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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Deep learning based plant part detection
    Barth, R. ; Afonso, M.V. ; Chauhan, A. - \ 2019
    In: EFITA Book of Abstracts. - EFITA - p. 40 - 40.
    A case study on prediction of sensitivity of tomato sepals to fungal infection using hyperspectral imaging
    Chauhan, A. ; Harchioui, Najim El; Hogeveen-van Echtelt, Esther ; Mensink, M.G.J. ; Brdar, Sanja ; Panic, Marko ; Grbovic, Zeljana - \ 2019
    In: Book of Abstracts European Federation for Information Technology in Agriculture, Food and the Environment (EFITA) 2019. - EFITA - p. 42 - 42.
    Plant disease detection based on multispectral and hyperspectral image analysis using deep learning techniques
    Chauhan, A. ; Polder, G. ; Villiers, H.A.C. de - \ 2019
    In: Book of Abstracts European Federation for Information Technology in Agriculture, Food and the Environment (EFITA) 2019. - EFITA - p. 37 - 37.
    Deep learning based plant part detection in Greenhouse settings
    Afonso, M.V. ; Barth, R. ; Chauhan, A. - \ 2019
    In: 12th EFITA International Conference. - Rhodes Island, Greece : EFITA - p. 48 - 53.
    Precision agriculture challenges such as automatic harvesting, phenotyping, and yield prediction require precise detection of plant parts such as the fruits, leaves or stems. Deep learning has emerged as the state-of-the-art technology for image segmentation and object detection in several domains, notably in self-driving vehicles and medical imaging. In recent years, deep learning methods are being increasingly adopted in vision-based applications for precision agriculture. In previous work, methods were investigated to segment the image for plant parts. However, such an approach did not yield object instances. In this work, we applied the state-of-the-art deep learning object detector, Mask RCNN, to the problem of detecting fruit and other plant parts, in the sweet pepper (capsicum annuum) plant. An extensive study was carried out where we investigated different transfer learning schemes, different convolutional neural network architectures, and varying numbers of training images. Experimentally, we found that Mask RCNN trained with the synthetic data and fine-tuned with very few empirical images is able to detect more than 95% of the sweet pepper fruit. It was also found that training on the synthetic data and then fine-tuning over a few empirical images led to a better performance in the detection of fruit, over training only on the limited set of empirical images. Furthermore, results show that the best model could successfully generalize to different imaging conditions. This work is a necessary step for applying deep learning in high-throughput robotics and phenotyping approaches and will open up many opportunities for smart farming and more efficient use of resources. Currently, training deep learning models is dependent on the knowledge and expertise of the scientists involved. The insights gained from this work should lead to more automatic training protocols, allowing widespread use in very different applications.
    Toward the improvement of total nitrogen deposition budgets in the United States
    Walker, J.T. ; Beachley, G. ; Amos, H.M. ; Baron, J.S. ; Bash, J. ; Baumgardner, R. ; Bell, M.D. ; Benedict, K.B. ; Chen, X. ; Clow, D.W. ; Cole, A. ; Coughlin, J.G. ; Cruz, K. ; Daly, R.W. ; Decina, S.M. ; Elliott, E.M. ; Fenn, M.E. ; Ganzeveld, L. ; Gebhart, K. ; Isil, S.S. ; Kerschner, B.M. ; Larson, R.S. ; Lavery, T. ; Lear, G.G. ; Macy, T. ; Mast, M.A. ; Mishoe, K. ; Morris, K.H. ; Padgett, P.E. ; Pouyat, R.V. ; Puchalski, M. ; Pye, H.O.T. ; Rea, A.W. ; Rhodes, M.F. ; Rogers, C.M. ; Saylor, R. ; Scheffe, R. ; Schichtel, B.A. ; Schwede, D.B. ; Sexstone, G.A. ; Sive, B.C. ; Sosa, R. ; Templer, P.H. ; Thompson, T. ; Tong, D. ; Wetherbee, G.A. ; Whitlow, T.H. ; Wu, Z. ; Yu, Z. ; Zhang, L. - \ 2019
    Science of the Total Environment 691 (2019). - ISSN 0048-9697 - p. 1328 - 1352.
    Ammonia - Dry deposition - Organic nitrogen - Oxidized nitrogen - Reactive nitrogen - Wet deposition

    Frameworks for limiting ecosystem exposure to excess nutrients and acidity require accurate and complete deposition budgets of reactive nitrogen (Nr). While much progress has been made in developing total Nr deposition budgets for the U.S., current budgets remain limited by key data and knowledge gaps. Analysis of National Atmospheric Deposition Program Total Deposition (NADP/TDep) data illustrates several aspects of current Nr deposition that motivate additional research. Averaged across the continental U.S., dry deposition contributes slightly more (55%) to total deposition than wet deposition and is the dominant process (>90%) over broad areas of the Southwest and other arid regions of the West. Lack of dry deposition measurements imposes a reliance on models, resulting in a much higher degree of uncertainty relative to wet deposition which is routinely measured. As nitrogen oxide (NOx) emissions continue to decline, reduced forms of inorganic nitrogen (NHx = NH3 + NH4 +) now contribute >50% of total Nr deposition over large areas of the U.S. Expanded monitoring and additional process-level research are needed to better understand NHx deposition, its contribution to total Nr deposition budgets, and the processes by which reduced N deposits to ecosystems. Urban and suburban areas are hotspots where routine monitoring of oxidized and reduced Nr deposition is needed. Finally, deposition budgets have incomplete information about the speciation of atmospheric nitrogen; monitoring networks do not capture important forms of Nr such as organic nitrogen. Building on these themes, we detail the state of the science of Nr deposition budgets in the U.S. and highlight research priorities to improve deposition budgets in terms of monitoring and flux measurements, leaf- to regional-scale modeling, source apportionment, and characterization of deposition trends and patterns.

    The water-land-food-energy-climate Nexus for a resource efficient Europe
    Laspidou, C. ; Witmer, M. ; Vamvakeridou-Lyroudia, L.S. ; Domingo, X. ; Brouwer, F.M. ; Howells, M. ; Sušnik, J. ; Blanco, M. ; Bonazountas, M. ; Fournier, M. ; Papadopoulou, M.P. - \ 2017
    - 5 p.
    Α novel methodology for addressing policy
    inconsistencies and knowledge gaps that hinder the
    transition to a greater resource efficiency Europe is
    proposed. We focus on the integration of all different
    sectors that interact and influence each other, namely the
    “water- energy- food- land use- climate nexus” and we
    develop tools for identifying and quantifying their complex
    interlinkages under the influence of climate change. In
    order to achieve this, we employ a series of sophisticated
    models (referred to as “thematic models”), each of which
    addresses a different nexus dimension, or a combination of
    a few, while none addresses all nexus dimensions in an
    integrative manner. We use dynamic systems modeling
    and other complexity science techniques in order to
    “merge” different thematic model outputs in a single
    coherent result, which is presented to the user in an easy to
    comprehend Serious Game environment. This way, the
    effect of policies that are designed to affect one field
    (nexus dimension) on others can be quantified and
    simulated, thus informing policy-makers for the
    unintended consequences of their policies, reducing
    uncertainties, covering knowledge gaps and leading to a
    resource efficient Europe faster.
    The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses
    Does, Dieuwertje Van der; Boutrot, Freddy ; Engelsdorf, Timo ; Rhodes, Jack ; McKenna, Joseph F. ; Vernhettes, Samantha ; Koevoets, Iko ; Tintor, Nico ; Veerabagu, Manikandan ; Miedes, Eva ; Segonzac, Cécile ; Roux, Milena ; Breda, Alice S. ; Hardtke, Christian S. ; Molina, Antonio ; Rep, Martijn ; Testerink, Christa ; Mouille, Grégory ; Höfte, Herman ; Hamann, Thorsten ; Zipfel, Cyril - \ 2017
    Plos Genetics 13 (2017)6. - ISSN 1553-7390

    Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.

    OSL-thermochronometry using bedrock quartz: A note of caution
    Guralnik, B. ; Ankjærgaard, C. ; Jain, M. ; Murray, A.S. ; Müller, A. ; Walle, M. ; Lowick, S.E. ; Preusser, F. ; Rhodes, E.J. ; Wu, T.S. ; Mathew, G. ; Herman, F. - \ 2015
    Quaternary Geochronology 25 (2015). - ISSN 1871-1014 - p. 37 - 48.
    Optically stimulated luminescence (OSL) thermochronometry is an emerging application, whose capability to record sub-Million-year thermal histories is of increasing interest to a growing number of subdisciplines of Quaternary research. However, several recent studies have encountered difficulties both in extraction of OSL signals from bedrock quartz, and in their thermochronometric interpretation, thus highlighting the need for a methodological benchmark. Here, we investigate the characteristic OSL signals from quartz samples across all major types of bedrock and covering a wide range of chemical purities. High ratios of infrared to blue stimulated luminescence (IRSL/BLSL), an insensitive ‘fast’ OSL component, and anomalously short recombination lifetimes seen in time-resolved luminescence (TROSL), are often encountered in quartz from crystalline (magmatic and metamorphic) bedrock, and may hamper successful OSL dating. Furthermore, even when the desirable signal is present, its concentration might be indistinguishable from its environmental steady-state prediction, thus preventing its conversion to a cooling or heating history. We explore the saturation properties and the thermal activation parameters of various OSL signals in quartz to outline the capabilities and limitations for their use in lowtemperature thermochronometry.
    OSL-thermochronometry of feldspar from the KTB borehole, Germany
    Guralnik, B. ; Jain, M. ; Herman, F. ; Ankjærgaard, C. ; Murray, A.S. ; Valla, P.G. ; Preusser, F. ; King, G.E. ; Chen, R. ; Lowick, S.E. ; Kook, M. ; Rhodes, E.J. - \ 2015
    Earth and Planetary Science Letters 423 (2015). - ISSN 0012-821X - p. 232 - 243.
    The reconstruction of thermal histories of rocks (thermochronometry) is a fundamental tool both in Earth science and in geological exploration. However, few methods are currently capable of resolving the low-temperature thermal evolution of the upper ~2km of the Earth’s crust. Here we introduce a new thermochronometer based on the infrared stimulated luminescence (IRSL) from feldspar, and validate the extrapolation of its response to artificial radiation and heat in the laboratory to natural environmental conditions. Specifically, we present a new detailed Na-feldspar IRSL thermochronology from a well-documented thermally-stable crustal environment at the German Continental Deep Drilling Program (KTB). There, the natural luminescence of Na-feldspar extracted from twelve borehole samples (0.1–2.3km depth, corresponding to 10–70¿C) can be either (i) predicted within uncertainties from the current geothermal gradient, or (ii) inverted into a geothermal palaeogradient of 29 ±2¿Ckm-1, integrating natural thermal conditions over the last ~65ka. The demonstrated ability to invert a depth–luminescence dataset into a meaningful geothermal palaeogradient opens new venues for reconstructing recent ambient temperatures of the shallow crust (200¿CMa-1range). Although Na-feldspar IRSL is prone to field saturation in colder or slower environments, the method’s primary relevance appears to be for borehole and tunnel studies, where it may offer remarkably recent (
    Exploiting natural variation in susceptibility of arabidopsis thaliana to meloidogyne incognita to breed broad-spectrum resistance to root knot nematodes m. incognita
    Warmerdam, S. ; Schaik, C.C. van; Lozano Torres, J.L. ; Finkers-Tomczak, A.M. ; Bakker, J. ; Goverse, A. ; Smant, G. - \ 2014
    The root knot nematode Meloidogyne incognita is able to parasitize hundreds of plant species and is a major threat in nearly all vegetable-growing regions in the world. Current resistance is based on major dominant resistance (R) genes, which are frequently overcome by the occurrence of resistance-breaking races. Allelic variation in genetic loci associated with susceptibility to plant-parasitic nematodes within a single plant species has thus far not been used to breed durable broad-spectrum resistance in crops. Susceptibility investigation of 364 different ecotypes of A. thaliana to the root-knot nematode M. incognita was performed. A nine-fold difference in susceptibility to M. incognita was found between the most and the least susceptible ecotypes. This allelic variation can be caused by polymorphic transcript levels, and by allelic variation within transcripts of essential susceptibility genes. Genome wide association mapping was performed to determine loci that are contributing to the susceptibility factors in A. thaliana. Genes underlying these loci were investigated with T-DNA insertion mutation to confirm their contribution to the susceptibility of A. thaliana to M. incognita. Genes involved in endoreduplication, mitosis and protein degradation showed to be involved in the process of forming feeding cells by M. incognita in A. thaliana. These data indicate that allelic variation can be used to breed for broad-spectrum nematode resistance.
    The role of effectors, pamps, and secondary metabolites in adaptation of Cladosporium fulvum to tomato
    Wit, P.J.G.M. de; Mesarich, C.H. ; Ökmen, B. ; Burgt, I.A. van der; Iida, Y. ; Battaglia, E. ; Beenen, H.G. ; Griffiths, S.A. ; Bradshaw, R.E. ; Collemare, J.A.R. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 41 - 41.
    CS-28.1 - The biotrophic tomato pathogen Cladosporium fulvum is a Dothideomycete fungus that is most related to the hemi-biotrophic fungal pine pathogen Dothistroma septosporum. We are interested in understanding genomic adaptations in these and related fungi that can explain host plant-specificity. The C. fulvum genomic sequence allowed us to identify and study its complete effector catalogue of which many effectors had been identified already and of which three represent core effectors (Avr4, Ecp2 and Ecp6) occurring in several genera and species of fungal plant pathogens. The genome of D. septosporum harbors the highest number of homologs of C. fulvum effectors, but some are pseudogenized. Both C. fulvum and D. septosporum contain a large number of genes involved in the production of secondary metabolites. C. fulvum seems to adapt its host plant tomato by down-regulation or pseudogenization of genes involved in the production of toxic secondary metabolites. One clear example is dothiostromin, a toxin produced by D. septosporum during colonization of pine needle. C. fulvum contains all genes required for the production of this toxin, but a few crucial genes have been pseudogenized. Apart from new effectors (Avr5) we have also identified new pathogen-associated molecular patterns (PAMPs), as well as damage-associated molecular patterns (MAMPs) from C. fulvum. Both represent cell-wall degrading enzymes produced by C. fulvum during infection of tomato. We also found that C. fulvum has adapted to grow on tomato by secreting the enzyme a- tomatinase that hydrolyses the antifungal tomato saponin a-tomatine into the non-toxic compounds lycotetraose and tomatidine.
    The parastagonospora nodorum necrotrophic effector SnTox1 elicits recognition in wheat and then protects from the resulting host defense response
    Friesen, T.L. ; Liu, Z. ; Kim, Y. ; Gao, Y. ; Wit, P.J.G.M. de; Faris, J.D. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 24 - 24.
    CS-9.5 - Parastagonospora nodorum (Synonym: Stagonospora nodorum) is a destructive pathogen of wheat that induces yield and quality losses by causing disease on the leaves and glumes of wheat. P. nodorum is a necrotrophic specialist pathogen that secretes an arsenal of necrotrophic effectors (NEs) involved in disease induction. SnTox1 was the first of seven NEs to be reported from P. nodorum and interacts directly or indirectly with the single dominant susceptibility gene Snn1. SnTox1 is recognized by Snn1 followed by the induction of a classical defense response involving programmed cell death (PCD), an oxidative burst, DNA laddering and up regulation of several PR proteins, however, this recognition results in susceptibility to P. nodorum rather than resistance. Interestingly, in addition to inducing PCD that results in susceptibility, we have shown that SnTox1 is important in defending the pathogen against wheat chitinases that attack the cell wall of fungi, reducing growth. To demonstrate that SnTox1 was involved in protection from host-produced chitinases, we cloned and expressed several wheat chitinase genes to assess the effects of chitinases in the presence and absence of the SnTox1, showing that SnTox1 is highly important in protecting the pathogen from wheat chitinases. Additionally, we transformed non-pathogens of wheat with SnTox1 and showed that not only did some of these fungi become pathogens of wheat but all had increased protection from wheat chitinases. The dual function of this protein explains the high prevalence of SnTox1 , relative to other NEs in the P. nodorum global population.
    Towards the identification of fungal effector molecules involved in host plant developmental reprogramming
    Thole, K. ; Reusche, M. ; Faino, L. ; Thomma, B.P.H.J. ; Thürmer, A. ; Daniel, R. ; Teichmann, T. ; Lipka, V. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 49 - 49.
    P033 - Typically, Verticillium infections lead to severe wilting symptoms on their respective host plants with the remarkable exception of V. longisporum infected Brassicaceae, which maintain their water status. Recently, we could show that water status maintenance correlates with a substantial V. longisporum-induced developmental reprogramming on its host plants Brassica napus and Arabidopsis thaliana. Together, our data suggest that de novo formation of xylem by hyperplasia and transdifferentiation in response to infection by V. longisporum is likely to be a compensatory plant response resulting in increase of plant water storage capacity. We recently analyzed symptom development induced by 22 diploid V. longisporum and 43 haploid V. dahliae strains on Arabidopsis ecotype Col-0. Interestingly, we identified five V. dahliae strains that phenocopy the disease symptoms typically associated with V. longisporum infection. Notably, these isolates are also able to cause defoliation on their respective host plants cotton or olive. In contrast, we found several V. dahliae strains which induce strong wilting phenotypes and lateral branching, but lack the capacity to induce developmental reprogramming of vascular tissues. In accordance to that, two cotton non-defoliating isolates induced mild wilting symptoms but no chlorosis or de novo xylem formation on Arabidopsis. This indicates that fungal molecules involved in V. longisporum associated symptom development on Arabidopsis might be primarily prevalent in V. dahliae isolates causing defoliation on their respective host plants. Comparative genomic sequence analyses of these haploid V. dahliae strains will be conducted to identify and functionally characterize effector molecule candidates underlying the distinct infection symptoms.
    Evolution and biological function of effectors of the vascular wilt pathogen Verticillium dahliae
    Berg, G.C.M. van den; Boshoven, J.C. ; Cook III, D.E. ; Damme, M.M.A. van; Ebert, M.K. ; Faino, L. ; Kombrink, A. ; Li, J. ; Rojas Padilla, J.E. ; Rövenich, H. ; Sánchez-Vallet, A. ; Seidl, M.F. ; Shi, X. ; Song, Y. ; Valkenburg, D.J. ; Thomma, B.P.H.J. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 17 - 17.
    PL-1.2 - Verticillium dahliae, causal agent of vascular wilt disease, is one of the most notorious plant pathogens of tomato. By comparative population genomics, we previously identified the race 1 specific effector that activates tomato immunity mediated by the cell surface receptor Ve1. Further comparative genomics revealed extensive genomic rearrangements between individual V. dahliae isolates that could be implicated in the occurrence of lineage-specific regions involved in niche adaptation and virulence. Remarkably, genes that reside in lineage-specific regions are over-represented during in planta expression. Moreover, in contrast to candidate effector genes that reside in the core genome, targeted disruptionof several lineage-specific effector genes resulted in compromised virulence. One of the lineage-specific effectors that could be implicated in virulence on tomato is a LysM effector, a homolog of the Ecp6 effector of the foliar tomato pathogen Cladosporium fulvum that functions in suppression of chitin-triggered host immunity. Structural analysis revealed a novel mechanism for chitin binding by Ecp6 through intrachain LysM dimerization, leading to a composite binding site that binds chitin with ultra-high affinity. Considering the importance of lineage-specific regions for V. dahliae aggressiveness, detailed genomic information on the recombination sites is required to understand how lineage-specific regions arise. To this end, we re-sequenced a V. dahliae genome with PacBio technology, leading to a gapless assembly of eight complete chromosomes. Our efforts to identify genomic signatures at the recombination sites and the molecular mechanism(s) that establish chromosomal rearrangements will be discussed.
    The receptor-like protein (RLP) Cf-4, present in a constitutive complex with SOBIR1/EVR, recruits BAK1 to mount plant immunity
    Liebrand, T.W.H. ; Postma, J. ; Evrard, A. ; Bye, R.R. ; Burgh, A.M. van der; Robatzek, S. ; Joosten, M.H.A.J. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 84 - 84.
    P382 - Plants perceive microbial patterns by cell surface receptors that are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs), usually containing extracellular leucine-rich repeats (LRRs). However, RLPs lack an intracellular kinase domain for activation of downstream signaling upon ligand perception. Recently, we showed that tomato (Solanum lycopersicum, Sl) Cf-4, an LRR-RLP mediating resistance to Avr4-expressing strains of the fungal pathogen Cladosporium fulvum, constitutively interacts with the RLK SOBIR1. SOBIR 1 is unrelated to the RLK BRI1-Associated Kinase (BAK1), which is an essential regulatory LRR-RLK for pattern recognition receptors (PRRs), such as the LRR-RLK FLS2. Interestingly,Arabidopsis thaliana (At) SOBIR1 complements a knock-down in Nicotiana benthamiana (Nb) of NbSOBIR1 by restoring the Cf-4/Avr4-triggered HR, whereas a kinase-dead version of At SOBIR1 does not. Furthermore, overexpression of SOBIR1 in N. benthamiana results in an HR, which does not take place with a kinase-dead version of this RLK. The Cf-4/SOBIR1 complex does not dissociate in the presence of Avr4, suggesting that the complex acts as a two-component RLK. We found that the Cf-4/SOBIR1 complex localizes at the ARA7/ARA6-positive late endosomes upon activation with Avr4. Similar trafficking takes place for FLS2 upon its activation, a process which is BAK1-d ependent. Strikingly, we discovered that the Cf-4/SOBIR1 complex also recruits BAK1, an interaction which is required for both Avr4-triggered HR and Cf-4/SOBIR1 endocytosis. Thus, Cf-4 immune signalling is initiated by the formation of at least a tripartite receptor complex, showing that RLPs, ina complex with SOBIR1, function like bona fide PRRs. Future studies will focus on (1) the requirements of tripartite complex formation, (2) possible differential phosphorylation of SOBIR1 before and after Avr4/Cf-4-triggered BAK1 recruitment and (3) identification of downstream events that take place upon Avr4-mediated activation of the Cf-4/SOBIR1 complex.
    Traffic control for receptors at the host-microbe interface
    Beck, M. ; Bourdais, G. ; Kopischke, M. ; Mbengue, M. ; Postma, J. ; Spallek, T. ; Liebrand, T.W.H. ; Joosten, M.H.A.J. ; Robatzek, S. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 17 - 17.
    PL-1.4 - Host membranes respond to microbial perception and different membrane domains enable host cells to perform the functions they require in mediating immunity. Focusing on how the membrane trafficking network responds to microbial attack, we have dissected the spatio - temporal redistribution of membranes cargoes such as the pattern recognition receptors (PRRs). FLS2 is a known plasma membrane-resident receptor kinase PRR that enters two distinct endosomal trafficking routes depending on its activation status. To this end, ligand - induced late endosomal trafficking of FLS2 requires an active receptor complex. FLS2 endocytosis mediated by clathrin and involves sorting by the ESCRT machinery. Ligand-induced endocytosis is conserved across different PRR protein families as well as across different plant species. By studying the EFR and PEPR1/2 receptor kinases, we identified a common endosomal pathway, which regulates the removal of activated PRRs from the plasma membrane. This expands into the family of receptor-like protein type PRRs, of which the Cf-4-SOBIR1 constitutive complex forms a tripartite interaction with BAK1/SERK3 upon Avr4 perception, required to induce the endocytosis of the complex. Our findings show that the mechanism of endocytosis is employed in regulating the spatio-temporal dynamics of PRRs, which can facilitate the uptake of microbial molecules as observed for lagellin, and is involved in specific immun e responses such as the closure of stomata.
    Third generation sequencing paves the way for rapid and complete fungal genome sequening
    Seidl, M.F. ; Faino, L. ; Berg, G. van den; Datema, E. ; Janssen, A. ; Wittenberg, A. ; Thomma, B.P.H.J. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 193 - 193.
    Verticillium dahliae, causal agent of vascular wilt disease, is one of the most notorious plant pathogens on tomato. By using population genome sequencing with the Illumina platform, we have recently shown that frequent genomic rearrangements drive the evolution of lineage-specific regions that establish virulence and niche adaptation in this species. Due to limitations associated with second generation sequencing, in particular the short read length and lack of coverage of repetitive sequence stretches, the exact genomic signatures at the site of the chromosomal rearrangements, and thus the molecular mechanism that establishes these modifications, remain largely unknown. In order to obtain detailed genomic information on the recombination sites, we re-sequenced the ~37 Mb genome of V. dahliae strain JR2 using single-molecule real time (SMRT) sequencing with PacBio technology. To this end, we generated ~8 Gb of sequencing data. De novo assembly was performed resulting in 66 contigs of which 16 covered 99% of the complete genome as inferred from an optical map. Subsequent manual and software-guided scaffolding resulted in a gapless assembly of all eight complete chromosomes. Thus, the re-assembled V. dahliae strain JR2 genome represents the first finished, gapless fungal genome. We subsequently applied comparative genomics and inferred the exact positions of the previously identified genomic rearrangements. Our approach to identify genomic signatures at the recombination sites will be discussed. This study highlights the superior genome assembly qualities of third generation sequencing technologies and exemplifies the pivotal role of a finished genome in understanding the evolution and biology of fungal pathogens. We anticipate that third generation sequencing will pave the way for rapid and affordable genome sequencing approaches aiming for finished assemblies in fungal and also other eukaryotic species.
    Proteomics and phosphoproteomics of P. infestans and potato
    Resjö, S. ; Ali, A. ; Meijer, H.J.G. ; Seidl, M.F. ; Snel, B. ; Sandin, M. ; Levander, F. ; Grenville-Briggs, L. ; Govers, F. ; Andreasson, E. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 58 - 58.
    P122 - We are using proteomics to study the underlying processes that govern pathogenicity and development in Phytophthora infestans and resistance to P. infestans in potato. In a large-scale phosphoproteomics study of six life stages, we obtained quantitative data for 2922 phosphopeptides. Life stage-specific phosphopeptides include ABC transporters and an appressorium specific kinase. We also identified 2179 phosphorylation sites and deduced 22 phosphomotifs. In addition, we detected tyrosine phosphopeptides that are potential targets of tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families.The latter are phosphorylated in several life stages, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. In a proteomic study of the same life stages, we have identified a number of proteins with expression patterns that are specific for life stages involved in (pre) infection. Among these are both proteins previously identified as being specifically expressed in these stages, as well as transport proteins, RXLR proteins, kinases and proteins putatively involved in cell wall synthesis. We are now analysing a selection of these proteins further using RNAi and other techniques. In addition, we have also used proteomics to study the apoplastic proteome of potato cultivars with different degrees of resistance to P. infestans in response to infection, identifying potential effector targets and proteins involved in pathogen resistance. Thus, we show that proteomic techniques are valuable tools to elucidate novel mechanisms of pathogenicity and resistance in oomycete-host interactions.
    The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and suppression of plant immunity
    Sheng, Yuting ; Wang, Yonglin ; Meijer, H.J.G. ; Yang, Xinyu ; Hua, C. ; Ye, Wenwu ; Tao, Kai ; Liu, Xiaoyun ; Govers, F. ; Wang, Yuanchao - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 49 - 49.
    P039 - In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defense. However, ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study we indentified the PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. PsHSF1 belongs to a large family of heat shock transcription factors (HSFs) that is conserved in Phytophthora spp. It is highly up-regulated under oxidative stress, heat shock stress and during cyst germination. Heterologous expression of PsHSF1 in a yeast knock-out mutant, that lacks a TF functioning in stress tolerance (¿skn7), rescued the aberrant oxidative stress response. Phenotypic analyses of P. sojae knock-down mutants revealed that PsHSF1 is required for stress tolerance and virulence, and likely functions as a transcriptional regulator of peroxidase and laccase genes. PsHSF1-silenced transformants showed reduced levels of extracellular peroxidase and laccase activities. Moreover, they were compromised in suppressing hydrogen peroxide accumulation and innate immunity in the host, phenotypes that were partially restored by adding the antioxidant glutathione. Silencing PsLAC4, one of the laccase genes regulated by PsHSF1, resulted in reduced virulence thereby illustrating the contribution of PsLAC4 and PsHSF1 to ROS depletion. Our results identify PsHSF1 as a TF that acts as a positive regulator of extracellular peroxidase and laccase genes which are important for ROS resistance of P. sojae.
    Effector-triggered defence against apoplastic fungal pathogens
    Stotz, H.U. ; Mitrousia, G.K. ; Wit, P.J.G.M. de; Fitt, B.D.L. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 93 - 93.
    P474 - The plant immune system was originally defined as including pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). After arrival of a potential pathogen, PTI is rapidly activated by recognition of PAMPs by pattern recognition receptors (PRRs) located on the cell surface. PTI has also been defined as PRR-triggered immunity. In resistant hosts, ETI is initiated by intracellular host recognition of pathogen effectors. Haustoria-forming pathogens generally deliver their effectors into the host cell, whereas apoplastic fungal pathogens deliver effectors into the apoplast to target extracellular or membrane proteins. However, it is not generally believed that resistance against apoplastic pathogens should be considered as PTI as some considerit as ETI. To explain interactions between apoplastic fungal pathogens and their host plants and reconcile these opposing views, it is proposed that this resistance is referred to as effector- triggered defence (ETD). EDT is mediated by R genes encoding cell surface-localized receptor-like proteins (RLPs) that engage the receptor-like kinase SOBIR1. In contrasts, intracellular effectors are directly or indirectly recognised by intracellular nucleotide binding site (NBS) leucine-rich repeat (LRR) receptors (NLRs). Whereas ETI is usually associated with fast, hypersensitive host cell death, ETD often triggers host cell death only after a period of endophytic pathogen growth. This resistance does not generally eliminate these apoplastic pathogens since there are cases that they are subsequently able to complete their sexual life cycle on the resistant host.
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