Functional stacking of three resistance genes against Phytophthora infestans in potato

Functional stacking of broad spectrum resistance (R) genes could potentially be an effective strategy for more durable disease resistance, for example, to potato late blight caused by Phytophthora infestans (Pi). For this reason, three broad spectrum potato R genes (Rpi), Rpi-sto1 (Solanum stoloniferum), Rpi-vnt1.1 (S. venturii) and Rpi-blb3 (S. bulbocastanum) were selected, combined into a single binary vector pBINPLUS and transformed into the susceptible cultivar Desiree. Among the 550 kanamycin resistant regenerants, 28 were further investigated by gene specific PCRs. All regenerants were positive for the nptII gene and 23 of them contained the three Rpi genes, referred to as triple Rpi gene transformants. Detached leaf assay and agro-infiltration of avirulence (Avr) genes showed that the 23 triple Rpi gene transformants were resistant to the selected isolates and showed HR with the three Avr effectors indicating functional stacking of all the three Rpi genes. It is concluded that Avr genes, corresponding to the R genes to be stacked, must be available in order to assay for functionality of each stack component. No indications were found for silencing or any other negative effects affecting the function of the inserted Rpi genes. The resistance spectrum of these 23 triple Rpi gene transformants was, as expected, a sum of the spectra from the three individual Rpi genes. This is the first example of a one-step approach for the simultaneous domestication of three natural R genes against a single disease by genetic transformation.     

A resistance gene against potato late blight originating from Solanum × michoacanum maps to potato chromosome VII

Solanum ×  michoacanum (Bitter.) Rydb. is a diploid, 1 EBN (Endosperm Balance Number) nothospecies, a relative of potato originating from the area of Morelia in Michoacán State of Mexico that is believed to be a natural hybrid of S. bulbocastanum × S. pinnatisectum. Both parental species and S. michoacanum have been described as sources of resistance to Phytophthora infestans (Mont.) de Bary. The gene for resistance to potato late blight, Rpi-mch1, originating from S. michoacanum was mapped to the chromosome VII of the potato genome. It confers high level of resistance since the plants possessing it showed only small necrotic lesions or no symptoms of the P. infestans infection and we could ascribe over 80% of variance observed in the late blight resistance test of the mapping population to the effect of the closest marker. Its localization on chromosome VII may correspond to the localization of the Rpi1 gene from S. pinnatisectum. When mapping Rpi-mch1, one of the first genetic maps made of 798 Diversity Array Technology (DArT) markers of a plant species from the Solanum genus and the first map of S. michoacanum, a 1EBN potato species was constructed. Particular chromosomes were identified using 48 sequence-specific PCR markers, originating mostly from the Tomato-EXPEN 2000 linkage map (SGN), but also from other sources. Recently, the first DArT linkage map of 2 EBN species Solanum phureja has been published and it shares 197 DArT markers with map obtained in this study, 88% of which are in the concordant positions.     

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.     

A novel approach to locate Phytophthora infestans resistance genes on the potato genetic map

Mapping resistance genes is usually accomplished by phenotyping a segregating population for the resistance trait and genotyping it using a large number of markers. Most resistance genes are of the NBS-LRR type, of which an increasing number is sequenced. These genes and their analogs (RGAs) are often organized in clusters. Clusters tend to be rather homogenous, viz. containing genes that show high sequence similarity with each other. From many of these clusters the map position is known. In this study we present and test a novel method to quickly identify to which cluster a new resistance gene belongs and to produce markers that can be used for introgression breeding. We used NBS profiling to identify markers in bulked DNA samples prepared from resistant and susceptible genotypes of small segregating populations. Markers co-segregating with resistance can be tested on individual plants and directly used for breeding. To identify the resistance gene cluster a gene belongs to, the fragments were sequenced and the sequences analyzed using bioinformatics tools. Putative map positions arising from this analysis were validated using markers mapped in the segregating population. The versatility of the approach is demonstrated with a number of populations derived from wild Solanum species segregating for P. infestans resistance. Newly identified P. infestans resistance genes originating from S. verrucosum, S. schenckii, and S. capsicibaccatum could be mapped to potato chromosomes 6, 4, and 11, respectively.     

Crossing relationships and chromosome numbers of Solanum section Solanum in Uganda

The ability of taxa to cross/hybridize is useful information for plant systematists and breeders. Crossability reflects reproductive isolation and the biological species concept stresses the need for reproductive isolation between species to maintain morphological distincness. For plant breeders knowledge on crossing ability facilitate selection of taxa for character improvement breeding. In this study, the crossing relationships and chromosome numbers within and among Ugandan species of Solanum sect. Solanum is studied by making 800 crosses involving 246 combinations. Less than half of these combinations were successful, producing F1 offspring. All studied accessions are self‐compatible and most accessions crossed readily with accessions of their own species. Interspecific crossings failed either to yield seeds, yielded F1 seeds that did not germinate, or resulted in F1s that did not have stainable pollen – implying a crossing barrier; or stainable pollen, but with chromosome numbers that indicated reproduction by apomixis. The results support the taxonomic treatment of Solanum based on classical, numerical and partly molecular evidences. The material studied represents eight Ugandan taxa: S. americanum, a diploid (2n = 2x = 24); five tetraploids (2n = 4x = 48) S. florulentum, S. memphiticum, S. tarderemotum, S. villosum ssp. villosum and S. villosum ssp. miniatum; and two hexaploids (2n = 6x = 72) S. scabrum subsp. scabrum and S. scabrum subsp. laevis. In addition to confirming the ploidy levels of the Ugandan accessions, the ploidy levels of S. florulentum, S. memphiticum and S. tarderemotum are reported for the first time. Non‐Ugandan material of Solanum sarrachoides was found to be diploid. Knowledge of the crossing behaviour and ploidy levels in Solanum will facilitate breeding for character improvement in these important species that are used commonly as food and/or medicine in eastern Africa.     

Tissue-specific signal(s) activate the promoter of a metallocarboxypeptidase inhibitor gene family in potato tuber and berry

The molecular basis of the differential expression of the GM7-type metallocarboxypeptidase inhibitor (MCPI) genes in tuberizing (StMCPI) and non-tuberizing Solanum species (SbMCPI) was investigated. It was shown that the StMCPI is encoded by a gene family in Solanum tuberosum (potato), but SbMCPI might be a single-copy gene in the non-tuberizing species Solanum brevidens. The StMCPI promoter shows evolutionary relatedness to the S. brevidens-derived SbMCPI and to the fruit-specific tomato promoter 2A11. Both StMCPI and SbMCPI promoter regions were able to confer tuber- and berry-specific expression for the -glucuronidase reporter gene in potato suggesting that the difference in MCPI gene expression is in trans regulatory factors between the tuberizing and the non-tuberizing Solanum species. The MCPI promoters did not respond to metabolic, environmental or hormonal signals in leaves. Thus, the MCPI genes are regulated in a different way than the other known tuber-specific genes and potentially are suitable for biotechnological application in potato to provide specific transgene expression in tuber and berry.     

Antixenosis phloem‐based resistance to aphids: is it the rule?

1. The concept of plant defence syndrome states that plant species growing in similar biotic or abiotic constraints should have convergent defensive traits. This article is a first step to test the prediction of this concept, by conducting experiments on wild Solanum species (or accessions) that originated from the Andes. The nature and the tissue localisation of the resistance of five wild Solanum species known to be resistant against the aphids Myzus persicae and Macrosiphum euphorbiae were determined by olfactometry and electrical penetration graph experiments. 2. Volatile organic compounds may contribute to wild Solanum resistance, depending on Solanum accessions and aphid species. Volatiles of S. bukasovii and S. stoloniferum PI 275248 were not attractive to M. persicae, whereas S. bukasovii was repulsive to M. euphorbiae. In contrast, volatiles of S. stoloniferum PI 275248 were attractive for M. euphorbiae. 3. Some wild Solanum species presented a generalised resistance in all plant tissues, so as for S. bukasovii and S. stoloniferum PI 275248 against M. persicae. However, except for S. bukasovii which was susceptible to M. euphorbiae, all tested Solanum species presented a phloem‐based antixenosis resistance against the two aphid species. 4. A review of articles focused on the nature of resistance of wild Solanum species against aphids corroborated with our results, i.e. a phloem‐based antixenosis resistance against aphids is the rule concerning the system aphids–wild Solanum species.     

Somatic hybrids of Solanum tuberosum – application to genetics and breeding

Cultivated potato (Solanum tuberosum L.) is one of the first agricultural crops successfully cultured in vitro and used for obtaining of somatic hybrids. The review presents the current state of knowledge of somatic hybridisation involving this and other species from the genus of Solanum. Methods of somatic hybridisation, in particular factors that must be considered during designing the experiments are presented and discussed. The main attention however is focused on processes that are responsible for somatic hybrid formation. Complex interactions between genomes and plasmones lead to formation of symmetric, asymmetric and cytoplasmic recombinants. The concept of alloplasmic incompatibility is presented and discussed in relation to Solanum hybrids. Selected examples of potato somatic hybrids with agronomically important traits derived from wild species are presented in the table and discussed.     

Epidermal hair morphology in Solanum L. section Solanum

Scanning electron microscopy was used to examine stem, leaf, staminal and stylar hairs on species belonging to Solanum L. section Solanum. The surface morphology of these hairs is illustrated. Simple, uniseriate hairs characterize the section Solanum, and these may have eglandular or glandular heads; they are usually multicellular, but in some species the stylar hairs appear to be unicellular. In addition, stalked glands, described here as spherical, four-celled glands, are universally present in species belonging to the section Solanum.     

Control of the expression of bacterial genes involved in symbiotic nitrogen fixation

Several genera of N₂-fixing bacteria establish symbiotic associations with plants. Among these, the genus Rhizobium has the most significant contribution, in terms of yield, in many important crop plants. The establishment of the Rhizobium-legume symbiosis is a very complex process involving many genes which need to be co-ordinately regulated. In the first instance, plant signal molecules, known to be flavonoids, trigger the expression of host-specific genes in the bacterial partner through the action of the regulatory NodD protein. In response to these signals, Rhizobium bacteria synthesize lipo-oligosaccharide molecules which in turn cause cell differentiation and nodule development. Once the nodule has formed, Rhizobium cells differentiate into bacteroids and another set of genes is activated. These genes, designated nif and fix, are responsible for N₂ fixation. In this system, several regulatory proteins are involved in a complex manner, the most important being NifA and a two component (FixK and FixL) regulatory system. Our knowledge about the establishment of these symbioses has advanced recently, although there are many questions yet to be solved.     

A locus conferring effective late blight resistance in potato cultivar Sárpo Mira maps to chromosome XI

Late blight of potato, caused by Phytophthora infestans, is one of the most economically important diseases worldwide, resulting in substantial yield losses when not adequately controlled by fungicides. Late blight was a contributory factor in The Great Irish Famine, and breeding for resistance to the disease began soon after. Several disease-resistant cultivars have subsequently been obtained, and amongst them Sárpo Mira is currently one of the most effective. The aim of this work was to extend the knowledge about the genetic basis of the late blight resistance in Sárpo Mira and to identify molecular markers linked to the resistance locus which would be useful for marker-assisted selection. A tetraploid mapping population from a Sárpo Mira × Maris Piper cross was phenotyped for foliar late blight resistance using detached leaflet tests. A locus with strong effect on late blight resistance was mapped at the end of chromosome XI in the vicinity of the R3 locus. Sárpo Mira’s genetic map of chromosome XI contained 11 markers. Marker 45/XI exhibited the strongest linkage to the resistance locus and accounted for between 55.8 and 67.9 % of variance in the mean resistance scores noted in the detached leaflet assays. This marker was used in molecular marker-facilitated gene pyramiding. Ten breeding lines containing a late blight resistance locus from cultivar Sárpo Mira and the Rpi-phu1 gene originating from the late blight resistant accession of Solanum phureja were obtained. These lines have extended the spectrum of late blight resistance compared with Sárpo Mira and it is expected that resistance in plants containing this gene pyramid will have enhanced durability.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Comparative next-generation mapping of the Phytophthora infestans resistance gene Rpi-dlc2 in a European accession of Solanum dulcamara

Phytophthora infestans, the causal agent of late blight, remains the main threat to potato production worldwide. Screening of 19 accessions of Solanum dulcamara with P. infestans isolate Ipo82001 in detached leaf assays revealed strong resistance in an individual belonging to accession A54750069-1. This plant was crossed with a susceptible genotype, and an F₁ population consisting of 63 individuals was obtained. This population segregated for resistance in 1:1 ratio, both in detached leaf assays and in an open-field experiment. Presence of the formerly mapped Rpi-dlc1 gene as the cause of the observed segregating resistance could be excluded. Subsequently, AFLP analyses using 128 primer combinations enabled identification of five markers linked to a novel resistance gene named Rpi-dlc2. AFLP markers did not show sequence similarity to the tomato and potato genomes, hampering comparative genetic positioning of the gene. For this reason we used next-generation mapping (NGM), an approach that exploits direct sequencing of DNA (in our case: cDNA) pools from bulked segregants to calculate the genetic distance between SNPs and the locus of interest. Plotting of these genetic distances on the tomato and potato genetic map and subsequent PCR-based marker analysis positioned the gene on chromosome 10, in a region overlapping with the Rpi-ber/ber1 and -ber2 loci from S. berthaultii. Pyramiding of Rpi-dlc2 and Rpi-dlc1 significantly increased resistance to P. infestans, compared with individuals containing only one of the genes, showing the usefulness of this strategy to enhance resistance against Phytophthora.     

Cloning and mapping multiple S-locus F-box genes in European pear (Pyrus communis L.)

European pear, as well as its close relatives Japanese pear and apple, exhibits S-RNase-based gametophytic self-incompatibility. The male determinant of this self-incompatibility mechanism is a pollen-expressed protein containing an F-box domain; in the genera Petunia (Solanaceae), Antirrhinum (Plantaginaceae), and Prunus (Rosaceae), a single F-box gene determines the pollen S. In apple and Japanese pear, however, multiple S-locus F-box genes were recently identified as candidates for the pollen S, and they were named S-locus F-Box Brothers. These genes were considered good candidates for the pollen S determinant since they exhibit S-haplotype-specific polymorphisms, pollen-specific expression, and linkage to the S-RNase. In the present study, S-locus F-Box Brothers homologs have been cloned from two of the most agronomically important European pear varieties, “Abbé Fétel” (S_104-2/S₁₀₅) and “Max Red Bartlett” (S₁₀₁/S₁₀₂), and they have been mapped on a genetic linkage map developed on their progeny. Our results suggest that the number of F-box genes linked to the S-locus of the European pear is higher than expected according with previous reports for apple and Japanese pear, since up to five genes were found to be linked to a single S-haplotype. Moreover, two of these genes exhibited an incomplete linkage to the S-RNase, allowing the identification of low-frequency recombinant haplotypes, generated by a crossing-over event between the two genes. These F-box genes are most likely placed in close proximity of the S-locus but do not belong to it, and they can thus be excluded from being responsible for the determination of pollen S function.