Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 regulates a multi-protein immune complex at chromatin
Llewelyn, Alex ; Townsend, Philip D. ; Fenyk, Stepan ; Dixon, Christopher ; Palsson, Lars-Olof ; Gawehns, Fleur ; Takken, F. ; Slootweg, E.J. ; Sukarta, O.C.A. ; Goverse, A. ; Cann, Martin J. - \ 2019
Exploring the Role of RanGTPase Activating Protein 2 (RanGAP2) in Recognition by the Potato CC NB LRR Immune Receptors Rx1 and Gpa2
Sukarta, O.C.A. ; Slootweg, E.J. ; Diaz Granados Muñoz, A. ; Martin, Eliza C. ; Roosien, J. ; Bakker, J. ; Smant, G. ; Petrescu, A. ; Goverse, A. - \ 2019
Balanced nuclear and cytoplasmic distribution is required for activation of Gpa2 mediated cell death induced by the potato cyst nematode effector RBP 1
Zheng, Qi ; Slootweg, E.J. ; Sukarta, O.C.A. ; Schaik, C.C. van; Smant, G. ; Goverse, A. - \ 2019
Exploring the Role of RanGAP2 in Recognition by the Potato CC-NB-LRR Immune Receptors Rx1 and Gpa2
Sukarta, O.C.A. - \ 2019
The Ran GTPase activating protein 2 (RanGAP2) is a co-factor of the potato CC-NB-LRR immune receptor Rx1. However, its role in defence remains to be fully elucidated. Artificial tethering of RanGAP2 to the nematode effector GpRbp-1 was shown to enhance the cell death response of Gpa2, a closely related immune receptor of Rx1 that also interacts with RanGAP2 (Sacco et al. 2009). This suggests that RanGAP2 may contribute to immunity by facilitating effector recognition. Here, we expand on this model using a combination of structure-informed approaches, including co-immunoprecipitation and cellular imaging studies. We show that RanGAP2 can in fact form complexes in planta with the cognate effectors of Rx1 and Gpa2, namely the PVX coat protein (CP) and GpRbp-1. Interestingly, this was noted for both the eliciting and non-eliciting effector variants, suggesting that the RanGAP2/effector interaction may not be sufficient to confer recognition. This is in line with existing data demonstrating that the C-terminal region of the Rx1 and Gpa2 LRR domains is required for successful pathogen perception (Slootweg et al. 2013). Currently, we are performing structural and biochemical analysis to discern whether RanGAP2 acts as a bait that facilitates specific effector recognition either by bridging or enhancing the effector/cognate LRR interaction. This model may explain the bifurcation of pathogen recognition specificities of these two closely related immune receptors during evolution.
RanGAP1 and RanGAP2 are common virulence targets of two independently evolved effectors from the potato cyst nematode Globodera pallida and Potato Virus X
Sukarta, O.C.A. ; Diaz Granados Muñoz, A. ; Slootweg, E.J. ; Pokare, Somnath ; Grundler, F. ; Smant, G. ; Goverse, A. - \ 2019
Nucleocytoplastic distribution is required for activation of Gpa-2 resistance ageinst Globodera pallida
Zheng, Qi ; Slootweg, E.J. ; Sukarta, O.C.A. ; Schaik, C.C. van; Smant, G. ; Goverse, A. - \ 2019
Identification and functional characterisation of host interactions of the potato CC-NB-LRR Rx1
Sukarta, O.C.A. ; Slootweg, E.J. ; Zheng, Qi ; Schoen, Ruben ; Pomp, H. ; Roosien, J. ; Smant, G. ; Goverse, A. - \ 2018
The potato Rx1 is an intracellular Nucleotide-binding Leucine Rich Repeat (NLR) immune receptor with an archetypical N-terminal coiled-coil (CC) domain. It confers extreme resistance against Potato Virus X (PVX) by gene-specific recognition of the viral coat protein (CP). Recent findings point to a role of Rx1 in the nucleus whereby it could directly bind host genetic material, though it remains unclear how this process eventually leads to defence. A possibility is that Rx1 recruits other host factors, for example via the CC domain, which is predicted to act as scaffolds for nuclear signalling. Here, we used the CC domains of Rx1 and the Rx1-like protein Gpa2 (mediates defence against the nematode Globodera pallida) as baits in a Co-IP/MS analysis after cell fractionation to co-purify putative interactors from Nicotiana benthamiana. Five hits (designated Rp01-Rp05) were further prioritized as candidate Rx1/Gpa2 interacting proteins. Similar pull-down experiments confirmed complex formation with the full-length immune receptors in plantae. Interestingly, co-expression of Rp05 alters the subcellular distribution of the Rx1-CC domain, hinting its role in Rx1-function. Transient overexpression experiments confirm that Rp05 could in fact potentiate defense against PVX. Interestingly, however, this occurs independently of Rx1. We substantiated this model by demonstrating that Rp05 could influence HR-responses by other NLR proteins (e.g. Gpa2, Sw5A/B and Mi-1) indicating that it may be a common downstream component in immune signaling. Currently, we focus on elucidating the detailed molecular underpinning of Rp05 function in R-gene mediated resistances using Rx1 as the principal model system.
Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1
Slootweg, Erik J. ; Spiridon, Laurentiu N. ; Martin, Eliza C. ; Tameling, Wladimir I.L. ; Townsend, Philip D. ; Pomp, Rikus ; Roosien, Jan ; Drawska, Olga ; Sukarta, Octavina C.A. ; Schots, Arjen ; Borst, Jan Willem ; Joosten, Matthieu H.A.J. ; Bakker, Jaap ; Smant, Geert ; Cann, Martin J. ; Petrescu, Andrei-Jose ; Goverse, Aska - \ 2018
Plant Physiology 178 (2018)3. - ISSN 0032-0889 - p. 1310 - 1331.
The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality.
Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.
Exploring the Resistosome of the Potato CC-NB-LRR Immune Receptor Rx1
Sukarta, O.C.A. ; Slootweg, E.J. ; Boeren, J.A. ; Roosien, J. ; Pomp, H. ; Bakker, J. ; Smant, G. ; Goverse, A. - \ 2018
The potato Rx1 is an intracellular Nucleotide-binding Leucine Rich Repeat (NLR) immune receptor with an archetypical N-terminal coiled-coil (CC) domain. It confers extreme resistance against Potato Virus X (PVX) by gene-specific recognition of the viral coat protein (CP). Recent findings point to a role of Rx1 in the nucleus whereby it could directly bind host genetic material, though it remains unclear how this process eventually leads to defence. A possibility is that Rx1 recruits other host factors, for example via the CC domain, which is predicted to act as scaffolds for nuclear signalling. Here, we used the CC domains of Rx1 and the Rx1-like protein Gpa2 (mediates defence against the nematode Globodera pallida) as baits in a Co-IP/MS analysis after cell fractionation to co-purify putative interactors from Nicotiana benthamiana. Five hits (designated Rp01-Rp05) were further prioritized as candidate Rx1/Gpa2 interacting proteins. Similar pull-down experiments confirmed complex formation with the full-length immune receptors in plantae. Interestingly, co-expression of Rp05 alters the subcellular distribution of the Rx1-CC domain, hinting its role in Rx1-function. Transient overexpression experiments confirm that Rp05 could in fact potentiate defense against PVX. Interestingly, however, this occurs independently of Rx1. We substantiated this model by demonstrating that Rp05 could influence HR-responses by other NLR proteins (e.g. Gpa2, Sw5A/B and Mi-1) indicating that it may be a common downstream component in immune signaling. Currently, we focus on elucidating the detailed molecular underpinning of Rp05 function in R-gene mediated resistances using Rx1 as the principal model system.
Identification and functional characterisation of host interactions of the potato CC-NB-LRR Rx1
Sukarta, Octavina - \ 2018
Genome-wide association mapping of the architecture of susceptibility to the root-knot nematode Meloidogyne incognita in Arabidopsis thaliana
Warmerdam, S. ; Sterken, M.G. ; Schaik, C.C. van; Oortwijn, M.E.P. ; Sukarta, O.C.A. ; Lozano Torres, J.L. ; Dicke, M. ; Helder, J. ; Kammenga, J.E. ; Goverse, A. ; Bakker, J. ; Smant, G. - \ 2018
Dissecting Gpa2-mediated immune signalling pathways involved in resistance to the potato cyst nematode Globodera Pallida
Zheng, Qi ; Slootweg, E.J. ; Sukarta, O.C.A. ; Schaik, C.C. van; Smant, G. ; Goverse, A. - \ 2018
Genome-wide association mapping of the architecture of susceptibility to the root-knot nematode Meloidogyne incognita in Arabidopsis thaliana
Warmerdam, Sonja ; Sterken, Mark G. ; Schaik, Casper van; Oortwijn, Marian E.P. ; Sukarta, Octavina C.A. ; Lozano-Torres, Jose L. ; Dicke, Marcel ; Helder, Johannes ; Kammenga, Jan E. ; Goverse, Aska ; Bakker, Jaap ; Smant, Geert - \ 2018
New Phytologist 218 (2018)2. - ISSN 0028-646X - p. 724 - 737.
Susceptibility to the root-knot nematode Meloidogyne incognita in plants is thought to be a complex trait based on multiple genes involved in cell differentiation, growth and defence. Previous genetic analyses of susceptibility to M. incognita have mainly focused on segregating dominant resistance genes in crops. It is not known if plants harbour significant genetic variation in susceptibility to M. incognita independent of dominant resistance. To study the genetic architecture of susceptibility to M. incognita, we analysed nematode reproduction on a highly diverse set of 340 natural inbred lines of Arabidopsis thaliana with genome-wide association mapping. We observed a surprisingly large variation in nematode reproduction among these lines. Genome-wide association mapping revealed four quantitative trait loci (QTLs) located on chromosomes 1 and 5 of A. thaliana significantly associated with reproductive success of M. incognita, none of which harbours typical resistance gene homologues. Mutant analysis of three genes located in two QTLs showed that the transcription factor BRASSINAZOLE RESISTANT1 and an F-box family protein may function as (co-)regulators of susceptibility to M. incognita in Arabidopsis.
Our data suggest that breeding for loss-of-susceptibility, based on allelic variants critically involved in nematode feeding, could be used to make crops more resilient to root-knot nematodes.
The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor
Townsend, Philip D. ; Dixon, Christopher H. ; Slootweg, Erik J. ; Sukarta, Octavina C.A. ; Yang, Ally W.H. ; Hughes, Timothy R. ; Sharples, Gary J. ; Palsson, Lars-Olof ; Takken, Frank L.W. ; Goverse, Aska ; Cann, Martin J. - \ 2018
Journal of Biological Chemistry 293 (2018)9. - ISSN 0021-9258 - p. 3218 - 3233.
Plant NLR proteins enable the immune system to recognise and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here we report a functional interaction between Rx1 and NbGlk1, a Golden2- like transcription factor. Rx1 binds to NbGlk1 in vitro and in planta. NbGlk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of NbGlk1 for DNA in vitro. NbGlk1 activates cellular responses to potato virus X, whereas Rx1 associates with NbGlk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can co-ordinate an immune signalling response at DNA following pathogen perception.
The potato Rx1 immune receptor interacting protein Ri-7 potentiates both Rx1 dependent and independent defenses against PVX
Sukarta, O.C.A. - \ 2017
The potato Rx1 immune receptor interacting protein Ri-7 potentiates both Rx1 dependent and independent defenses against PVX
Sukarta, O.C.A. ; Slootweg, E.J. ; Boeren, J.A. ; Roosien, J. ; Pomp, H. ; Bakker, J. ; Smant, G. ; Goverse, A. - \ 2017
1. Background

The Rx1 immune receptor, introgressed from the wild potato cultivar Solanum andigena, confers resistance against Potato Virus X (PVX) (Bendahmane et al., 1999). However, the underlying mechanisms of Rx1-mediated resistance are not fully understood, prompting the need to identify signaling components of Rx1-immunity. In a recent Co-IP/MS-analysis, we identified Ri-7 as a candidate interactor of Rx1 in Nicotiana benthamiana. The current research aims to determine the functional role for the interaction of Ri-7 with Rx1. We hypothesize that Ri-7 forms a complex with Rx1 to regulate immunity against PVX.
2. Methods

To functionally characterize Ri-7, a combination of reverse genetics and advanced microscopy techniques was performed in the model plant specie Nicotiana benthamiana. This included transient overexpression and silencing assays as well as in vivo imaging by confocal laser scanning microscopy.

3. Results

Interestingly, transient overexpression of Ri-7 was demonstrated to enhance both Rx1-dependant and independent defenses against PVX (as demonstrated by PVX virulence assays). Additional expression analysis by qRT-PCR indicates that Ri-7 regulates defense gene expressions upon PVX infection and resistance by Rx1. More strikingly, we demonstrate that Ri-7 could also enhance defense responses by other structurally similar immune receptors. Collectively, our data pinpoint the integral role of Ri-7 as a central hub in defense signaling.

Keywords: Plant immunity, immune receptors, Rx1, PVX

4. References

Bendahmane, A., Kanyuka, K., and Baulcombe, D.C. (1999). The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11, 781.
Inter- and intramolecular interactions regulating the activity of the CNL immune receptors Rx1 and Gpa2 in complex with RanGAP2
Slootweg, E.J. ; Pomp, H. ; Roosien, J. ; Voogt, Laurens ; Sukarta, O.C.A. ; Goverse, A. - \ 2017
The activities of the coiled coil (CC), nucleotide-binding (NB), leucine-rich repeat (LRR) resistance protein Gpa2 from potato and its close homolog Rx1 are regulated by interdependent interactions of the LRR and CC with the NB domain. Both proteins require a nucleocytoplasmic distribution in the cell for proper functioning. Their CC influences this distribution by association with either nuclear components or with the cytoplasmic co-factor RanGTPase Activating Protein 2 (RanGAP2) which retains the proteins in the cytoplasm. This study is focused on the role of the CC and its interaction with RanGAP2 in the regulation of activation of resistance and cell death responses. Intra- and intermolecular interactions of the R protein have been visualised using Fluorescence Lifetime Imaging Microscopy. Aside from the EDVID motif a few aromatic residues in the N-terminal half of the CC are required for the interaction of the CC with the NB-LRR, but not RanGAP2. Mutations in one region of the CC affect cell death more than resistance signaling, suggesting that the CC plays different roles in regulating either pathway. In addition, mutations in the CC affect the nucleocytoplasmic distribution, underscoring the close link between protein structure, complex formation and subcellular localisation. Disruption of the structure of the CC leads to a more cytoplasmic localisation whereas mutants affected in their interaction with RanGAP2 are no longer sequestered in the cytoplasm by RanGAP2.
Exploring the Resistosome of the Potato CC-NB-LRR Immune Receptor Rx1
Sukarta, O.C.A. ; Slootweg, E.J. ; Boeren, J.A. ; Roosien, J. ; Pomp, H. ; Bakker, J. ; Smant, G. ; Goverse, A. - \ 2017
The potato Rx1 is an intracellular Nucleotide-binding Leucine Rich Repeat (NLR) immune receptor with an archetypical N-terminal coiled-coil (CC) domain. It confers extreme resistance against Potato Virus X (PVX) by gene-specific recognition of the viral coat protein (CP). Recent findings point to a role of Rx1 in the nucleus whereby it could directly bind host genetic material, though it remains unclear how this process eventually leads to defence. A possibility is that Rx1 recruits other host factors, for example via the CC domain, which is predicted to act as scaffolds for nuclear signalling. Here, we used the CC domains of Rx1 and the Rx1-like protein Gpa2 (mediates defence against the nematode Globodera pallida) as baits in a Co-IP/MS analysis after cell fractionation to co-purify putative interactors from Nicotiana benthamiana. Five hits (designated Rp01-Rp05) were further prioritized as candidate Rx1/Gpa2 interacting proteins. Similar pull-down experiments confirmed complex formation with the full-length immune receptors in plantae. Interestingly, co-expression of Rp05 alters the subcellular distribution of the Rx1-CC domain, hinting its role in Rx1-function. Transient overexpression experiments confirm that Rp05 could in fact potentiate defense against PVX. Interestingly, however, this occurs independently of Rx1. We substantiated this model by demonstrating that Rp05 could influence HR-responses by other NLR proteins (e.g. Gpa2, Sw5A/B and Mi-1) indicating that it may be a common downstream component in immune signaling. Currently, we focus on elucidating the detailed molecular underpinning of Rp05 function in R-gene mediated resistances using Rx1 as the principal model system.
Exploring the Resistosome of the Potato CC-NB-LRR Immune Receptor Rx1
Sukarta, O.C.A. - \ 2016
Structure-informed insights for NLR functioning in plant immunity
Sukarta, Octavina Citra Ayudhany ; Slootweg, Erik J. ; Goverse, Aska - \ 2016
Seminars in Cell and Developmental Biology 56 (2016). - ISSN 1084-9521 - p. 134 - 149.
To respond to foreign invaders, plants have evolved a cell autonomous multilayered immune system consisting of extra- and intracellular immune receptors. Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) mediate recognition of pathogen effectors inside the cell and trigger a host specific defense response, often involving controlled cell death. NLRs consist of a central nucleotide-binding domain, which is flanked by an N-terminal CC or TIR domain and a C-terminal leucine-rich repeat domain (LRR). These multidomain proteins function as a molecular switch and their activity is tightly controlled by intra and inter-molecular interactions. In contrast to metazoan NLRs, the structural basis underlying NLR functioning as a pathogen sensor and activator of immune responses in plants is largely unknown. However, the first crystal structures of a number of plant NLR domains were recently obtained. In addition, biochemical and structure-informed analyses revealed novel insights in the cooperation between NLR domains and the formation of pre- and post activation complexes, including the coordinated activity of NLR pairs as pathogen sensor and executor of immune responses. Moreover, the discovery of novel integrated domains underscores the structural diversity of NLRs and provides alternative models for how these immune receptors function in plants. In this review, we will highlight these recent advances to provide novel insights in the structural, biochemical and molecular aspects involved in plant NLR functioning
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