Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broad-spectrum nematode resistance Hero gene

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.     

Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.     

Mapping QTLs for resistance to the cyst nematode Globodera pallida derived from the wild potato species Solanum vernei

Resistance to the potato cyst nematode (PCN) species Globodera pallida, derived from the wild diploid potato species Solanum vernei, has been investigated. This source of resistance, which is effective against all of the major pathotypes of G. pallida and Globodera rostochiensis, has been assumed to be due to several genetic factors, but it has proved difficult to deploy effectively in breeding strategies for potato cultivars. Diploid and tetraploid potato populations segregating for 'vernei' resistance were analysed. At the tetraploid level, a bulk segregant analysis (BSA) approach was employed and detected AFLP markers linked to a resistance QTL on potato linkage group V. Conventional linkage analysis of a diploid population identified QTL on linkage groups V and IX. A marker linked to a QTL on linkage group V has been converted to a single-locus PCR-based marker, which can be used to detect the presence of the QTL in diploid and tetraploid potato germplasm. Moreover, there is evidence that one of the AFLPs detected by BSA appears to be specific to an introgressed segment of DNA from S. vernei. These results are compared with those obtained from other studies on resistance to the PCN species G. pallida.     

Comparison of British populations of potato cyst nematodes with populations from continental Europe and South America using RAPDs.

Genetic variation between populations of Globodera pallida, primarily from Britain but including populations from continental Europe and South America and two Globodera rostochiensis populations, was examined using random amplified polymorphic DNA (RAPD). Fourteen primers were used and 250 amplification products observed. A comparison was made of the similarities between the species and, within G. pallida, between populations from Britain, The Netherlands, Germany, and Switzerland, of the pathotypes Pa2 and Pa3. In addition, one Pa1 population and two others from South America were included. On the basis of the RAPD analysis, all the Pa2-Pa3 populations, except one from Scotland (Luffness), constituted a single group with no clear distinction based on pathotype designation. The Luffness population is known to be distinct in its virulence. The data indicated that the main Pa2-Pa3 group could be subdivided based on geographic origin, but this is not well supported by bootstrap analysis. The Pa1 population and the two populations from South America all formed distinct groups.     

Genetic basis for the hierarchical interaction between Tobamovirus spp. and L resistance gene alleles from different pepper species

The pepper L gene conditions the plant's resistance to Tobamovirus spp. Alleles L(1), L(2), L(3), and L(4) confer a broadening spectra of resistance to different virus pathotypes. In this study, we report the genetic basis for the hierarchical interaction between L genes and Tobamovirus pathotypes. We cloned L(3) using map-based methods, and L(1), L(1a), L(1c), L(2), L(2b), and L(4) using a homology-based method. L gene alleles encode coiled-coil, nucleotide-binding, leucine-rich repeat (LRR)-type resistance proteins with the ability to induce resistance response to the viral coat protein (CP) avirulence effectors by themselves. Their different recognition spectra in original pepper species were reproduced in an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana. Chimera analysis with L(1), which showed the narrowest recognition spectrum, indicates that the broader recognition spectra conferred by L(2), L(2b), L(3), and L(4) require different subregions of the LRR domain. We identified a critical amino acid residue for the determination of recognition spectra but other regions also influenced the L genes' resistance spectra. The results suggest that the hierarchical interactions between L genes and Tobamovirus spp. are determined by the interaction of multiple subregions of the LRR domain of L proteins with different viral CP themselves or some protein complexes including them.     

Rapid identification of cyst (Heterodera spp., Globodera spp.) and root-knot (Meloidogyne spp.) nematodes on the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) in cultures and field samples

Cyst and root-knot nematodes show high levels of gross morphological similarity. This presents difficulties for the study of their ecology in natural ecosystems. In this study, cyst and root-knot nematode species, as well as some ectoparasitic nematode species, were identified using the second internal transcribed spacer (ITS2) sequence variation detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). The ITS2 region was sufficiently variable within the taxa investigated to allow species to be separated on the basis of minor sequence variation. The PCR primers used in this study were effective for 12 species with three genera within the Heteroderinae (Globodera pallida, G. rostochiensis, Heterodera arenaria/avenae, H. ciceri, H. daverti, H. hordecalis, H. mani, H. schachtii, H. trifolii, Meloidogyne ardenensis, M. duytsi and M. maritima). However, pathotypes of Globodera pallida and G. rostochiensis could not be distinguished. The method was tested at two coastal dune locations in The Netherlands (one in the lime-poor dunes of the north and one in calcareous dunes of the south) to determine the population structure of cyst nematodes. At each site, cyst nematodes were associated with three plant species: two plant species on the foredune (Elymus farctus and Ammophila arenaria) and one plant species occurring further inland (Calamagrostis epigejos). Two species of cyst nematodes, H. arenaria and H. hordecalis, were found. H. arenaria associated with vigorous A. arenaria and H. hordecalis in association with degenerating A. arenaria and C. epigejos. The field survey demonstrated that in coastal dunes abiotic factors may be the important affecting the distribution of cyst nematodes.     

Recombination between paralogues at the Rp1 rust resistance locus in maize

Rp1 is a complex rust resistance locus of maize. The HRp1-D haplotype is composed of Rp1-D and eight paralogues, seven of which also code for predicted nucleotide binding site-leucine rich repeat (NBS-LRR) proteins similar to the Rp1-D gene. The paralogues are polymorphic (DNA identities 91-97%), especially in the C-terminal LRR domain. The remaining family member encodes a truncated protein that has no LRR domain. Seven of the nine family members, including the truncated gene, are transcribed. Sequence comparisons between paralogues provide evidence for past recombination events between paralogues and diversifying selection, particularly in the C-terminal half of the LRR domain. Variants selected for complete or partial loss of Rp1-D resistance can be explained by unequal crossing over that occurred mostly within coding regions. The Rp1-D gene is altered or lost in all variants, the recombination breakpoints occur throughout the genes, and most recombinant events (9/14 examined) involved the same untranscribed paralogue with the Rp1-D gene. One recombinant with a complete LRR from Rp1-D, but the amino-terminal portion from another homologue, conferred the Rp1-D specificity but with a reduced level of resistance.     

Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and arabidopsis

The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.     

Multiplication of English isolates of potato pale cyst- nematode, Globodera pallida, on potatoes of differing nematode resistance and the identity of potato cyst- nematode populations

In pots, 25 populations of potato pale cyst-nematode, Globodera pallida Stone, differed significantly in their ability to multiply on potato clones P55/7 and ZC83/ 6, both fully resistant to G. pallida pathotype Pal. Neither clone was fully resistant to any of the populations. For 21 populations common to this and an earlier experiment, increase on the more resistant potatoes (P55/7, ZC83/6, cvs Sante, Paladin and Glenna) was correlated with their increase on less resistant potatoes (cvs Morag, Fingal and Valiant). Variation in virulence on these partially resistant potatoes was not matched by differences in the electrophoretic patterns of the nematodes' proteins. The identification of populations of G. rostochiensis (Woll.) Skarbilovich used in these experiments was confirmed by electrophoresis. All populations of G. pallida Stone, appeared to contain very small numbers of G. rostochiensis after subculture on susceptible potatoes (cv. Désirée).