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2.
Mol Plant Pathol ; 21(9): 1240-1247, 2020 09.
Article in English | MEDLINE | ID: mdl-32672422

ABSTRACT

The soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pathogens of soybeans. SCN is an obligate and sedentary parasite that transforms host plant root cells into an elaborate permanent feeding site, a syncytium. Formation and maintenance of a viable syncytium is an absolute requirement for nematode growth and reproduction. In turn, sensing pathogen attack, plants activate defence responses and may trigger programmed cell death at the sites of infection. For successful parasitism, H. glycines must suppress these host defence responses to establish and maintain viable syncytia. Similar to other pathogens, H. glycines engages in these molecular interactions with its host via effector proteins. The goal of this study was to conduct a comprehensive screen to identify H. glycines effectors that interfere with plant immune responses. We used Nicotiana benthamiana plants infected by Pseudomonas syringae and Pseudomonas fluorescens strains. Using these pathosystems, we screened 51 H. glycines effectors to identify candidates that could inhibit effector-triggered immunity (ETI) and/or pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We identified three effectors as ETI suppressors and seven effectors as PTI suppressors. We also assessed expression modulation of plant immune marker genes as a function of these suppressors.


Subject(s)
Glycine max/parasitology , Plant Diseases/parasitology , Plant Immunity , Tylenchoidea/genetics , Animals , Host-Parasite Interactions , Pathogen-Associated Molecular Pattern Molecules/metabolism , Plant Diseases/immunology , Pseudomonas syringae/genetics , Pseudomonas syringae/pathogenicity , Pseudomonas syringae/physiology , Glycine max/genetics , Glycine max/immunology , Nicotiana/genetics , Nicotiana/immunology , Nicotiana/microbiology , Tylenchoidea/pathogenicity , Tylenchoidea/physiology
3.
Database (Oxford) ; 2019(1)2019 01 01.
Article in English | MEDLINE | ID: mdl-31680133

ABSTRACT

Soybean is an important worldwide crop, and farmers continue to experience significant yield loss due to the soybean cyst nematode (SCN), Heterodera glycines. This soil-borne roundworm parasite is rated the most important pathogen problem in soybean production. The infective nematodes enter into complex interactions with their host plant by inducing the development of specialized plant feeding cells that provide the parasites with nourishment. Addressing the SCN problem will require the development of genomic resources and a global collaboration of scientists to analyze and use these resources. SCNBase.org was designed as a collaborative hub for the SCN genome. All data and analyses are downloadable and can be analyzed with three integrated genomic tools: JBrowse, Feature Search and BLAST. At the time of this writing, a number of genomic and transcriptomic data sets are already available, with 43 JBrowse tracks and 21 category pages describing SCN genomic analyses on gene predictions, transcriptome and read alignments, effector-like genes, expansion and contraction of genomic repeats, orthology and synteny with related nematode species, Single Nucleotide Polymorphism (SNPs) from 15 SCN populations and novel splice sites. Standard functional gene annotations were supplemented with orthologous gene annotations using a comparison to nine related plant-parasitic nematodes, thereby enabling functional annotations for 85% of genes. These annotations led to a greater grasp on the SCN effectorome, which include over 3324 putative effector genes. By designing SCNBase as a hub, future research findings and genomic resources can easily be uploaded and made available for use by others with minimal needs for further curation. By providing these resources to nematode research community, scientists will be empowered to develop novel, more effective SCN management tools.


Subject(s)
Databases, Nucleic Acid , Gene Expression Regulation , Genome, Helminth , Molecular Sequence Annotation , Polymorphism, Single Nucleotide , Rhabditida/genetics , Animals , Gene Ontology , Glycine max
4.
Plant J ; 98(6): 1000-1014, 2019 06.
Article in English | MEDLINE | ID: mdl-30801789

ABSTRACT

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector-coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two-hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain-like cysteine protease (PLCP) 'Responsive to Dehydration 21A' (RD21A), which has been shown to function in the plant defense response. Activity-based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re-localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two-hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector-mediated re-localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense-inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Cysteine Proteases/metabolism , Helminth Proteins/metabolism , Host-Parasite Interactions , Plant Diseases/parasitology , Tylenchoidea/physiology , Animals , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis/parasitology , Arabidopsis Proteins/genetics , Beta vulgaris/parasitology , Cell Nucleus/metabolism , Cell Wall/metabolism , Cysteine Proteases/genetics , Cytoplasm/metabolism , Female , Helminth Proteins/genetics , Plant Diseases/immunology , Plant Immunity , Protein Transport , Two-Hybrid System Techniques , Vacuoles/metabolism
5.
BMC Genomics ; 20(1): 119, 2019 Feb 07.
Article in English | MEDLINE | ID: mdl-30732586

ABSTRACT

BACKGROUND: Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock. RESULTS: Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123 Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7 Mb), and horizontal gene transfer events (151 genes). A large number of putative effectors (431 genes) were identified in the genome, many of which were found in transposons. CONCLUSIONS: This advance provides a glimpse into the host and parasite interplay by revealing a diversity of mechanisms that give rise to virulence genes in the soybean cyst nematode, including: tandem duplications containing over a fifth of the total gene count, virulence genes hitchhiking in transposons, and 107 horizontal gene transfers not reported in other plant parasitic nematodes thus far. Through extensive characterization of the H. glycines genome, we provide new insights into H. glycines biology and shed light onto the mystery underlying complex host-parasite interactions. This genome sequence is an important prerequisite to enable work towards generating new resistance or control measures against H. glycines.


Subject(s)
Evolution, Molecular , Gene Duplication , Genomics , Glycine max/parasitology , Tylenchoidea/genetics , Tylenchoidea/physiology , Animals , Genotype , Host-Parasite Interactions , Molecular Sequence Annotation , Plant Diseases/parasitology , Polymorphism, Single Nucleotide , Sequence Analysis, DNA
7.
J Exp Bot ; 67(8): 2339-51, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26917556

ABSTRACT

Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1.


Subject(s)
Arabidopsis/metabolism , Carbonic Anhydrases/metabolism , Chlamydomonas/enzymology , Plant Proteins/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Amino Acids/metabolism , Arabidopsis/genetics , Carbonic Anhydrases/chemistry , Disulfides/metabolism , Peptides/chemistry , Plant Proteins/chemistry , Plants, Genetically Modified , Protein Subunits/chemistry , Protein Subunits/metabolism , Sequence Deletion
8.
Mol Plant Pathol ; 17(6): 832-44, 2016 08.
Article in English | MEDLINE | ID: mdl-26575318

ABSTRACT

Cyst nematodes are plant-parasitic roundworms that are of significance in many cropping systems around the world. Cyst nematode infection is facilitated by effector proteins secreted from the nematode into the plant host. The cDNAs of the 25A01-like effector family are novel sequences that were isolated from the oesophageal gland cells of the soybean cyst nematode (Heterodera glycines). To aid functional characterization, we identified an orthologous member of this protein family (Hs25A01) from the closely related sugar beet cyst nematode H. schachtii, which infects Arabidopsis. Constitutive expression of the Hs25A01 CDS in Arabidopsis plants caused a small increase in root length, accompanied by up to a 22% increase in susceptibility to H. schachtii. A plant-expressed RNA interference (RNAi) construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii. These data document that Hs25A01 has physiological functions in planta and a role in cyst nematode parasitism. In vivo and in vitro binding assays confirmed the specific interactions of Hs25A01 with an Arabidopsis F-box-containing protein, a chalcone synthase and the translation initiation factor eIF-2 ß subunit (eIF-2bs), making these proteins probable candidates for involvement in the observed changes in plant growth and parasitism. A role of eIF-2bs in the mediation of Hs25A01 virulence function is further supported by the observation that two independent eIF-2bs Arabidopsis knock-out lines were significantly more susceptible to H. schachtii.


Subject(s)
Helminth Proteins/metabolism , Plant Diseases/parasitology , Plant Proteins/metabolism , Plant Roots/parasitology , Tylenchoidea/metabolism , Amino Acid Sequence , Animals , Arabidopsis/genetics , Arabidopsis/parasitology , Beta vulgaris , Cytoplasm/metabolism , DNA, Bacterial/genetics , Disease Susceptibility , Gene Expression Profiling , Gene Expression Regulation, Plant , Gene Silencing , Helminth Proteins/chemistry , In Situ Hybridization , Mutagenesis, Insertional/genetics , Oligonucleotide Array Sequence Analysis , Plants, Genetically Modified , Protein Binding , Reproducibility of Results , Sequence Alignment
9.
Plant Cell ; 27(3): 891-907, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25715285

ABSTRACT

Plant-parasitic cyst nematodes synthesize and secrete effector proteins that are essential for parasitism. One such protein is the 10A07 effector from the sugar beet cyst nematode, Heterodera schachtii, which is exclusively expressed in the nematode dorsal gland cell during all nematode parasitic stages. Overexpression of H. schachtii 10A07 in Arabidopsis thaliana produced a hypersusceptible phenotype in response to H. schachtii infection along with developmental changes reminiscent of auxin effects. The 10A07 protein physically associates with a plant kinase and the IAA16 transcription factor in the cytoplasm and nucleus, respectively. The interacting plant kinase (IPK) phosphorylates 10A07 at Ser-144 and Ser-231 and mediates its trafficking from the cytoplasm to the nucleus. Translocation to the nucleus is phosphorylation dependent since substitution of Ser-144 and Ser-231 by alanine resulted in exclusive cytoplasmic accumulation of 10A07. IPK and IAA16 are highly upregulated in the nematode-induced syncytium (feeding cells), and deliberate manipulations of their expression significantly alter plant susceptibility to H. schachtii in an additive fashion. An inactive variant of IPK functioned antagonistically to the wild-type IPK and caused a dominant-negative phenotype of reduced plant susceptibility. Thus, exploitation of host processes to the advantage of the parasites is one mechanism by which cyst nematodes promote parasitism of host plants.


Subject(s)
Arabidopsis/metabolism , Arabidopsis/parasitology , Cell Nucleus/metabolism , Host-Parasite Interactions , Protein Processing, Post-Translational , Protozoan Proteins/metabolism , Tylenchoidea/metabolism , Amino Acid Sequence , Animals , Arabidopsis Proteins/metabolism , Beta vulgaris/parasitology , Indoleacetic Acids/metabolism , Models, Biological , Molecular Sequence Data , Mutation/genetics , Nuclear Localization Signals , Phosphorylation , Phosphoserine/metabolism , Plant Diseases/parasitology , Protein Kinases/metabolism , Protein Transport , Up-Regulation
10.
BMC Res Notes ; 6: 255, 2013 Jul 06.
Article in English | MEDLINE | ID: mdl-23830484

ABSTRACT

BACKGROUND: Bean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed. FINDINGS: Here we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for analyzing soybean genes involved in resistance to soybean cyst nematode (SCN). A detailed SCN screening pipeline is described. CONCLUSIONS: The VIGS method described here provides a new tool to identify genes involved in soybean-nematode interactions. This method could be adapted to study genes associated with any root pathogenic or symbiotic associations.


Subject(s)
Comovirus/metabolism , Gene Silencing , Genetic Vectors , Glycine max/genetics , Glycine max/parasitology , Nematoda/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/parasitology , RNA Interference , Animals , Gene Expression Regulation, Plant , Host-Parasite Interactions/genetics , Plant Roots
11.
Nature ; 492(7428): 256-60, 2012 Dec 13.
Article in English | MEDLINE | ID: mdl-23235880

ABSTRACT

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.


Subject(s)
Glycine max/genetics , Glycine max/parasitology , Host-Parasite Interactions , Nematoda/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Amino Acid Sequence , Animals , DNA Mutational Analysis , Gene Order , Gene Silencing , Genetic Complementation Test , Glycine Hydroxymethyltransferase/genetics , Glycine Hydroxymethyltransferase/metabolism , Haplotypes , Models, Molecular , Molecular Sequence Data , Plant Proteins/chemistry , Polymorphism, Genetic/genetics , Protein Structure, Tertiary , Quantitative Trait Loci/genetics , Glycine max/enzymology
12.
Mol Plant Pathol ; 13(9): 1140-8, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22738403

ABSTRACT

Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)-based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in-depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert orientation and targeted region in the coding sequence. A 3' sequence in reverse orientation produced the strongest silencing phenotypes. Furthermore, we documented that BPMV VIGS can achieve widespread silencing in a broad range of tissues, including leaves, stems, flowers and roots. Near-complete silencing was attained in leaves and flowers. Although weaker than in shoots, the observed gene silencing in soybean roots will also allow reverse genetics studies in this tissue. When GFP fluorescence was assayed in cross-sections of stems and leaf petioles, near-complete and uniform silencing was observed in all cell types. Silencing was observed from as early as 2 weeks post-virus inoculation in leaves to 7 weeks post-virus inoculation in flowers, suggesting that this system can induce and maintain silencing for significant durations.


Subject(s)
Comovirus/physiology , Gene Silencing , Glycine max/genetics , Glycine max/virology , Flowers/metabolism , Flowers/virology , Green Fluorescent Proteins/metabolism , Plant Leaves/metabolism , Plant Leaves/virology , Time Factors , Transgenes/genetics
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