Broad-spectrum Late Blight Resistance Research Articles

Comparative analysis of late blight resistance R genes and their coding proteins in some potato genotypes

Late blight (LB) disease can cause potato yield losses in both Egypt and the world. Therefore, the structural analysis of resistance (R) genes responsible for LB resistance will help in understanding their functions. This work aimed to identify the variations between the dominant and recessive alleles of two genes, R3a and R8 at the nucleotide and amino acid levels in five potato genotypes. Two genes of R3a and R8 representing the broad-spectrum LB resistance were amplified by specific primers, which gave one amplicon of 194 and 220 bp of each gene, respectively. Two fragments were sequenced after purification using an ABI 3730xl System DNA Sequencer. The DNA sequence alignments of two genes, R3a and R8, were determined among five selected potato genotypes. The percentage of genetic similarity of the nucleotide sequences of the R3a and R8 genes ranged between (82–83%) and (86–87%), respectively, in comparison to the reference sequences in the nucleotide BLAST. We report on the existence of positional differences in the nucleotide sequences, and base-pair substitutions of two fragments, resulting in amino acid changes between the resistant and susceptible potato genotypes. On the other hand, the highest total number of base-pair substitutions was recorded as 16 in the recessive allele r8 of the varieties Bellini and Cara. However, the lowest number was recorded as four in the dominant allele R3a of the variety Cara. The dendrograms of the five potato genotypes were made up of phylogenetically different clusters, separate from all the other named potato accessions of the two genes. The results of this study will create a solid base for the further understanding of the mechanism of plant-pathogen interactions and supply a theoretical reference for durable resistance to late blight diseases in the potato.

Identification and rapid mapping of a gene conferring broad-spectrum late blight resistance in the diploid potato species Solanum verrucosum through DNA capture technologies

Key messageA broad-spectrum late blight disease-resistance gene from Solanum verrucosum has been mapped to potato chromosome 9. The gene is distinct from previously identified-resistance genes.We have identified and characterised a broad-spectrum resistance to Phytophthora infestans from the wild Mexican species Solanum verrucosum. Diagnostic resistance gene enrichment (dRenSeq) revealed that the resistance is not conferred by previously identified nucleotide-binding, leucine-rich repeat genes. Utilising the sequenced potato genome as a reference, two complementary enrichment strategies that target resistance genes (RenSeq) and single/low-copy number genes (Generic-mapping enrichment Sequencing; GenSeq), respectively, were deployed for the rapid, SNP-based mapping of the resistance through bulked-segregant analysis. Both approaches independently positioned the resistance, referred to as Rpi-ver1, to the distal end of potato chromosome 9. Stringent post-enrichment read filtering identified a total of 64 informative SNPs that corresponded to the expected ratio for significant polymorphisms in the parents as well as the bulks. Of these, 61 SNPs are located on potato chromosome 9 and reside within 27 individual genes, which in the sequenced potato clone DM locate to positions 45.9 to 60.9 Mb. RenSeq- and GenSeq-derived SNPs within the target region were converted into allele-specific PCR-based KASP markers and further defined the position of the resistance to a 4.3 Mb interval at the bottom end of chromosome 9 between positions 52.62–56.98 Mb.

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

Key messageThe potato late blight resistance geneR8has been cloned.R8is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato.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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-016-2740-0) contains supplementary material, which is available to authorized users.

Development of late blight resistant potatoes by cisgene stacking.

BackgroundPhytophthora 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.ResultsWe 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.ConclusionsWe 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.

High-Resolution Mapping of Two Broad-Spectrum Late Blight Resistance Genes from Two Wild Species of the Solanum circaeifolium Group

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.

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

A Novel Class of Simple PCR Markers with SNP-Level Sensitivity for Mapping and Haplotype Characterization in Solanum Species

The RB gene from wild potato Solanum bulbocastanum imparts broad-spectrum late blight resistance to cultivated potato. To explore marker associations and haplotype frequencies near RB, we developed, optimized, validated, and employed a set of markers specific to the haplotype associated with the RB resistance allele. Our markers, developed using a mismatch amplification mutation assay (MAMA)-PCR approach, have single nucleotide polymorphism-level sensitivity. MAMA markers were validated via linkage mapping and applied to a collection of 60 S. bulbocastanum genotypes representing the geographic distribution of the species. Considerable marker disassociation within a 100 kb region encompassing RB was revealed. Physical distance between markers was not an effective predictor of marker disassociation. Applied to a collection of four S. bulbocastanum genotypes of known RB allelic status, a set of six MAMA markers accurately predicted the presence of the RB allele. Implications for marker aided genotype selection and association mapping in wild potato species are explored.

Competition between Phytophthora infestans effectors leads to increased aggressiveness on plants containing broad-spectrum late blight resistance.

BackgroundThe destructive plant disease potato late blight is caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary. This disease has remained particularly problematic despite intensive breeding efforts to integrate resistance into cultivated potato, largely because of the pathogen's ability to quickly evolve to overcome major resistance genes. The RB gene, identified in the wild potato species S. bulbocastanum, encodes a protein that confers broad-spectrum resistance to most P. infestans isolates through its recognition of highly conserved members of the corresponding pathogen effector family IPI-O. IpiO is a multigene family of effectors and while the majority of IPI-O proteins are recognized by RB to elicit host resistance, some variants exist that are able to elude detection (e.g. IPI-O4).Methods and FindingsIn the present study, analysis of ipiO variants among 40 different P. infestans isolates collected from Guatemala, Thailand, and the United States revealed a high degree of complexity within this gene family. Isolate aggressiveness was correlated with increased ipiO diversity and especially the presence of the ipiO4 variant. Furthermore, isolates expressing IPI-O4 overcame RB-mediated resistance in transgenic potato plants even when the resistance-eliciting IPI-O1 variant was present. In support of this finding, we observed that expression of IPI-O4 via Agrobacterium blocked recognition of IPI-O1, leading to inactivation of RB-mediated programmed cell death in Nicotiana benthamiana.ConclusionsIn this study we definitively demonstrate and provide the first evidence that P. infestans can defeat an R protein through inhibition of recognition of the corresponding effector protein.

The Rpi‐blb2 gene from Solanum bulbocastanum is an Mi‐1 gene homolog conferring broad‐spectrum late blight resistance in potato

The necessity to develop potato and tomato crops that possess durable resistance against the oomycete pathogen Phytophthora infestans is increasing as more virulent, crop-specialized and pesticide resistant strains of the pathogen are rapidly emerging. Here, we describe the positional cloning of the Solanum bulbocastanum-derived Rpi-blb2 gene, which even when present in a potato background confers broad-spectrum late blight resistance. The Rpi-blb2 locus was initially mapped in several tetraploid backcross populations, derived from highly resistant complex interspecific hybrids designated ABPT (an acronym of the four Solanum species involved:S. acaule, S. bulbocastanum, S. phureja and S. tuberosum), to the same region on chromosome 6 as the Mi-1 gene from tomato, which confers resistance to nematodes, aphids and white flies. Due to suppression of recombination in the tetraploid material, fine mapping was carried out in a diploid intraspecific S. bulbocastanum F1 population. Bacterial artificial chromosome (BAC) libraries, generated from a diploid ABPT-derived clone and from the resistant S. bulbocastanum parent clone, were screened with markers linked to resistance in order to generate a physical map of the Rpi-blb2 locus. Molecular analyses of both ABPT- and S. bulbocastanum-derived BAC clones spanning the Rpi-blb2 locus showed it to harbor at least 15 Mi-1 gene homologs (MiGHs). Of these, five were genetically determined to be candidates for Rpi-blb2. Complementation analyses showed that one ABPT- and one S. bulbocastanum-derived MiGH were able to complement the susceptible phenotype in both S. tuberosum and tomato. Sequence analyses of both genes showed them to be identical. The Rpi-blb2 protein shares 82% sequence identity to the Mi-1 protein. Significant expansion of the Rpi-blb2 locus compared to the Mi-1 locus indicates that intrachromosomal recombination or unequal crossing over has played an important role in the evolution of the Rpi-blb2 locus. The contrasting evolutionary dynamics of the Rpi-blb2/Mi-1 loci in the two related genomes may reflect the opposite evolutionary potentials of the interacting pathogens.

An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato.

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease for potato cultivation. Here, we describe the positional cloning of the Rpi-blb1 gene from the wild potato species Solanum bulbocastanum known for its high levels of resistance to late blight. The Rpi-blb1 locus, which confers full resistance to complex isolates of P. infestans and for which race specificity has not yet been demonstrated, was mapped in an intraspecific S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial artificial chromosome (BAC) clone spanning the Rpi-blb1 locus identified a cluster of four candidate resistance gene analogues of the coiled coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) class of plant resistance (R) genes. One of these candidate genes, designated the Rpi-blb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspecific transfer of broad-spectrum late blight resistance to cultivated Solanaceae from sexually incompatible host species. Paired comparisons of synonymous and non-synonymous nucleotide substitutions between different regions of Rpi-blb1 paralogues revealed high levels of synonymous divergence, also in the LRR region. Although amino acid diversity between Rpi-blb1 homologues is centred on the putative solvent exposed residues of the LRRs, the majority of nucleotide differences in this region have not resulted in an amino acid change, suggesting conservation of function. These data suggest that Rpi-blb1 is relatively old and may be subject to balancing selection.

Race nonspecific resistance for potato late blight

The late blight fungus ( Phytophthora infestans) rots susceptible species of potato plants. None of the major varieties of potato ( Solanum tuberosum) grown in the USA is resistant to US-8, the most prevalent genotype of the fungus. Now, Junqi Song, James Bradeen and colleagues have cloned the RB gene from the wild diploid potato species, Solanum bulbocastanum, using a map-based approach in combination with long-range PCR. Transgenic plants containing the gene, normally fully susceptible, displayed broad-spectrum late blight resistance.

Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight.

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating potato disease in the world. Control of late blight in the United States and other developed countries relies extensively on fungicide application. We previously demonstrated that the wild diploid potato species Solanum bulbocastanum is highly resistant to all known races of P. infestans. Potato germplasm derived from S. bulbocastanum has shown durable and effective resistance in the field. Here we report the cloning of the major resistance gene RB in S. bulbocastanum by using a map-based approach in combination with a long-range (LR)-PCR strategy. A cluster of four resistance genes of the CC-NBS-LRR (coiled coil-nucleotide binding site-Leu-rich repeat) class was found within the genetically mapped RB region. Transgenic plants containing a LR-PCR product of one of these four genes displayed broad spectrum late blight resistance. The cloned RB gene provides a new resource for developing late blight-resistant potato varieties. Our results also demonstrate that LR-PCR is a valuable approach to isolate genes that cannot be maintained in the bacterial artificial chromosome system.

Concomitant reiterative BAC walking and fine genetic mapping enable physical map development for the broad-spectrum late blight resistance region, RB.

The wild potato species Solanum bulbocastanum is a source of genes for potent late blight resistance. We previously mapped resistance to a single region of the S. bulbocastanum chromosome 8 and named the region RB (for "Resistance from S. Bulbocastanum"). We now report physical mapping and contig construction for the RB region via a novel reiterative method of BAC walking and concomitant fine genetic mapping. BAC walking was initiated using RFLP markers previously shown to be associated with late blight resistance. Subcontig extension was accomplished using new probes developed from BAC ends. Significantly, BAC end and partial BAC sequences were also used to develop PCR-based markers to enhance map resolution in the RB region. As they were developed from BAC clones of known position relative to RB, our PCR-based markers are known a priori to be physically closer to the resistance region. These markers allowed the efficient screening of large numbers of segregating progeny at the cotyledon stage, and permitted us to assign the resistance phenotype to a region of approximately 55 kb. Our markers also directed BAC walking efforts away from regions distantly related to RB in favor of the 55-kb region. Because the S. bulbocastanum genotype used in BAC library construction is heterozygous for RB (RB/rb), codominant PCR-based markers, originally developed for fine-scale mapping, were also used to determine homolog origins for individual BAC clones. Ultimately, BAC contigs were constructed for the RB region from both resistant (RB) and susceptible (rb) homologs.