Molecular contest between potato and the potato cyst nematode Globodera pallida: modulation of Gpa2-mediated resistance

K.B. Koropacka

Research output: Thesisinternal PhD, WU

Abstract

Gpa2 recognition specificity
Among all the multicellular animals, nematodes are the most numerous. In soil, a high variety
of free living nematodes feeding on bacteria can be found as well as species that parasitize
insects, animals or plants. The potato cyst nematode (PCN) Globodera pallida is an important
pest of cultivated potato. Upon infection of the roots, the nematode induces a feeding cell
complex or so-called syncytium, on which the immobilized nematode fully depends for its
development and reproduction. Due to the sophisticated feeding manner and ability to survive
for a long time in the absence of a host plant, the best way to control these soil-born
pathogens is the exploitation host resistance. Natural resistance to nematodes is based on
single dominant resistance genes (R) or quantitative trait loci (QTL). Several nematode
resistance genes have been identified and mapped. This includes the potato gene Gpa2 (Van
der Vossen et al., 2000) that confers resistance against the population D383 of G. pallida. The
Gpa2 gene is highly homologous to Rx1, which confers resistance against potato virus X
(Bendahmane et al., 1999). Both genes encode a protein with a nucleotide-binding leucinerich
repeat (NB-LRR) domains and a short coiled-coil domain at the N-terminus, which are in
88% identical at the amino acid level. The vast majority of the differences between Gpa2 and
Rx1 is found in the predicted solvent exposed regions of the LRR domain. In chapter 2, we
have shown that the LRR domain is essential for the recognition specificities of Gpa2 and
Rx1, whereas the CC-NBS domains can be exchanged without affecting the specificity. In
chapter 5, we have used a series of chimeric constructs in which segments of the Gpa2 LRR
were replaced by the corresponding segments from Rx1. These constructs allowed us to
narrow down the region required for nematode recognition to a stretch of residues between
808 and 912 amino acid residues in Gpa2, including 10 amino acids that differ between Gpa2
and Rx1. Furthermore, a computer-aided 3D model of the LRR domain is presented in which
7 of the Gpa2 specific amino acid residues map in a cluster onto the concave surface of the
horseshoe-like structure of the LRR domain.
Gpa2-mediated nematode resistance
The research described in chapter 3 aimed to understand the mechanisms underlying Gpa2-
mediated resistance to the potato cyst nematode G. pallida. The extreme resistance response
conferred by the close homologue Rx1 results in the blocking of the potato virus X (PVX) at
the infection sites and hence, the prevention of systemic spreading throughout the plant.
Surprisingly, an entirely different defense mechanism was observed for resistant potato plants
infected with juveniles of the avirulent Globodera pallida population D383. In susceptible
plants, both the virulent population Rookmaker and the avirulent population D383 formed
normal developing syncytia and nematodes were able to complete their life cycle as described
in previous studies. Infection of resistant plants with the avirulent population showed no
differences between susceptible and resistant potato plants in the early stages of G.pallida
parasitism (root entering, migration, syncytium initiation). Syncytium induction took place in
parenchyma cells, but rarely in other tissues. In samples collected 7 days later, however, the
first necrotic cells in the surrounding of the syncytium were noticed including symptoms of
degradation in the ultra structure of the syncytium itself in case of resistant plants infected
with avirulent nematodes. Samples collected 10 days post infection had already a layer of
necrotic cells, which separates the syncytium from the vascular bundle. At 14 days post
infection, it was observed that the parenchyma cells not incorporated directly in the syncytia
started to divide fast. Groups of hyperplastic cells surrounding the degrading syncytium
resulted in pushing it away to the outer part of the root. This unique phenomenon, which was
not observed before, can be part of the Gpa2-mediated defense response or a secondary
reaction to the presence of necrotic, dead cells and a way to exclude them from the healthy
conductive tissue of the root.
Transcriptional regulation of the Gpa2 promoter
To look in more details into the transcriptional regulation and expression of Gpa2, the native
promoter was fused to the reporter gene GUS and this construct was introduced into
susceptible potato. In chapter 3, the activity of the Gpa2 promoter was observed and shown to
be restricted to the vascular system and the root tips in uninfected plants. Roots were
challenged with G.pallida and the localization of the GUS expression was observed at the
infection sites at different parasitic stages. During infection with virulent nematodes - but not
the avirulent ones - this activity seems to be down regulated in vicinity of the syncytium.
Such a local inhibition of Gpa2 promoter activity is in line with observations made on
resistant roots when necrotic cells were only present around the feeding cell complex,
distantly from the feeding nematode.
The effector protein RBP-1 elicits a Gpa2 dependent HR
Recently, a RBP-1protein with strong similarity to the SPRY domain of the Ran-binding
protein RanBPM in juveniles of G. pallida was identified as a putative Gpa2 elicitor.
Transient expression of RBP-1 in N. benthamiana leaves elicits a Gpa2-dependent cell death
typical for the R-gene associated hypersensitive response (HR). Total RNA isolated from two
populations of G.pallida, D383 (avr to Gpa2) and Rookmaker (vir to Gpa2) was converted
into cDNA and screened for the presence of RBP-1s. This screening allowed the identification
of in total 10 classes of closely related homologs of RBP-1. All identified classes were tested
for their ability to elicit the Gpa2-dependent HR in an agroinfiltration assay. The capacity to
induce an Gpa2-dependent HR was shown to correlate with a single amino acid substitution in
RBP-1. No response was observed for two classes, which were obtained from the virulent
population (RBP-1ROOK2, RBP-1ROOK4). For the other homologous RBP-1 classes – both
deriving from the virulent and avirulent population - the response was ranging from a mild to
a strong and fast HR. Both in-active RBP-1 variants have a serine substitution at position 166
(S166P) within the SPRY domain. When this residue was projected on a computer aided 3D
model of RBP, we noticed that this amino acid is in a loop extending from the protein core.
Replacing the proline into a serine is predicted to change the shape of the loop and hence, to
affect the potential surface for protein-protein interactions.
Non-eliciting RBP-1 variants suppress RBP-induced Gpa2 activation
It was shown that the non-eliciting variants (RBP-1ROOK2 and RBP-1ROOK4) can suppress the
activation of a Gpa2-mediated HR by the eliciting RBP-1 variants. This effect was specific
for the Gpa2-mediated HR, and not observed with a Rx1-induced HR. As autoactive mutants
of Gpa2 and Rx1-mediated cell death are not blocked by the inactive variants of RBP-1, the
mechanism of suppression or inhibition likely operates on a functional Gpa2 protein, instead
of downstream Gpa2-activated signaling pathways. Further research is required to resolve the
mechanism underlying the possible competitive interactions of the active and the inactive
RBP-1 variants on the Gpa2-mediated HR. Essentially, two possible models that could
explain this phenomenon. First, the inactive variants could physically out compete the active
RBP-1s. The binding target of active and inactive variants of RBP-1 variants could be directly
in the Gpa2 protein or in the virulence target monitored by Gpa2. Alternatively, the inactive
variants of RBP-1 may intercept active RBP-1 variants by forming an inactive heterodimer
complex rendering it essentially undetectable for the Gpa2 protein.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Bakker, Jaap, Promotor
  • Goverse, Aska, Co-promotor
  • Smant, Geert, Co-promotor
Award date5 Feb 2010
Place of Publication[S.l.
Publisher
Print ISBNs9789085856061
Publication statusPublished - 2010

Fingerprint

Globodera pallida
cyst nematodes
giant cells
hypersensitive response
Nematoda
potatoes
infection
cells
amino acids
promoter regions
Potato virus X
genes
proteins
serine
cell death
agroinfiltration
free-living nematodes
plant vascular system
surface proteins
vascular bundles

Keywords

  • solanum tuberosum
  • globodera pallida
  • disease resistance
  • induced resistance
  • genes
  • genetic analysis
  • quantitative trait loci
  • genetic mapping

Cite this

@phdthesis{e7b0a9959d6545458473e5e3b9a8aa26,
title = "Molecular contest between potato and the potato cyst nematode Globodera pallida: modulation of Gpa2-mediated resistance",
abstract = "Gpa2 recognition specificity Among all the multicellular animals, nematodes are the most numerous. In soil, a high variety of free living nematodes feeding on bacteria can be found as well as species that parasitize insects, animals or plants. The potato cyst nematode (PCN) Globodera pallida is an important pest of cultivated potato. Upon infection of the roots, the nematode induces a feeding cell complex or so-called syncytium, on which the immobilized nematode fully depends for its development and reproduction. Due to the sophisticated feeding manner and ability to survive for a long time in the absence of a host plant, the best way to control these soil-born pathogens is the exploitation host resistance. Natural resistance to nematodes is based on single dominant resistance genes (R) or quantitative trait loci (QTL). Several nematode resistance genes have been identified and mapped. This includes the potato gene Gpa2 (Van der Vossen et al., 2000) that confers resistance against the population D383 of G. pallida. The Gpa2 gene is highly homologous to Rx1, which confers resistance against potato virus X (Bendahmane et al., 1999). Both genes encode a protein with a nucleotide-binding leucinerich repeat (NB-LRR) domains and a short coiled-coil domain at the N-terminus, which are in 88{\%} identical at the amino acid level. The vast majority of the differences between Gpa2 and Rx1 is found in the predicted solvent exposed regions of the LRR domain. In chapter 2, we have shown that the LRR domain is essential for the recognition specificities of Gpa2 and Rx1, whereas the CC-NBS domains can be exchanged without affecting the specificity. In chapter 5, we have used a series of chimeric constructs in which segments of the Gpa2 LRR were replaced by the corresponding segments from Rx1. These constructs allowed us to narrow down the region required for nematode recognition to a stretch of residues between 808 and 912 amino acid residues in Gpa2, including 10 amino acids that differ between Gpa2 and Rx1. Furthermore, a computer-aided 3D model of the LRR domain is presented in which 7 of the Gpa2 specific amino acid residues map in a cluster onto the concave surface of the horseshoe-like structure of the LRR domain. Gpa2-mediated nematode resistance The research described in chapter 3 aimed to understand the mechanisms underlying Gpa2- mediated resistance to the potato cyst nematode G. pallida. The extreme resistance response conferred by the close homologue Rx1 results in the blocking of the potato virus X (PVX) at the infection sites and hence, the prevention of systemic spreading throughout the plant. Surprisingly, an entirely different defense mechanism was observed for resistant potato plants infected with juveniles of the avirulent Globodera pallida population D383. In susceptible plants, both the virulent population Rookmaker and the avirulent population D383 formed normal developing syncytia and nematodes were able to complete their life cycle as described in previous studies. Infection of resistant plants with the avirulent population showed no differences between susceptible and resistant potato plants in the early stages of G.pallida parasitism (root entering, migration, syncytium initiation). Syncytium induction took place in parenchyma cells, but rarely in other tissues. In samples collected 7 days later, however, the first necrotic cells in the surrounding of the syncytium were noticed including symptoms of degradation in the ultra structure of the syncytium itself in case of resistant plants infected with avirulent nematodes. Samples collected 10 days post infection had already a layer of necrotic cells, which separates the syncytium from the vascular bundle. At 14 days post infection, it was observed that the parenchyma cells not incorporated directly in the syncytia started to divide fast. Groups of hyperplastic cells surrounding the degrading syncytium resulted in pushing it away to the outer part of the root. This unique phenomenon, which was not observed before, can be part of the Gpa2-mediated defense response or a secondary reaction to the presence of necrotic, dead cells and a way to exclude them from the healthy conductive tissue of the root. Transcriptional regulation of the Gpa2 promoter To look in more details into the transcriptional regulation and expression of Gpa2, the native promoter was fused to the reporter gene GUS and this construct was introduced into susceptible potato. In chapter 3, the activity of the Gpa2 promoter was observed and shown to be restricted to the vascular system and the root tips in uninfected plants. Roots were challenged with G.pallida and the localization of the GUS expression was observed at the infection sites at different parasitic stages. During infection with virulent nematodes - but not the avirulent ones - this activity seems to be down regulated in vicinity of the syncytium. Such a local inhibition of Gpa2 promoter activity is in line with observations made on resistant roots when necrotic cells were only present around the feeding cell complex, distantly from the feeding nematode. The effector protein RBP-1 elicits a Gpa2 dependent HR Recently, a RBP-1protein with strong similarity to the SPRY domain of the Ran-binding protein RanBPM in juveniles of G. pallida was identified as a putative Gpa2 elicitor. Transient expression of RBP-1 in N. benthamiana leaves elicits a Gpa2-dependent cell death typical for the R-gene associated hypersensitive response (HR). Total RNA isolated from two populations of G.pallida, D383 (avr to Gpa2) and Rookmaker (vir to Gpa2) was converted into cDNA and screened for the presence of RBP-1s. This screening allowed the identification of in total 10 classes of closely related homologs of RBP-1. All identified classes were tested for their ability to elicit the Gpa2-dependent HR in an agroinfiltration assay. The capacity to induce an Gpa2-dependent HR was shown to correlate with a single amino acid substitution in RBP-1. No response was observed for two classes, which were obtained from the virulent population (RBP-1ROOK2, RBP-1ROOK4). For the other homologous RBP-1 classes – both deriving from the virulent and avirulent population - the response was ranging from a mild to a strong and fast HR. Both in-active RBP-1 variants have a serine substitution at position 166 (S166P) within the SPRY domain. When this residue was projected on a computer aided 3D model of RBP, we noticed that this amino acid is in a loop extending from the protein core. Replacing the proline into a serine is predicted to change the shape of the loop and hence, to affect the potential surface for protein-protein interactions. Non-eliciting RBP-1 variants suppress RBP-induced Gpa2 activation It was shown that the non-eliciting variants (RBP-1ROOK2 and RBP-1ROOK4) can suppress the activation of a Gpa2-mediated HR by the eliciting RBP-1 variants. This effect was specific for the Gpa2-mediated HR, and not observed with a Rx1-induced HR. As autoactive mutants of Gpa2 and Rx1-mediated cell death are not blocked by the inactive variants of RBP-1, the mechanism of suppression or inhibition likely operates on a functional Gpa2 protein, instead of downstream Gpa2-activated signaling pathways. Further research is required to resolve the mechanism underlying the possible competitive interactions of the active and the inactive RBP-1 variants on the Gpa2-mediated HR. Essentially, two possible models that could explain this phenomenon. First, the inactive variants could physically out compete the active RBP-1s. The binding target of active and inactive variants of RBP-1 variants could be directly in the Gpa2 protein or in the virulence target monitored by Gpa2. Alternatively, the inactive variants of RBP-1 may intercept active RBP-1 variants by forming an inactive heterodimer complex rendering it essentially undetectable for the Gpa2 protein.",
keywords = "solanum tuberosum, globodera pallida, ziekteresistentie, ge{\"i}nduceerde resistentie, genen, genetische analyse, loci voor kwantitatief kenmerk, genetische kartering, solanum tuberosum, globodera pallida, disease resistance, induced resistance, genes, genetic analysis, quantitative trait loci, genetic mapping",
author = "K.B. Koropacka",
note = "WU thesis 4775",
year = "2010",
language = "English",
isbn = "9789085856061",
publisher = "S.n.",
school = "Wageningen University",

}

Molecular contest between potato and the potato cyst nematode Globodera pallida: modulation of Gpa2-mediated resistance. / Koropacka, K.B.

[S.l. : S.n., 2010. 131 p.

Research output: Thesisinternal PhD, WU

TY - THES

T1 - Molecular contest between potato and the potato cyst nematode Globodera pallida: modulation of Gpa2-mediated resistance

AU - Koropacka, K.B.

N1 - WU thesis 4775

PY - 2010

Y1 - 2010

N2 - Gpa2 recognition specificity Among all the multicellular animals, nematodes are the most numerous. In soil, a high variety of free living nematodes feeding on bacteria can be found as well as species that parasitize insects, animals or plants. The potato cyst nematode (PCN) Globodera pallida is an important pest of cultivated potato. Upon infection of the roots, the nematode induces a feeding cell complex or so-called syncytium, on which the immobilized nematode fully depends for its development and reproduction. Due to the sophisticated feeding manner and ability to survive for a long time in the absence of a host plant, the best way to control these soil-born pathogens is the exploitation host resistance. Natural resistance to nematodes is based on single dominant resistance genes (R) or quantitative trait loci (QTL). Several nematode resistance genes have been identified and mapped. This includes the potato gene Gpa2 (Van der Vossen et al., 2000) that confers resistance against the population D383 of G. pallida. The Gpa2 gene is highly homologous to Rx1, which confers resistance against potato virus X (Bendahmane et al., 1999). Both genes encode a protein with a nucleotide-binding leucinerich repeat (NB-LRR) domains and a short coiled-coil domain at the N-terminus, which are in 88% identical at the amino acid level. The vast majority of the differences between Gpa2 and Rx1 is found in the predicted solvent exposed regions of the LRR domain. In chapter 2, we have shown that the LRR domain is essential for the recognition specificities of Gpa2 and Rx1, whereas the CC-NBS domains can be exchanged without affecting the specificity. In chapter 5, we have used a series of chimeric constructs in which segments of the Gpa2 LRR were replaced by the corresponding segments from Rx1. These constructs allowed us to narrow down the region required for nematode recognition to a stretch of residues between 808 and 912 amino acid residues in Gpa2, including 10 amino acids that differ between Gpa2 and Rx1. Furthermore, a computer-aided 3D model of the LRR domain is presented in which 7 of the Gpa2 specific amino acid residues map in a cluster onto the concave surface of the horseshoe-like structure of the LRR domain. Gpa2-mediated nematode resistance The research described in chapter 3 aimed to understand the mechanisms underlying Gpa2- mediated resistance to the potato cyst nematode G. pallida. The extreme resistance response conferred by the close homologue Rx1 results in the blocking of the potato virus X (PVX) at the infection sites and hence, the prevention of systemic spreading throughout the plant. Surprisingly, an entirely different defense mechanism was observed for resistant potato plants infected with juveniles of the avirulent Globodera pallida population D383. In susceptible plants, both the virulent population Rookmaker and the avirulent population D383 formed normal developing syncytia and nematodes were able to complete their life cycle as described in previous studies. Infection of resistant plants with the avirulent population showed no differences between susceptible and resistant potato plants in the early stages of G.pallida parasitism (root entering, migration, syncytium initiation). Syncytium induction took place in parenchyma cells, but rarely in other tissues. In samples collected 7 days later, however, the first necrotic cells in the surrounding of the syncytium were noticed including symptoms of degradation in the ultra structure of the syncytium itself in case of resistant plants infected with avirulent nematodes. Samples collected 10 days post infection had already a layer of necrotic cells, which separates the syncytium from the vascular bundle. At 14 days post infection, it was observed that the parenchyma cells not incorporated directly in the syncytia started to divide fast. Groups of hyperplastic cells surrounding the degrading syncytium resulted in pushing it away to the outer part of the root. This unique phenomenon, which was not observed before, can be part of the Gpa2-mediated defense response or a secondary reaction to the presence of necrotic, dead cells and a way to exclude them from the healthy conductive tissue of the root. Transcriptional regulation of the Gpa2 promoter To look in more details into the transcriptional regulation and expression of Gpa2, the native promoter was fused to the reporter gene GUS and this construct was introduced into susceptible potato. In chapter 3, the activity of the Gpa2 promoter was observed and shown to be restricted to the vascular system and the root tips in uninfected plants. Roots were challenged with G.pallida and the localization of the GUS expression was observed at the infection sites at different parasitic stages. During infection with virulent nematodes - but not the avirulent ones - this activity seems to be down regulated in vicinity of the syncytium. Such a local inhibition of Gpa2 promoter activity is in line with observations made on resistant roots when necrotic cells were only present around the feeding cell complex, distantly from the feeding nematode. The effector protein RBP-1 elicits a Gpa2 dependent HR Recently, a RBP-1protein with strong similarity to the SPRY domain of the Ran-binding protein RanBPM in juveniles of G. pallida was identified as a putative Gpa2 elicitor. Transient expression of RBP-1 in N. benthamiana leaves elicits a Gpa2-dependent cell death typical for the R-gene associated hypersensitive response (HR). Total RNA isolated from two populations of G.pallida, D383 (avr to Gpa2) and Rookmaker (vir to Gpa2) was converted into cDNA and screened for the presence of RBP-1s. This screening allowed the identification of in total 10 classes of closely related homologs of RBP-1. All identified classes were tested for their ability to elicit the Gpa2-dependent HR in an agroinfiltration assay. The capacity to induce an Gpa2-dependent HR was shown to correlate with a single amino acid substitution in RBP-1. No response was observed for two classes, which were obtained from the virulent population (RBP-1ROOK2, RBP-1ROOK4). For the other homologous RBP-1 classes – both deriving from the virulent and avirulent population - the response was ranging from a mild to a strong and fast HR. Both in-active RBP-1 variants have a serine substitution at position 166 (S166P) within the SPRY domain. When this residue was projected on a computer aided 3D model of RBP, we noticed that this amino acid is in a loop extending from the protein core. Replacing the proline into a serine is predicted to change the shape of the loop and hence, to affect the potential surface for protein-protein interactions. Non-eliciting RBP-1 variants suppress RBP-induced Gpa2 activation It was shown that the non-eliciting variants (RBP-1ROOK2 and RBP-1ROOK4) can suppress the activation of a Gpa2-mediated HR by the eliciting RBP-1 variants. This effect was specific for the Gpa2-mediated HR, and not observed with a Rx1-induced HR. As autoactive mutants of Gpa2 and Rx1-mediated cell death are not blocked by the inactive variants of RBP-1, the mechanism of suppression or inhibition likely operates on a functional Gpa2 protein, instead of downstream Gpa2-activated signaling pathways. Further research is required to resolve the mechanism underlying the possible competitive interactions of the active and the inactive RBP-1 variants on the Gpa2-mediated HR. Essentially, two possible models that could explain this phenomenon. First, the inactive variants could physically out compete the active RBP-1s. The binding target of active and inactive variants of RBP-1 variants could be directly in the Gpa2 protein or in the virulence target monitored by Gpa2. Alternatively, the inactive variants of RBP-1 may intercept active RBP-1 variants by forming an inactive heterodimer complex rendering it essentially undetectable for the Gpa2 protein.

AB - Gpa2 recognition specificity Among all the multicellular animals, nematodes are the most numerous. In soil, a high variety of free living nematodes feeding on bacteria can be found as well as species that parasitize insects, animals or plants. The potato cyst nematode (PCN) Globodera pallida is an important pest of cultivated potato. Upon infection of the roots, the nematode induces a feeding cell complex or so-called syncytium, on which the immobilized nematode fully depends for its development and reproduction. Due to the sophisticated feeding manner and ability to survive for a long time in the absence of a host plant, the best way to control these soil-born pathogens is the exploitation host resistance. Natural resistance to nematodes is based on single dominant resistance genes (R) or quantitative trait loci (QTL). Several nematode resistance genes have been identified and mapped. This includes the potato gene Gpa2 (Van der Vossen et al., 2000) that confers resistance against the population D383 of G. pallida. The Gpa2 gene is highly homologous to Rx1, which confers resistance against potato virus X (Bendahmane et al., 1999). Both genes encode a protein with a nucleotide-binding leucinerich repeat (NB-LRR) domains and a short coiled-coil domain at the N-terminus, which are in 88% identical at the amino acid level. The vast majority of the differences between Gpa2 and Rx1 is found in the predicted solvent exposed regions of the LRR domain. In chapter 2, we have shown that the LRR domain is essential for the recognition specificities of Gpa2 and Rx1, whereas the CC-NBS domains can be exchanged without affecting the specificity. In chapter 5, we have used a series of chimeric constructs in which segments of the Gpa2 LRR were replaced by the corresponding segments from Rx1. These constructs allowed us to narrow down the region required for nematode recognition to a stretch of residues between 808 and 912 amino acid residues in Gpa2, including 10 amino acids that differ between Gpa2 and Rx1. Furthermore, a computer-aided 3D model of the LRR domain is presented in which 7 of the Gpa2 specific amino acid residues map in a cluster onto the concave surface of the horseshoe-like structure of the LRR domain. Gpa2-mediated nematode resistance The research described in chapter 3 aimed to understand the mechanisms underlying Gpa2- mediated resistance to the potato cyst nematode G. pallida. The extreme resistance response conferred by the close homologue Rx1 results in the blocking of the potato virus X (PVX) at the infection sites and hence, the prevention of systemic spreading throughout the plant. Surprisingly, an entirely different defense mechanism was observed for resistant potato plants infected with juveniles of the avirulent Globodera pallida population D383. In susceptible plants, both the virulent population Rookmaker and the avirulent population D383 formed normal developing syncytia and nematodes were able to complete their life cycle as described in previous studies. Infection of resistant plants with the avirulent population showed no differences between susceptible and resistant potato plants in the early stages of G.pallida parasitism (root entering, migration, syncytium initiation). Syncytium induction took place in parenchyma cells, but rarely in other tissues. In samples collected 7 days later, however, the first necrotic cells in the surrounding of the syncytium were noticed including symptoms of degradation in the ultra structure of the syncytium itself in case of resistant plants infected with avirulent nematodes. Samples collected 10 days post infection had already a layer of necrotic cells, which separates the syncytium from the vascular bundle. At 14 days post infection, it was observed that the parenchyma cells not incorporated directly in the syncytia started to divide fast. Groups of hyperplastic cells surrounding the degrading syncytium resulted in pushing it away to the outer part of the root. This unique phenomenon, which was not observed before, can be part of the Gpa2-mediated defense response or a secondary reaction to the presence of necrotic, dead cells and a way to exclude them from the healthy conductive tissue of the root. Transcriptional regulation of the Gpa2 promoter To look in more details into the transcriptional regulation and expression of Gpa2, the native promoter was fused to the reporter gene GUS and this construct was introduced into susceptible potato. In chapter 3, the activity of the Gpa2 promoter was observed and shown to be restricted to the vascular system and the root tips in uninfected plants. Roots were challenged with G.pallida and the localization of the GUS expression was observed at the infection sites at different parasitic stages. During infection with virulent nematodes - but not the avirulent ones - this activity seems to be down regulated in vicinity of the syncytium. Such a local inhibition of Gpa2 promoter activity is in line with observations made on resistant roots when necrotic cells were only present around the feeding cell complex, distantly from the feeding nematode. The effector protein RBP-1 elicits a Gpa2 dependent HR Recently, a RBP-1protein with strong similarity to the SPRY domain of the Ran-binding protein RanBPM in juveniles of G. pallida was identified as a putative Gpa2 elicitor. Transient expression of RBP-1 in N. benthamiana leaves elicits a Gpa2-dependent cell death typical for the R-gene associated hypersensitive response (HR). Total RNA isolated from two populations of G.pallida, D383 (avr to Gpa2) and Rookmaker (vir to Gpa2) was converted into cDNA and screened for the presence of RBP-1s. This screening allowed the identification of in total 10 classes of closely related homologs of RBP-1. All identified classes were tested for their ability to elicit the Gpa2-dependent HR in an agroinfiltration assay. The capacity to induce an Gpa2-dependent HR was shown to correlate with a single amino acid substitution in RBP-1. No response was observed for two classes, which were obtained from the virulent population (RBP-1ROOK2, RBP-1ROOK4). For the other homologous RBP-1 classes – both deriving from the virulent and avirulent population - the response was ranging from a mild to a strong and fast HR. Both in-active RBP-1 variants have a serine substitution at position 166 (S166P) within the SPRY domain. When this residue was projected on a computer aided 3D model of RBP, we noticed that this amino acid is in a loop extending from the protein core. Replacing the proline into a serine is predicted to change the shape of the loop and hence, to affect the potential surface for protein-protein interactions. Non-eliciting RBP-1 variants suppress RBP-induced Gpa2 activation It was shown that the non-eliciting variants (RBP-1ROOK2 and RBP-1ROOK4) can suppress the activation of a Gpa2-mediated HR by the eliciting RBP-1 variants. This effect was specific for the Gpa2-mediated HR, and not observed with a Rx1-induced HR. As autoactive mutants of Gpa2 and Rx1-mediated cell death are not blocked by the inactive variants of RBP-1, the mechanism of suppression or inhibition likely operates on a functional Gpa2 protein, instead of downstream Gpa2-activated signaling pathways. Further research is required to resolve the mechanism underlying the possible competitive interactions of the active and the inactive RBP-1 variants on the Gpa2-mediated HR. Essentially, two possible models that could explain this phenomenon. First, the inactive variants could physically out compete the active RBP-1s. The binding target of active and inactive variants of RBP-1 variants could be directly in the Gpa2 protein or in the virulence target monitored by Gpa2. Alternatively, the inactive variants of RBP-1 may intercept active RBP-1 variants by forming an inactive heterodimer complex rendering it essentially undetectable for the Gpa2 protein.

KW - solanum tuberosum

KW - globodera pallida

KW - ziekteresistentie

KW - geïnduceerde resistentie

KW - genen

KW - genetische analyse

KW - loci voor kwantitatief kenmerk

KW - genetische kartering

KW - solanum tuberosum

KW - globodera pallida

KW - disease resistance

KW - induced resistance

KW - genes

KW - genetic analysis

KW - quantitative trait loci

KW - genetic mapping

M3 - internal PhD, WU

SN - 9789085856061

PB - S.n.

CY - [S.l.

ER -