[Show abstract][Hide abstract] 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 esophageal 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 22% increase in susceptibility to H. schachtii. A plant-expressed 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 as well as in vitro binding assays confirmed specific interactions of Hs25A01 with an Arabidopsis F-box containing protein, a chalcone synthase, and the translation initiation factor eIF-2 beta subunit (eIF-2bs), making these proteins likely candidates for being involved in the observed changes in plant growth and parasitism. A role of eIF-2bs in mediating the 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. This article is protected by copyright. All rights reserved.
Full-text · Article · Nov 2015 · Molecular Plant Pathology
[Show abstract][Hide abstract] ABSTRACT: Plant-parasitic cyst nematodes induce the formation of hypermetabolic feeding sites, termed syncytia, as their sole source of nutrients. The formation of the syncytium is orchestrated by the nematode in part by modulation of phytohormone responses, including cytokinin. In response to infection by the nematode Heterodera schachtii, cytokinin signaling is transiently induced at the site of infection and in the developing syncytium. Arabidopsis lines with reduced cytokinin sensitivity show reduced susceptibility to nematode infection, indicating that cytokinin signaling is required for optimal nematode development. Furthermore, lines with increased cytokinin sensitivity also exhibit reduced nematode susceptibility. To ascertain why cytokinin hypersensitivity reduces nematode parasitism, we examined the transcriptomes in wild-type and a cytokinin-hypersensitive type-A arr Arabidopsis mutant in response to H. schachtii infection. Genes involved in the response to biotic stress and defense response were elevated in the type-A arr mutant in the absence of nematodes and were hyper-induced following H. schachtii infection, which suggests that the Arabidopsis type-A arr mutants impede nematode development because they are primed to respond to pathogen infection. These results suggest that cytokinin signaling is required for optimal H. schachtii parasitism of Arabidopsis, but that elevated cytokinin signaling triggers a heightened immune response to nematode infection.
Full-text · Article · Oct 2015 · Molecular Plant-Microbe Interactions
[Show abstract][Hide abstract] ABSTRACT: Heterodera glycines, the soybean cyst nematode, is the number one pathogen of soybean (Glycine max). This nematode infects soybean roots and forms an elaborate feeding site in the vascular cylinder. H. glycines produces an arsenal of effector proteins in the secretory esophageal gland cells. More than sixty H. glycines candidate effectors were identified in previous gland cell-mining projects. However, it is likely that additional candidate effectors remained unidentified. With the goal of identifying remaining H. glycines candidate effectors, we constructed and sequenced a large gland cell cDNA library resulting in 11,814 ESTs. After bioinformatic filtering for candidate effectors using a number of criteria, in situ hybridizations were performed in H. glycines whole mount specimens to identify candidate effectors whose mRNA exclusively accumulated in the esophageal gland cells, which is a hallmark of many nematode effectors. This approach resulted in the identification of eighteen new H. glycines esophageal gland cell-specific candidate effectors. Of these candidate effectors, eleven sequences were pioneers without similarities to known proteins while seven sequences had similarities to functionally annotated proteins in databases. These putative homologies provided the bases for the development of hypotheses about potential functions in the parasitism process.
[Show abstract][Hide abstract] ABSTRACT: Sedentary plant-parasitic nematodes induce the formation of elaborate feeding sites in their host plants. The cell-specific gene expression analysis of nematode feeding sites yielded valuable information about the transcriptional regulation and gene regulatory networks associated with compatible and incompatible interactions of sedentary plant-parasitic nematodes and their host plants. Host endogenous small RNAs and RNA silencing pathways appear to play key roles in regulating gene expression changes and reprogramming infected root cells into specific syncytial or giant cell types. We highlight the wide gene silencing events that are associated with the initiation and formation of the nematode feeding sites. Also, we emphasize different types of transcriptional and posttranscriptional small RNA-mediated gene silencing in the feeding sites, highlighting their regulations and mode of actions. Finally, the implementation of two artificial RNA interference systems, namely host-induced gene silencing and virus-induced gene silencing as powerful tools for functional genomics of plant-nematode interactions is discussed.
No preview · Article · Mar 2015 · Advances in Botanical Research
[Show abstract][Hide abstract] ABSTRACT: The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs.
Full-text · Article · Dec 2014 · Frontiers in Plant Science
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Sedentary plant-parasitic nematodes engage in complex interactions with their host plants by secreting effector proteins. Some effectors of both root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera and Globodera spp.) mimic plant ligand proteins. Most prominently, cyst nematodes secrete effectors that mimic plant CLAVATA3/ESR-related (CLE) ligand proteins. However, only cyst nematodes have been shown to secrete such effectors and to utilize CLE ligand mimicry in their interactions with host plants. Here, we document the presence of ligand-like motifs in bona fide root-knot nematode effectors that are most similar to CLE peptides from plants and cyst nematodes. We have identified multiple tandem CLE-like motifs conserved within the previously identified Meloidogyne avirulence protein (MAP) family that are secreted from root-knot nematodes and have been shown to function in planta. By searching all 12 MAP family members from multiple Meloidogyne spp., we identified 43 repetitive CLE-like motifs composing 14 unique variants. At least one CLE-like motif was conserved in each MAP family member. Furthermore, we documented the presence of other conserved sequences that resemble the variable domains described in Heterodera and Globodera CLE effectors. These findings document that root-knot nematodes appear to use CLE ligand mimicry and point toward a common host node targeted by two evolutionarily diverse groups of nematodes. As a consequence, it is likely that CLE signaling pathways are important in other phytonematode pathosystems as well.
[Show abstract][Hide abstract] ABSTRACT: Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins, which M. incognita secretes into its host plants during infection, is an important step towards finding new ways to manage this pest. In this study we have identified the cDNAs for 18 putative effectors, i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants. These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that in the plant cells, these putative effectors are likely to localize to cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically up regulated during different stages of the nematode's life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of Meloidogyne hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed, and reproduce on their host plants. Future studies investigating the roles these proteins play in planta will help mitigate the effects of this damaging pest.
Full-text · Article · May 2014 · Molecular Plant-Microbe Interactions
[Show abstract][Hide abstract] ABSTRACT: Growth regulating factors (GRFs) are a conserved class of transcription factor in seed plants. GRFs are involved in various aspects of tissue differentiation and organ development. The implication of GRFs in biotic stress response has also been recently reported, suggesting a role of these transcription factors in coordinating the interaction between developmental processes and defense dynamics. However, the molecular mechanisms by which GRFs mediate the overlaps between defense signaling and developmental pathways are elusive. Here, we report large scale identification of putative target candidates of Arabidopsis GRF1 and GRF3 by comparing mRNA profiles of the grf1/grf2/grf3 triple mutant and those of the transgenic plants overexpressing miR396-resistant version of GRF1 or GRF3. We identified 1,098 and 600 genes as putative targets of GRF1 and GRF3, respectively. Functional classification of the potential target candidates revealed that GRF1 and GRF3 contribute to the regulation of various biological processes associated with defense response and disease resistance. GRF1 and GRF3 participate specifically in the regulation of defense-related transcription factors, cell-wall modifications, cytokinin biosynthesis and signaling, and secondary metabolites accumulation. GRF1 and GRF3 seem to fine-tune the crosstalk between miRNA signaling networks by regulating the expression of several miRNA target genes. In addition, our data suggest that GRF1 and GRF3 may function as negative regulators of gene expression through their association with other transcription factors. Collectively, our data provide new insights into how GRF1 and GRF3 might coordinate the interactions between defense signaling and plant growth and developmental pathways.
[Show abstract][Hide abstract] ABSTRACT: Plant-parasitic cyst nematodes induce the formation of multinucleated feeding site in the infected roots, termed syncytium. Recent studies pointed to key roles of the phytohormone auxin in the regulation of gene expression and establishment of the syncytium. Nevertheless, information about the spatiotemporal expression patterns of the transcription factors that mediate auxin transcriptional responses during syncytium formation is limited. Here, we provide a gene expression map of 22 auxin response factors (ARFs) during initiation, formation and maintenance stages of the syncytium induced by the cyst nematode Heterodera schachtii in Arabidopsis. We observed distinct and overlapping expression patterns of ARFs throughout syncytium development phases. We identified a set of ARFs whose expression is predominantly located inside the developing syncytium, while others are expressed in the neighboring cells, presumably to initiate specific transcriptional programs required for their incorporation with the developing syncytium. Our analyses also point to a role of certain ARFs in determining the maximum size of the syncytium. In addition, several ARFs were found to be highly expressed in fully-developed syncytia, suggesting a role in maintaining the functional phenotype of mature syncytia. The dynamic distribution and overlapping expression patterns of various ARFs seem to be essential characteristics of ARF activity during syncytium development.
No preview · Article · Jan 2014 · Molecular Plant Pathology
[Show abstract][Hide abstract] ABSTRACT: Nematode effector proteins originating from the esophageal gland cells play central roles in suppressing plant defenses and in the formation of the plant feeding cells required for growth and development of cyst nematodes. A gene (GrUBCEP12) encoding a unique ubiquitin carboxyl extension protein (UBCEP) that consists of a signal peptide (SP) for secretion, a monoubiquitin domain, and a 12-amino acid carboxyl extension protein (CEP12) domain was cloned from the potato cyst nematode Globodera rostochiensis. This GrUBCEP12 gene was expressed exclusively within the nematode's dorsal esophageal gland cell and was upregulated in the parasitic second-stage juvenile correlating to the time when feeding cell formation is initiated. We showed that specific GrUBCEP12 knockdown via RNA interference reduced nematode parasitic success and that overexpression of the secreted Gr(ΔSP) UBCEP12 protein in potato resulted in increased nematode susceptibility, providing direct evidence that this secreted effector is involved in plant parasitism. Using transient expression assays in Nicotiana benthamiana, we discovered that Gr(ΔSP) UBCEP12 was processed into free ubiquitin and a CEP12 peptide (GrCEP12) in planta and that GrCEP12 could suppress resistance gene-mediated cell death. A target search showed that RPN2a, a gene encoding a subunit of the 26S proteasome was dramatically suppressed in Gr(ΔSP) UBCEP12 but not GrCEP12 overexpression plants when compared with control plants. Together, these results suggest that when delivered into host plant cells, Gr(ΔSP) UBCEP12 becomes two functional units, one acting to suppress plant immunity and the other potentially affecting the host 26S proteasome, to promote feeding cell formation. Published 2013. This article is a US Government work and is in the public domain in the USA.
Full-text · Article · Jan 2013 · The Plant Journal
[Show abstract][Hide abstract] ABSTRACT: Introduction Root Infection and Feeding Cell Ontogenesis Genome-Wide Analysis of the Plant Response to Infection The Plant Cytoskeleton Is Targeted by Root Pathogens Root Pathogens Hijack Cell Cycle Regulators Severe Cell Wall Remodeling Is Associated with Feeding Site Formation Phytohormones Regulating Development and Defense May Control Feeding Site Formation Role of miRNAs in Feeding Site Formation and Function Nematode Effectors That Alter Root Cell Development during Parasitism Conclusion Acknowledgments References
[Show abstract][Hide abstract] ABSTRACT: Esophageal glands of plant-parasitic nematodes are highly specialized cells whose gene expression products include secreted effector proteins, which govern nematode parasitism of host plants. Elucidating the transcriptomes of esophageal glands with the goal of identifying nematode effectors, therefore, is a promising avenue to understanding nematode parasitism and its evolutionary origins as well as to devising nematode control strategies. We have developed a method to separate and isolate individual esophageal gland cells from multiple species of plant-parasitic nematodes while preserving RNA quality. We have used such isolated gland cells for transcriptome analysis via high throughput DNA sequencing. This method relies on the differential histochemical staining of the gland cells after homogenization of phytonematode tissues. Total RNA was extracted from whole gland cells isolated from eight different plant-parasitic nematode species. To validate this approach, the isolated RNA from three plant-parasitic nematode species, Globodera rostochiensis, Pratylenchus penetrans and Radopholus similis, was amplified, gel purified and used for 454 sequencing. We obtained 456,801 total reads with an average read length of 409 bases. Sequence analyses revealed the presence of homologs of previously known nematode effectors in these libraries thus validating our approach. These data provide compelling evidence that this technical advance can be used to relatively easily and expediently discover effector repertoires of plant-parasitic nematodes.
No preview · Article · Aug 2012 · Molecular Plant-Microbe Interactions
[Show abstract][Hide abstract] ABSTRACT: A key feature of sedentary plant-parasitic nematodes is the release of effector proteins from their esophageal gland cells through their stylets into host roots. These proteinaceous stylet secretions have been shown to be crucial for successful parasitism by mediating the transition of normal root cells into specialized feeding sites and by negating plant defenses. Recent technical advances of purifying mRNA from esophageal gland cells of plant-parasitic nematodes coupled with emerging sequencing technologies is steadily expanding our knowledge of nematode effector repertoires. Host targets and biological activities of a number of nematode effectors are continuously being reported, and by now a first picture of the complexity of sedentary nematode parasitism at the molecular level is starting to take shape. In this review, we highlight effector mechanisms that recently have been uncovered by studying the host-pathogen interactome. These mechanisms range from mediating susceptibility of host plants to the actual triggering of defense responses. In particular, we portray and discuss the mechanisms by which nematode effectors modify plant cell walls, negate host defense responses, alter auxin and polyamine signaling, mimic plant molecules, regulate stress signaling and activate hypersensitive responses. Continuous molecular characterization of newly discovered nematode effectors will be needed to determine how these effectors orchestrate host signaling pathways and biological processes leading to successful parasitism.
[Show abstract][Hide abstract] ABSTRACT: The beet cyst nematode, Heterodera schachtii, is a sedentary root parasite that induces the formation of a specialized root feeding structure, the syncytium. We previously have shown that coordinated regulation of miR396 and its target genes GRF1 and GRF3 in the syncytium is required for proper formation. To gain a better understanding of this coordinated regulation, we used quantitative real-time PCR to assess the abundance of primary (pri)-miRNA396a, pri-miRNA396b and mature miRNA396 in transgenic Arabidopsis plants overexpressing either wild-type variants of the GRF1 or GRF3 coding sequences or miR396-resistant variants. We also included a grf1/grf2/grf3 triple mutant in these analyses. We observed significant decreases in the abundance of pri-miRNA396a, pri-miRNA396b and mature miR396 in the transgenic plants overexpressing GRF1 or GRF3, particularly with the miRNA396-resistant variants. In contrast, the primary transcripts and mature miRNA396 abundance were significantly increased in the grf1/grf2/grf3 triple knockout mutant. These results demonstrate that homeostasis between miR396 and the target genes GRF1 and GRF3 is established through reciprocal feedback regulation, in which GRF1/GRF3 and miR396 negatively regulate each other's expression. In addition, we found that constitutive expression of GRF1 or GRF3 decreases the mRNA abundance of other GRFs, even those that are not targeted by miR396, as well as their own endogenous transcripts, which documents further regulatory facets of this equilibrium.
[Show abstract][Hide abstract] 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.
No preview · Article · Jun 2012 · Molecular Plant Pathology
[Show abstract][Hide abstract] ABSTRACT: Phytoparasitic nematodes secrete an array of effector proteins to modify selected recipient plant cells into elaborate and
essential feeding sites. The biological function of the novel 30C02 effector protein of the soybean cyst nematode, Heterodera glycines, was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachtii, which contains a homologue of the 30C02 gene. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but increased plant susceptibility to infection by H. schachtii. The 30C02 protein interacted with a specific (AT4G16260) host plant β-1,3-endoglucanase in both yeast and plant cells, possibly
to interfere with its role as a plant pathogenesis-related protein. Interestingly, the peak expression of 30C02 in the nematode and peak expression of At4g16260 in plant roots coincided at around 3–5 d after root infection by the nematode, after which the relative expression of At4g16260 declined significantly. An Arabidopsis At4g16260 T-DNA mutant showed increased susceptibility to cyst nematode infection, and plants that overexpressed At4g16260 were reduced in nematode susceptibility, suggesting a potential role of host β-1,3-endoglucanase in the defence response
against H. schachtii infection. Arabidopsis plants that expressed dsRNA and its processed small interfering RNA complementary to the Hg30C02 sequence were not phenotypically different from non-transformed plants, but they exhibited a strong RNA interference-mediated
resistance to infection by H. schachtii. The collective results suggest that, as with other pathogens, active suppression of host defence is a critical component
for successful parasitism by nematodes and a vulnerable target to disrupt the parasitic cycle.
Preview · Article · Mar 2012 · Journal of Experimental Botany