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.     

Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.     

Evolutionary meta-analysis of solanaceous resistance gene and solanum resistance gene analog sequences and a practical framework for cross-species comparisons

Cross-species comparative genomics approaches have been employed to map and clone many important disease resistance (R) genes from Solanum species-especially wild relatives of potato and tomato. These efforts will increase with the recent release of potato genome sequence and the impending release of tomato genome sequence. Most R genes belong to the prominent nucleotide binding site-leucine rich repeat (NBS-LRR) class and conserved NBS-LRR protein motifs enable survey of the R gene space of a plant genome by generation of resistance gene analogs (RGA), polymerase chain reaction fragments derived from R genes. We generated a collection of 97 RGA from the disease-resistant wild potato S. bulbocastanum, complementing smaller collections from other Solanum species. To further comparative genomics approaches, we combined all known Solanum RGA and cloned solanaceous NBS-LRR gene sequences, nearly 800 sequences in total, into a single meta-analysis. We defined R gene diversity bins that reflect both evolutionary relationships and DNA cross-hybridization results. The resulting framework is amendable and expandable, providing the research community with a common vocabulary for present and future study of R gene lineages. Through a series of sequence and hybridization experiments, we demonstrate that all tested R gene lineages are of ancient origin, are shared between Solanum species, and can be successfully accessed via comparative genomics approaches.     

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.     

Overexpression of the wild potato eIF4E-1 variant Eva1 elicits Potato virus Y resistance in plants silenced for native eIF4E-1

Potato virus Y (PVY) is the most important viral pathogen of cultivated potato (Solanum tuberosum) from a commercial perspective, causing severe losses in both tuber quality and yield worldwide. Specific accessions of wild potato species exhibit resistance against PVY but efforts to transfer the trait to cultivated material have not yielded widely adopted varieties. Because amino acid substitutions at specific domains of host factor eIF4E-1 often confer resistance to various crops, we sequenced the associated genes expressed in wild potato plants. A novel eIF4E-1 variant, designated here as Eva1, was identified in S. chacoense, S. demissum, and S. etuberosum. The protein contains amino acid substitutions at ten different positions when compared to its cultivated potato (S. tuberosum) homolog. In the yeast two-hybrid system, Eva1 failed to bind VPg, a viral protein required for infectivity. Overexpression of the associated cDNA conferred PVY resistance to transgenic potato plants silenced for the native eIF4E-1 gene. Because the gene sources of Eva1 are sexually compatible with potato, the molecular strategies described can be employed to develop 'intragenic' potato cultivars.     

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.     

Application of somatic hybrids between dihaploids of potato Solanum tuberosum L. and wild diploid species from Mexico in breeding: generation and backcrossing of dihaploids of somatic hybrids

The efficiency of an original approach to involvement of the valuable genetic pool of wild diploid potato species from Mexico is estimated. The essence of this method is in generation of dihaploids (2n = 2x = 24) of tetraploid somatic hybrids (2n = 4x = 48) followed by backcrossing with dihaploids of Solanum tuberosum. A haploid producer, S. phureja IvP35, was used to generate ten dihaploids of S. tuberosum + S. pinnatisectum, all of which crossed with fertile S. tuberosum dihaploids and developed plump viable seeds. This gives the possibility of an efficient introgression of the genes valuable for breeding from wild species to the bred plants at a diploid level, which has several advantages compared with the corresponding procedure at a tetraploid level. A part of the dihaploids produced was compatible (the pollen tubes reached the ovary) with diploid and tetraploid forms of S. pinnatisectum; however, no viable seeds were developed. The attempt to generate the dihaploids of S. tuberosum + S. bulbocastanum somatic hydrides using the haploid producer S. phureja IvP35 was unsuccessful.     

Genetic and physical mapping of homologues of the virus resistance gene <Emphasis Type="Italic">Rx1</Emphasis> and the cyst nematode resistance gene <Emphasis Type="Italic">Gpa2</Emphasis> in potato

Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.     

Quantitative Resistance to Phytophthora Infestans in Potato: A Case Study for Qtl Mapping in an Allogamous Plant Species

Phytophthora infestans is the most important fungal pathogen in the cultivated potato (Solanum tuberosum). Dominant, race-specific resistance alleles and quantitative resistance-the latter being more important for potato breeding- are found in the germplasm of cultivated and wild potato species. Quantitative trait loci (QTLs) for resistance to two races of P. infestans have been mapped in an F(1) progeny of a cross between non-inbred diploid potato parents with multiple alleles. Interval mapping methods based on highly informative restriction fragment length polymorphism markers revealed 11 chromosome segments on 9 potato chromosomes showing significant contrasts between marker genotypic classes. Whereas phenotypically no difference in quantitative resistance response was observed between the two fungal races, QTL mapping identified at least one race specific QT locus. Two QT regions coincided with two small segments on chromosomes V and XII to which the dominant alleles R1, conferring race specific resistance to P. infestans, Rx1 and Rx2, both inducing extreme resistance to potato virus X, have been allocated in independent mapping experiments. Some minor QTLs were correlated with genetic loci for specific proteins related to pathogenesis, the expression of which is induced after infection with P. infestans.     

Diploid hybrids between allotetraploid wild potato species Solanum acaule Bitt., S. stoloniferum Schltdl. and dihaploids of S. tuberosum L

The possibility to obtain diploid hybrids by pollination of allotetraploid wild potato species Solanum acaule and S. stoloniferum plants with fertile pollen of S. tuberosum dihaploids was demonstrated for the first time. Dihaploid hybrids have arisen with comparatively high frequency (from 12.5 to 33.3%). They were characterized by high regularity of meiosis and high fertility. They easily crossed with S. tuberosum dihaploids, forming viable progeny. This seems prospective for effective introgression of valuable genetic gene pool of wild allotetraploid potato species in breeding material of S. tuberosum on the diploid level.     

The late blight resistance locus Rpi-bib3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.     

吖啶橙失活处理应用于不对称融合初步探讨

马铃薯栽培种和野生种叶肉原生质体经过吖啶橙（ＡＯ）处理后,暗培养两天,再光照４ｈ而失活．失活程度取决于ＡＯ处理时间、ＡＯ浓度、光照时机等．四倍体失活浓度高于二倍体,处理后马上光照失活效果最强烈,８ｄ后光照其小细胞团同未光照处理相差不大。ＡＯ失活的栽培种ＮＯ７同罗丹明６Ｇ（Ｒ６Ｇ）失活的Ｓｏｌａｎｕｍｂｕｌｂｏｃａｓｔａｎｕｍ融合后可以发生代谢互补恢复分裂,已得到假定的杂种小愈伤组织．     

Development of SCAR markers to the PVY resistance gene Ryadg based on a common feature of plant disease resistance genes.

Sequence-characterized amplified regions (SCARs) were developed, based on nucleotide differences within resistance gene-like fragments isolated from a potato plant carrying the Ryadg gene, which confers extreme resistance to potato Y potyvirus (PVY). It originates from Solanum tuberosum subsp. andigena, and a susceptible potato plant. SCARs were tested using 103 potato breeding lines and cultivars with diverse genetic backgrounds derived from Europe, North America, and Japan. Two markers showed high accuracy for detection of the Ryadg gene. The SCAR marker RYSC3 was generated only in genotypes carrying Ryadg. The SCAR marker RYSC4 was detected in all genotypes carrying Ryadg but also in four PVY-susceptible genotypes. Neither marker was detected in genotypes carrying other Ry genes originating from different species than S. tuberosum subsp. andigena. Therefore, these SCAR markers should be powerful tools in marker-assisted selection for Ryadg in potato breeding programs, and should also be useful for cloning of the Ryadg gene.     

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.     

Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato

Rx2 confers resistance against potato virus X (PVX). To clone Rx2, we developed a system based on Agrobacterium-mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2-mediated resistance. Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified. Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2. The transient expression system is potentially adaptable to cloning of any other resistance gene. The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII. Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2. All three proteins are in the leucine zipper-nucleotide binding site-leucine rich repeat class of resistance gene products. Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein. In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein. However, in other regions these proteins are more similar to Rxh1 than to each other. Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion.     

Potato virus X-induced gene silencing in leaves and tubers of potato

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.     

Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants

Snakin-1 (SN1), a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro, was evaluated for its ability to confer resistance to pathogens in transgenic potatoes. Genetic variants of this gene were cloned from wild and cultivated Solanum species. Nucleotide sequences revealed highly evolutionary conservation with 91-98% identity values. Potato plants (S. tuberosum subsp. tuberosum cv. Kennebec) were transformed via Agrobacterium tumefaciens with a construct encoding the S. chacoense SN1 gene under the regulation of the ubiquitous CaMV 35S promoter. Transgenic lines were molecularly characterized and challenged with either Rhizoctonia solani or Erwinia carotovora to analyse whether constitutive in vivo overexpression of the SN1 gene may lead to disease resistance. Only transgenic lines that accumulated high levels of SN1 mRNA exhibited significant symptom reductions of R. solani infection such as stem cankers and damping-off. Furthermore, these overexpressing lines showed significantly higher survival rates throughout the fungal resistance bioassays. In addition, the same lines showed significant protection against E. carotovora measured as: a reduction of lesion areas (from 46.5 to 88.1% with respect to the wild-type), number of fallen leaves and thickened or necrotic stems. Enhanced resistance to these two important potato pathogens suggests in vivo antifungal and antibacterial activity of SN1 and thus its possible biotechnological application.     

Allele Mining in Solanum Germplasm: Cloning and Characterization of RB-Homologous Gene Fragments from Late Blight Resistant Wild Potato Species

Plant Molecular Biology Reporter - The late blight disease can be managed by introduction of resistance (R) genes from the wild Solanum species into the cultivated potato. The R genes are mostly...