental assay, which is both comparable to the natural situation, and assures a high percentage of successful infections. To this end, we developed a resistance assay with detached leaves under controlled conditions in the laboratory and compared this assay with a field trial. The tested growing conditions of the plants did not affect the resistance to P. infestans. Leaves on intact plants however, were more resistant than detached leaves. The incubation conditions of the detached leaves in the laboratory assay rather than the detachment itself appeared to affect the resistance expression. However, on intact plants the infection frequency was too low for molecular studies. Since the ranking of resistance levels within a set of twenty plant clones was similar under laboratory and field conditions, the laboratory assay proved adequate to study the Solanum - P. infestans interaction.A cytological survey of the interaction between twenty Solanum clones and three P. infestans isolates provided the first impression of the nature of the resistance responses. Potato cultivars with race-specific resistance ( R ) genes displayed the hypersensitive response (HR), a programmed cell death of plant cells, upon inoculation with P. infestans . Through this rapid cell death, the biotrophic pathogen became localized between dead cells, and was prevented from further growth. Also durably resistant potato cultivars without known R genes, wild Solanum species, and nonhosts displayed the HR. Interestingly, in highly resistant Solanum species such as S. berthaultii and S circaeifolium, and nonhosts such as Arabidopsis thaliana and S. nigrum (black nightshade), the HR was extremely fast and effective, resulting in very localized cell death. In partially resistant plants, the HR was delayed, and resulted in larger HR lesions. Occasionally, hyphae were able to escape from these lesions and established a biotrophic interaction with the host. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones, and correlated with resistance levels. In addition to the HR, local depositions of callose and phenolic compounds occurred, which may function as physical barriers. Although these responses did not correlate with resistance levels, they may influence the balance between growth of the pathogen and induction of the HR. Ultimately, this fine balance may determine resistance at the cellular level, and illustrates the quantitative nature of the resistance to P. infestans at the plant or field level .The HR is initiated upon recognition of pathogen elicitors by plant cell receptors or R gene products as suggested by the gene-for-gene hypothesis. Several types of R genes are recognized in plants, including the nucleotide binding site leucine-rich repeat (NBS-LRR) type, and the Pto-like serine/threonine protein kinase type. The Pto gene was originally identified from wild relatives of tomato. We exploited our Solanum collection to identify Pto -like sequences, and studied evolutionary scenarios for Pto -like genes. Polymerase chain reaction (PCR) amplifications using primers based on conserved and variable regions of Pto yielded 32 intact Pto -like sequences from six Solanum species, and revealed an extensive Pto family. Pto -like transcripts were also detected in leaf tissue of all tested plants. The kinase consensus and autophosphorylation residues were highly conserved, in contrast to the kinase activation domain which is involved in ligand recognition in Pto. Phylogenetic analyses distinguished nine classes of Pto -like genes, and revealed that orthologues (homologues separated by a speciation process) were more similar than paralogues (homologues generated by a gene duplication event). This suggests that the Pto gene family evolved through a series of ancient gene duplication events prior to speciation in Solanum . The phylogenetic data are in line with recent results on the NBS-LRR class of R genes, and suggest that Pto- like genes are ancient, and highly diverse.Various levels of nonspecific resistance were revealed in Solanum species after inoculation with five P. infestans isolates. In partially resistant plants where hyphal escape occurred, the lesions expanded often slower than in susceptible plants. Here, defense mechanisms other than HR are thought to operate; this might for example involve systemic acquired resistance (SAR). SAR can be induced by various signals, but also basal levels of SAR may vary between plants. When we monitored basal expression levels of SAR marker genes in healthy leaves , we found variation between the Solanum clones in constitutive mRNAs levels of the pathogenesis-related ( PR ) genes PR-1 , PR-2 , and PR-5 . At the genus level, there was no correlation between basal PR mRNA levels and nonspecific resistance to P. infestans . In contrast, a positive correlation was found at the species level in S. arnezii x hondelmannii , S. microdontum , S. sucrense and S. tuberosum . In S. tuberosum cultivars, the levels of PR gene expression were the highest in resistant 'Robijn', intermediate in partially resistant 'Première', 'Estima' and 'Ehud', and the lowest in susceptible 'Bintje'. These results suggest that constitutive expression of PR genes may contribute to nonspecific resistance to P. infestans in Solanum . Therefore, PR mRNA levels could serve as molecular markers in potato breeding programs.In conclusion, diverse resistance reactions to P. infestans operate at various levels in Solanum species, including specific and nonspecific mechanisms. The ubiquitous association of the HR in all types of resistance suggests that numerous R genes are present in Solanum against the oomycete P. infestans . A remaining challenge is the identification and transfer of these R genes into commercially grown potato cultivars.</p

Allele mining in solanum: conserved homologues of Rpi-blb 1 are identified in Solanum stoloniferum

Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding

High resolution mapping of a novel late blight resistance gene Rpi-avll, from the wild Bolivian species Solanum avilesii

Both Mexico and South America are rich in Solanum species that might be valuable sources of resistance (R) genes to late blight (Phytophthora infestans). Here, we focus on an R gene present in the diploid Bolivian species S. avilesii. The genotype carrying the R gene was resistant to eight out of 10 Phytophthora isolates of various provenances. The identification of a resistant phenotype and the generation of a segregating population allowed the mapping of a single dominant R gene, Rpi-avl1, which is located in an R gene cluster on chromosome 11. This R gene cluster is considered as an R gene “hot spot”, containing R genes to at least five different pathogens. High resolution mapping of the Rpi-avl1 gene revealed a marker co-segregating in 3890 F1 individuals, which may be used for marker assisted selection in breeding programs and for further cloning of Rpi-avl

Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato

Identification of resistance (R) genes to Phytophthora infestans is an essential step in molecular breeding of potato. We identified three specific R genes segregating in a diploid mapping population. One of the R genes is located on chromosome 4 and proved phenotypically indistinguishable from the Solanum demissum-derived R2, although S. demissum is not directly involved in the pedigree of the population. By bulked segregant analysis combined with a resistance assay, a genetic linkage map of the R2-like locus was constructed with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking the R2-like locus were applied to screen an extended population of 1,586 offspring. About 103 recombinants were selected, and an accurate high-resolution map was constructed. The R2-like resistance was localized in a 0.4 cM interval and was found co-segregating with four AFLP markers, which can be used to isolate the R2-like gene by map-based gene cloning. By analyzing race-specificity and R gene-specific molecular markers, we also found that an R1-like gene and an additional unknown R gene are segregating in the populatio

SolRgene: an online database to explore disease resistance genes in tuber-bearing Solanum species

Background The cultivated potato (Solanum tuberosum L.) is an important food crop, but highly susceptible to many pathogens. The major threat to potato production is the Irish famine pathogen Phytophthora infestans, which causes the devastating late blight disease. Potato breeding makes use of germplasm from wild relatives (wild germplasm) to introduce resistances into cultivated potato. The Solanum section Petota comprises tuber-bearing species that are potential donors of new disease resistance genes. The aim of this study was to explore Solanum section Petota for resistance genes and generate a widely accessible resource that is useful for studying and implementing disease resistance in potato. Description The SolRgene database contains data on resistance to P. infestans and presence of R genes and R gene homologues in Solanum section Petota. We have explored Solanum section Petota for resistance to late blight in high throughput disease tests under various laboratory conditions and in field trials. From resistant wild germplasm, segregating populations were generated and assessed for the presence of resistance genes. All these data have been entered into the SolRgene database. To facilitate genetic and resistance gene evolution studies, phylogenetic data of the entire SolRgene collection are included, as well as a tool for generating phylogenetic trees of selected groups of germplasm. Data from resistance gene allele-mining studies are incorporated, which enables detection of R gene homologs in related germplasm. Using these resources, various resistance genes have been detected and some of these have been cloned, whereas others are in the cloning pipeline. All this information is stored in the online SolRgene database, which allows users to query resistance data, sequences, passport data of the accessions, and phylogenic classifications. Conclusion Solanum section Petota forms the basis of the SolRgene database, which contains a collection of resistance data of an unprecedented size and precision. Complemented with R gene sequence data and phylogenetic tools, SolRgene can be considered the primary resource for information on R genes from potato and wild tuber-bearing relatives

Population dynamics of Phytophthora infestans in the Netherlands reveals expansion and spread of dominant clonal lineages and virulence in sexual offspring

For a comprehensive survey of the structure and dynamics of the Dutch Phytophthora infestans population, 652 P. infestans isolates were collected from commercial potato fields in the Netherlands during the 10-year period 2000–2009. Genotyping was performed using 12 highly informative microsatellite markers and mitochondrial haplotypes. In addition, for each isolate, the mating type was determined. STRUCTURE analysis grouped the 322 identified genotypes in three clusters. Cluster 1 consists of a single clonal lineage NL-001, known as “Blue_13”; all isolates in this cluster have the A2 mating type and the Ia mitochondrial haplotype. Clusters 2 and 3 display a more elaborate substructure containing many unique genotypes. In Cluster 3, several distinct clonal lineages were also identified. This survey witnesses that the Dutch population underwent dramatic changes in the 10 years under study. The most notable change was the emergence and spread of A2 mating type strain NL-001 (or “Blue_13”). The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation and dispersal mechanism for successful genotypes. Isolates were also screened for absence of the Avrblb1/ipiO class I gene, which is indicative for virulence on Rpi-blb1. This is also the first report of Rpi-blb1 breakers in the Netherlands. Superimposing the virulence screening on the SSR genetic backbone indicates that lack the Avrblb1/ipiO class I gene only occurred in sexual progeny. So far, the asexual spread of the virulent isolates identified has been limited

Mycorrhizal symbiosis: ancient signalling mechanisms co-opted

Mycorrhizal root endosymbiosis is an ancient property of land plants. Two parallel studies now provide novel insight into the mechanism driving this interaction and how it is used by other filamentous microbes like pathogenic oomycetes

Snel plantmonitoren met nieuwe cameratechniek (interview met Dr. V.G.A.A. Vleeshouwers)

De onderzoekers Henk Jalink en Rob van der Schoor van Wageningen UR hebben een innovatieve MIPS-LED camera ontwikkeld, waarmee collega-onderzoekers van de universiteit snel fotosynthese in planten kunnen vastleggen. Vivianne Vleeshouwers, is werkzaam bij Wageningen UR Plantenveredeling, en is een van de onderzoekers die de camera beproeft. Zij hoopt het apparaat te kunnen inzetten voor onderzoek naar phytophthoraresistentie in (nieuwe) aardappelrassen en wilde soorten