Identification of plant genes regulated in resistant potato Solanum sparsipilum during the early stages of infection by Globodera pallida

Using a complementary (c)DNA-amplified fragment length polymorphism (AFLP) approach, we investigated differential gene expression linked to resistance mechanisms during the incompatible potato – Globodera pallida interaction. Expression was compared between a resistant and a susceptible potato clone, inoculated or not inoculated with G. pallida. These clones were issued from a cross between the resistant Solanum sparsipilum spl329.18 accession and the susceptible dihaploid S. tuberosum Caspar H3, and carried, respectively, resistant and susceptible alleles at the resistance quantitative trait loci (QTLs). Analysis was done on root fragments picked up at 4 time points, during a period of 6 days after infection, from penetration of the nematode in the root to degradation of the feeding site in resistant plants. A total of 2560 transcript-derived fragments (TDFs) were analyzed, resulting in the detection of 46 TDFs that were up- or downregulated. The number of TDFs that were up- or downregulated increased with time after inoculation. The majority of TDFs were upregulated at only 1 or 2 time points in response to infection. After isolation and sequencing of the TDFs of interest, a subset of 36 sequences were identified, among which 22 matched plant sequences and 2 matched nematode sequences. Some of the TDFs that matched plant genes showed clear homologies to genes involved in cell-cycle regulation, transcription regulation, resistance downstream signalling pathways, and defense mechanisms. Other sequences with homologies to plant genes of unknown function or without any significant similarity to known proteins were also found. Although not exhaustive, these results represent the most extensive list of genes with altered RNA levels after the incompatible G. pallida–potato interaction that has been published to date. The function of these genes could provide insight into resistance or plant defense mechanisms during incompatible potato-cyst nematode interactions.

Resistance Quantitative Trait Loci Originating from Solanum sparsipilum Act Independently on the Sex Ratio of Globodera pallida and Together for Developing a Necrotic Reaction

Plant resistance to nematodes is related to the ability of the host to reduce the development of nematode juveniles into females. Resistance to the potato cyst nematode (PCN) Globodera pallida, originating from the wild species Solanum sparsipilum, was dissected by a quantitative trait loci (QTL) approach. Two QTL explained 89% of the phenotypic variation. The QTL GpaVsspl on chromosome V displayedthe major effect on the cyst number (coefficient of determination [R2] = 76.6%). It restricted G. pallida development to 16.2% of juveniles, 81.5% of males, and 2.3% of females. The QTL GpaXIsspl chromosome XI displayed a lower effect on the cyst number (R2 = 12.7%). It restricted G. pallida development to 13.8% of juveniles, 35.4% of males, and 50.8% of females. Clones carrying both QTL restricted the nematode development to 58.1% juveniles, 41.1% of males, and 0.8% of females. We demonstrated that potato clones carrying both QTL showed a strong necrotic reaction in roots infected by nematodes, while no such reaction was observed in clones carrying a single QTL. This result underlines the importance to introgress together GpaVsspl and GpaXIsspl into potato cultivars, in order to reduce the density of this quarantine pest in soil and to decrease the risk of selecting overcoming G. pallida subpopulations.

Genetics and phenotypic characterisation of the hypersensitive resistance of Solanum sparsipilum to Meloidogyne incognita

The hypersensitive resistance of the genotype '385.484.13' of Solanum sparsipilum to Meloidogyne incognita, one of the southern root-knot nematodes and their Mi virulent populations was analysed. Genetic control of the hypersensitive reaction was assessed based on segregation of the necrotic reaction in infected roots of diploid F1 plants obtained from the cross of the genotype '385.484.13' with the dihaploid susceptible potato genotype 'Keltia H12'. Two distinct tests showed a distorted segregation compared to the hypothetical ratio expected for a monogenic control. We hypothesised that the resistance is based on one dominant gene designated Mh and linked to the self incompatibility locus-S. Hypersensitive plants prevented juveniles from feeding, developing and reproducing. Ninety percent of invading juveniles remained undeveloped 6 weeks after inoculation. Almost all the adults were males and only very rarely were females observed. Inhibition of the development of juveniles into females showed a bimodal distribution of the genotypes: hypersensitive ones allowed almost no development of females whilst all the juveniles became females in non-hypersensitive plants. That result supports the hypothesis of a monogenic control of the resistance. The resistance was broken when plants were grown at 30°C. The histology of the hypersensitive reaction was very similar to that of the Mi gene of tomato and of some Me genes of pepper. Necrotic cells were localised not only around the head of the juveniles but also surrounding their bodies. In addition, healthy cells adjacent to necrotised ones underwent divisions parallel to the juveniles. Intercellular spaces between these cells and necrotised ones enlarged markedly, which is consistent with a process leading to isolation of the necrotic area from healthy tissue.

Resistance to the root-knot nematode Meloidogyne fallax in Solanum sparsipilum: analysis of the mechanisms

The genotype 88S.329.15 of Solanum sparsipilum was studied in order to analyse the genetic basis and the mechanisms of its resistance to Meloidogyne fallax. In infected plants grown at 20°C, juveniles invaded the root system with a clear delay and a lower infection rate in comparison to the susceptible S. tuberosum genotype BF15 H1. No defence reaction occurred during root invasion and migration toward the vascular cylinder. The juveniles induced development of feeding sites usually composed of several giant cells, which contained condensed cytoplasm, only small vacuoles, enlarged nuclei with pronounced nucleoli and almost no endoplasmic reticulum. Abundant necrosis of surrounding parenchymatous vascular cylinder cells lead to the degeneration of the giant cells. More than 90% of the invading juveniles failed to develop. The others developed as males. The resistance inheritance was analysed on 128 F1 hybrids obtained using the susceptible line BF15 H1 as the female parent and 88S.329.15 as the male parent. Among the progenies, 68 genotypes produced a necrotic reaction to nematode infection and 60 produced no necrosis. This 1 : 1 segregation pattern suggests a monogenic control of this defence reaction. Unlike the resistant parent 88S.329.15, some M. fallax females developed in the roots of necrotically responding hybrids. There was a normal distribution of mean numbers of adult females found in the roots of these genotypes. This result suggests that the ability of the resistant genotype 88S.329.15 to suppress development of females is quantitatively inherited and likely to be controlled by more than one locus. These data indicate that the mechanism of resistance is different from the resistance to Meloidogyne incognita conferred by the Mi gene of tomato.