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Sedentary plant parasitic nematodes have evolved sophisticated strategies that allow them to transform host cells in the roots of host plants into feeding structures. These complex structures enable the nematodes to complete their life cycle inside a single host plant. Feeding structure initiation and maintenance are thought to be determined by the concerted action of effectors produced by the esophageal glands of the nematodes. However, the molecular mechanisms underlying the transformation of host cells into feeding structures and the role of the effectors in this process are poorly understood. For example, it is generally thought that virulent nematodes also use effectors to protect their complex feeding structures from plant innate immune responses. However, nematode effectors suppressing plant innate immunity have not been identified to date.
The host ranges of sedentary plant parasitic nematodes can vary from very wide (e.g. root-knot nematodes) to relatively narrow, limited to single plant families (e.g. cyst nematodes). Potato cyst nematodes (PCN) are able to parasitize only Solanaceous plants. Every year, they cause substantial yield losses in potato production areas. PCN are very difficult to control by the use of cultivation methods or the application of pesticides alone. The introduction of novel nematode resistant potato cultivars to the market is therefore of great importance for potato growers all over the world. Resistances, however, can be overcome by the emergence of virulent nematode populations. The aim of this thesis is to study incompatible interactions between the potato cyst nematode Globodera rostochiensis and potato (Solanum tuberosum), by analyzing resistance loci that make the plant immune to potato cyst nematodes as well as nematode effectors that suppress plant innate immunity.
This thesis begins with an extensive review of the literature on molecular and cellular aspects of plant resistance to sedentary endoparasitic nematodes, including pre-infectional, non-host and host resistance (Chapter 2). Most research in this field has focused on host resistance to nematodes, which is determined by single (e.g.H1 and Gpa2) or multiple (e.g. GpaVSsplandGpaXISspl) resistance gene loci. Two potato cyst nematode resistance loci were studied in this thesis (i.e. Grp1 in Chapter 3 and H1 in Chapter 4) and the results of these studies are summarized below.
Grp1 locus confers broad-spectrum quantitative resistance to the potato cyst nematode species Globodera pallida and G. rostochiensis in potato. It was previously mapped to a 3 cM interval on the short arm of potato chromosome V between the markers GP21 and GP179 in a hot spot for resistance (Rouppe Van Der Voort et al., 1998). The aim of the work described in chapter 3 was to fine map the Grp1 locus. First, a diploid mapping population RHAM026, comprising 1536 genotypes was screened with the flanking markers GP21 and GP179, resulting in the identification of 61 recombinants in this region. Next, thirteen new markers were developed using the genomic sequence information available from the same region of Solanum demissum. Together with markers available from the literature, these thirteen markers were used to screen a subset of 54 recombinants. Finally, these recombinants were tested for resistance to G. pallida Pa2 and G. rostochiensis Ro5. This mapping of both resistance specificities resulted in two nearly identical LOD graphs with the highest score just north of marker TG432. We conclude that the resistances to both G. pallida and G. rostochiensis map to the same 1.08 cM interval between the markers SPUD838 and TG432. Other studies have revealed that this locus in potato harbors several gene clusters encoding classical NB-ARC-LRR resistance proteins. This finding led us to the hypothesis that the Grp1 resistance depends on one, or perhaps several tightly linked major genes.
Near-absolute resistance to G. rostochiensis pathotypes 1 and 4 is conferred by the H1 resistance locus at the distal end of chromosome V of the diploid S. tuberosum ssp. andigena genotype SH83-92-488 (SH). The H1 resistance involves a hypersensitive response in the cells surrounding the nematode feeding structure, so that it becomes isolated from the vascular tissues in the host. A high-resolution map of H1 locus was generated previously using SHxRH mapping population (Bakker et al., 2004). In chapter 4, we used markers from thismap to screen a BAC library of SH. The BAC inserts identified with the markers were used to construct a physical map covering this region in the resistant haplotype. Further sequencing of the BAC inserts, included in the physical map, revealed a genomic fragment of 341 Kb harboring a large cluster of CC-NB-ARC-LRR genes. We compared this cluster of resistance gene homologs with the sequences of the corresponding regions in the two susceptible haplotypes from the diploid genotype RH89-039-16 (S. tuberosum ssp. tuberosum/ S. phureja), spanning 700 and 319 Kb respectively. The genomic regions in all three haplotypes harbor from 17 up to 23 resistance gene homologs interspersed with numerous transposable elements, genes coding for extensin-like proteins, and an amino acid transporter. Strikingly, the three haplotypes do not reveal gene order conservation and the overall sequence homology is only confined to the coding sequences of the resistance gene homologs. These findings suggest that extensive rearrangements have shaped the H1 locus. Sequence data and marker information gained from this study will benefit future efforts to clone the H1 nematode resistance gene.
At the start of the research described in this thesis no suppressor of plant immune response had been found in plant parasitic nematodes. In chapter 5, we report the first identification and functional characterization of a G. rostochiensis effector suppressing plant innate immune responses. The Nematode Suppressors of Immunity 1 (NSI-1) are specifically expressed in the dorsal esophageal gland of the nematodes, and their expression is upregulated in stages that feed on host cells. We identified many variants of NSI-1 in the Ro1-Mierenbos field population, and showed that this gene family is under diversifying selection. Knocking-down NSI-1 transcription by RNA interference strongly reduced the number of nematodes developing into full-grown cysts. Overexpression of four NSI-1 variants in susceptible potato plants resulted in enhanced susceptibility to nematodes. Moreover, overexpression of three other variants enhanced the susceptibility of potato plants to the fungus Verticillium dahliae. Down-regulation of the potato homologs of the Arabidopsis thaliana transcription factors WRKY22 and WRKY53 in these plants indicated that NSI-1 target immune signaling in plants. In an agroinfiltration assay in leaves of Nicotiana benthamiana several NSI-1 variants suppressed the hypersensitive response caused by the co-expression of specific resistance proteins and matching pathogen effectors (i.e. RBP-1/Gpa2 and AvrBlb2/Rpi-blb2) and by autoactive mutants of the resistance protein Mi-1.2 and an H1 resistance gene homolog RGH10-H1. Interestingly, other NSI-1 effector variant suppressed the hypersensitivity response induced by an autoactive mutant of the immune signaling protein NRC1. These findings altogether lead to the conclusion that potato cyst nematodes secrete suppressors of plant immunity, most likely to protect their feeding structures.
In the final chapter of this thesis, we discuss our most important findings within the broader context of recent developments in the field of molecular plant-microbe interactions. First, we argue that quantitative nematode resistance Grp1 is encoded by one or more NB-ARC-LRR genes located in one of the resistance gene clusters mapped to the GP21-GP179 interval on potato chromosome V. We further examine obstacles and offer possible solutions with regard to future cloning of the H1 nematode resistance gene. Lastly, we elaborate on possible functions, activities, and evolution of NSI-1 effectors as suppressors of plant innate immunity.
|Qualification||Doctor of Philosophy|
|Award date||6 Sep 2011|
|Place of Publication||[S.l.]|
|Publication status||Published - 2011|
- solanum tuberosum
- pest resistance
- quantitative trait loci
- plant parasitic nematodes
- globodera rostochiensis
- globodera pallida
- genetic mapping