[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.
[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.
[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.
[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.
Journal of Experimental Botany 03/2012; 63(10):3683-95. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: The syncytium is a unique plant root organ whose differentiation is induced by plant-parasitic cyst nematodes to create a source of nourishment. Syncytium formation involves the redifferentiation and fusion of hundreds of root cells. The underlying regulatory networks that control this unique change of plant cell fate are not understood. Here, we report that a strong down-regulation of Arabidopsis (Arabidopsis thaliana) microRNA396 (miR396) in cells giving rise to the syncytium coincides with the initiation of the syncytial induction/formation phase and that specific miR396 up-regulation in the developed syncytium marks the beginning of the maintenance phase, when no new cells are incorporated into the syncytium. In addition, our results show that miR396 in fact has a role in the transition from one phase to the other. Expression modulations of miR396 and its Growth-Regulating Factor (GRF) target genes resulted in reduced syncytium size and arrested nematode development. Furthermore, genome-wide expression profiling revealed that the miR396-GRF regulatory system can alter the expression of 44% of the more than 7,000 genes reported to change expression in the Arabidopsis syncytium. Thus, miR396 represents a key regulator for the reprogramming of root cells. As such, this regulatory unit represents a powerful molecular target for the parasitic animal to modulate plant cells and force them into novel developmental pathways.
[show abstract][hide abstract] ABSTRACT: Cyst nematodes establish and maintain feeding sites (syncytia) in the roots of host plants by altering expression of host genes. Among these genes are members of the large gene family of class III peroxidases, which have reported functions in a variety of biological processes. In this study, we used Arabidopsis-Heterodera schachtii as a model system to functionally characterize peroxidase 53 (AtPRX53). Promoter assays showed that under non-infected conditions AtPRX53 is expressed mainly in the root, the hypocotyl and the base of the pistil. Under infected conditions, the AtPRX53 promoter showed upregulation at the nematode penetration sites and in their migration paths. Interestingly, strong GUS activity was observed in H. schachtii-induced syncytia during the early stage of infection and remained strong in the syncytia of third-stage juveniles. Also, AtPRX53 showed upregulation in response to wounding and jasmonic acid treatments. Manipulation of AtPRX53 expression through overexpression and knockout mutation affected both plant morphology and nematode susceptibility. While AtPRX53 overexpression lines exhibited short hypocotyls, aberrant flower development and reduced nematode susceptibility to H. schachtii, the atprx53 mutant showed long hypocotyls and a 3-carpel silique phenotype as well as a non significant increase of nematode susceptibility. Taken together these data, therefore, indicate diverse roles of AtPRX53 in the wound response, flower development and syncytium formation.
[show abstract][hide abstract] ABSTRACT: Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.
[show abstract][hide abstract] ABSTRACT: Successful cyst nematode parasitism depends on the formation and maintenance of feeding sites (syncytia) in host roots, and these processes are highly regulated by the interaction between the cyst nematode and the host. Using an integrated research approach and the Arabidopsis-Beta vulgaris (sugar beet) cyst nematode (Heterodera schachtii) pathosystem, we have determined that the two Arabidopsis basic helix-loop-helix transcription factors bHLH25 and bHLH27 positively influence cyst nematode parasitism. Promoter studies indicated that as early as 1 day post-inoculation, both transcription factor genes were upregulated in developing syncytia, whereas in non-infected plants, these two promoters were not found to be active in the same cells. By using yeast two-hybrid analyses and bimolecular fluorescence complementation assays, we documented that the two bHLH transcription factors can dimerize in planta. Transgenic Arabidopsis plants overexpressing either one or both of the bHLH genes exhibited altered morphology of roots and shoots, as well as an increased susceptibility to H. schachtii. bhlh25 or bhlh27 single mutants were without strong phenotypes, presumably because of functional redundancies in this gene family. However, the bhlh25 bhlh27 double mutant was less susceptible to H. schachtii, confirming an important conducive role of the co-expression of both transcription factor genes for cyst nematode parasitism. Our results document an example of pathogen-induced ectopic co-expression of two regulatory genes to enhance pathogen success, although these transcription factors apparently do not function in concert in non-infected plants. This is an intriguing biological phenomenon that highlights the complexity of obligate biotrophic plant-pathogen interactions, like those of cyst nematodes.
The Plant Journal 01/2011; 65(2):319-28. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: The soybean cyst nematode (Heterodera glycines) and the closely related sugar beet cyst nematode (Heterodera schachtii) are devastating pathogens of plant roots that use secreted effector proteins to engage in sophisticated host-parasite interactions. While H. schachtii infects and reproduces readily on the roots of Arabidopsis thaliana, H. glycines rarely is able to infect this model plant. The molecular basis for differing host ranges remains obscure but likely involves differences between nematode effector proteins and the recognition of host factors. Recently we reported that constitutive expression of the H. schachtii 10A06 effector protein gene (Hs-10A06) in Arabidopsis affected plant morphology and increased susceptibility to H. schachtii and that the 10A06 protein functions through its interaction with Arabidopsis spermidine synthase 2 (SPDS2). Therefore, we investigated whether differences between cyst nematode effector protein orthologs in two nematode species have a role in mediating host specificity. Here, we show that, similar to Hs-10A06, ectopic expression of H. glycines 10A06 (Hg-10A06) in Arabidopsis affected leaf number and root length, however, to a much lesser extent. More importantly, no effect of Hg-10A06 overexpression on Arabidopsis susceptibility to H. schachtii was observed. While we found that Hg-10A06 can weakly interact with Arabidopsis SPDS2 in yeast-two hybrid assays, this ability to interact with SPDS2 was decreased approximately 5-fold compared with Hs-10A06. Collectively, these data suggest that sequence divergence between cyst nematode effector protein orthologs could contribute in determining cyst nematode host range.
[show abstract][hide abstract] ABSTRACT: Cyst nematodes are sedentary plant parasites that cause dramatic cellular changes in the plant root to form feeding cells, so-called syncytia. 10A06 is a cyst nematode secretory protein that is most likely secreted as an effector into the developing syncytia during early plant parasitism. A homolog of the uncharacterized soybean cyst nematode (Heterodera glycines), 10A06 gene was cloned from the sugar beet cyst nematode (Heterodera schachtii), which is able to infect Arabidopsis (Arabidopsis thaliana). Constitutive expression of 10A06 in Arabidopsis affected plant morphology and increased susceptibility to H. schachtii as well as to other plant pathogens. Using yeast two-hybrid assays, we identified Spermidine Synthase2 (SPDS2), a key enzyme involved in polyamine biosynthesis, as a specific 10A06 interactor. In support of this protein-protein interaction, transgenic plants expressing 10A06 exhibited elevated SPDS2 mRNA abundance, significantly higher spermidine content, and increased polyamine oxidase (PAO) activity. Furthermore, the SPDS2 promoter was strongly activated in the nematode-induced syncytia, and transgenic plants overexpressing SPDS2 showed enhanced plant susceptibility to H. schachtii. In addition, in planta expression of 10A06 or SPDS2 increased mRNA abundance of a set of antioxidant genes upon nematode infection. These data lend strong support to a model in which the cyst nematode effector 10A06 exerts its function through the interaction with SPDS2, thereby increasing spermidine content and subsequently PAO activity. Increasing PAO activity results in stimulating the induction of the cellular antioxidant machinery in syncytia. Furthermore, we observed an apparent disruption of salicylic acid defense signaling as a function of 10A06. Most likely, increased antioxidant protection and interruption of salicylic acid signaling are key aspects of 10A06 function in addition to other physiological and morphological changes caused by altered polyamines, which are potent plant signaling molecules.
[show abstract][hide abstract] ABSTRACT: Nematode parasitism genes encode secreted effector proteins that play a role in host infection. A homologue of the expressed Hg4F01 gene of the root-parasitic soybean cyst nematode, Heterodera glycines, encoding an annexin-like effector, was isolated in the related Heterodera schachtii to facilitate use of Arabidopsis thaliana as a model host. Hs4F01 and its protein product were exclusively expressed within the dorsal oesophageal gland secretory cell in the parasitic stages of H. schachtii. Hs4F01 had a 41% predicted amino acid sequence identity to the nex-1 annexin of C. elegans and 33% identity to annexin-1 (annAt1) of Arabidopsis, it contained four conserved domains typical of the annexin family of calcium and phospholipid binding proteins, and it had a predicted signal peptide for secretion that was present in nematode annexins of only Heterodera spp. Constitutive expression of Hs4F01 in wild-type Arabidopsis promoted hyper-susceptibility to H. schachtii infection. Complementation of an AnnAt1 mutant by constitutive expression of Hs4F01 reverted mutant sensitivity to 75 mM NaCl, suggesting a similar function of the Hs4F01 annexin-like effector in the stress response by plant cells. Yeast two-hybrid assays confirmed a specific interaction between Hs4F01 and an Arabidopsis oxidoreductase member of the 2OG-Fe(II) oxygenase family, a type of plant enzyme demonstrated to promote susceptibility to oomycete pathogens. RNA interference assays that expressed double-stranded RNA complementary to Hs4F01 in transgenic Arabidopsis specifically decreased parasitic nematode Hs4F01 transcript levels and significantly reduced nematode infection levels. The combined data suggest that nematode secretion of an Hs4F01 annexin-like effector into host root cells may mimic plant annexin function during the parasitic interaction.
Journal of Experimental Botany 11/2009; 61(1):235-48. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: The study deals with the quantitative expression pattern of genes involved in signaling transduction pathways in response to water stress in leaves and embryos of a water stress tolerant genotype compared to a non-tolerant genotype using real-time quantitative PCR. The experiment was conducted in the field. The results showed a high quantitative up-regulation of genes belonging to protein kinase, phosphatase and transcription factor pathways (from two to 70 fold) only in leaves of the tolerant genotype compared to the non-tolerant genotype. Moreover, genes related to the protein kinase pathway were down-regulated in leaves of the non-tolerant genotype. On the contrary, in seeds, our study showed that the positive regulation of genes related to the signal transduction pathway observed in leaves of the tolerant genotype is turned off, suggesting different transcriptional control of signaling water stress in reproductive organs compared to vegetative organs.
[show abstract][hide abstract] ABSTRACT: Plant-parasitic cyst nematodes induce the formation of specialized feeding cells in infected roots, which involves plant developmental processes that have been shown to be influenced by microRNAs (miRNAs) and other small RNAs. This observation provided the foundation to investigate the potential involvement of small RNAs in plant-cyst nematode interactions. First, we examined the susceptibilities of Arabidopsis DICER-like (dcl) and RNA-dependent RNA polymerase (rdr) mutants to the sugar beet cyst nematode Heterodera schachtii. The examined mutants exhibited a trend of decreased susceptibility, suggesting a role of small RNAs mediating gene regulation processes during the plant-nematode interaction. Second, we generated two small RNA libraries from aseptic Arabidopsis roots harvested at 4 and 7 days after infection with surface-sterilized H. schachtii. Sequences of known miRNAs as well as novel small interfering (si)RNAs were identified. Following this discovery, we used real-time reverse-transcriptase polymerase chain reaction to quantify a total of 15 Arabidopsis transcripts that are known targets of six of the different miRNA families found in our study (miR160, miR164, miR167, miR171, miR396, and miR398) in inoculated and noninoculated Arabidopsis roots. Our analyses showed mostly negative correlations between miRNA accumulation and target gene mRNA abundance, suggesting regulatory roles of these miRNAs during parasitism. Also, we identified a total of 125 non-miRNA siRNAs. Some of these siRNAs perfectly complement protein-coding mRNAs or match transposon or retrotransposon sequences in sense or antisense orientations. We further quantified a group of siRNAs in H. schachtii-inoculated roots. The examined siRNAs exhibited distinct expression patterns in infected and noninfected roots, providing additional evidence for the implication of small RNAs in cyst nematode parasitism. These data lay the foundation for detailed analyses of the functions of small RNAs during phytonematode parasitism.
[show abstract][hide abstract] ABSTRACT: Plant-parasitic cyst nematodes secrete a complex of cell wall-digesting enzymes, which aid in root penetration and migration. The soybean cyst nematode Heterodera glycines also produces a cellulose binding protein (Hg CBP) secretory protein. To determine the function of CBP, an orthologous cDNA clone (Hs CBP) was isolated from the sugar beet cyst nematode Heterodera schachtii, which is able to infect Arabidopsis thaliana. CBP is expressed only in the early phases of feeding cell formation and not during the migratory phase. Transgenic Arabidopsis expressing Hs CBP developed longer roots and exhibited enhanced susceptibility to H. schachtii. A yeast two-hybrid screen identified Arabidopsis pectin methylesterase protein 3 (PME3) as strongly and specifically interacting with Hs CBP. Transgenic plants overexpressing PME3 also produced longer roots and exhibited increased susceptibility to H. schachtii, while a pme3 knockout mutant showed opposite phenotypes. Moreover, CBP overexpression increases PME3 activity in planta. Localization studies support the mode of action of PME3 as a cell wall-modifying enzyme. Expression of CBP in the pme3 knockout mutant revealed that PME3 is required but not the sole mechanism for CBP overexpression phenotype. These data indicate that CBP directly interacts with PME3 thereby activating and potentially targeting this enzyme to aid cyst nematode parasitism.
The Plant Cell 12/2008; 20(11):3080-93. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although high light (HL) and high temperature (HT) stresses have been extensively investigated, a global analysis of their combined effects on the transcriptome of any plant species has not yet been described. Sunflower is an agronomically important oil crop frequently subjected to these stress factors. Because results in model plants may not always translate well to crop plants, responses of sunflower (Helianthus annuus) to HL, HT and a combination of both stresses were analysed by profiling gene expression in leaves and immature seeds.
Plants were grown in HL (600 microE m(-2) s(-1)), HT (35 degrees C) and a combination of HL and HT (HL + HT), and gene expression in leaves and immature seeds was profiled using cDNA microarrays containing more than 8000 putative unigenes.
Using two-way analysis of variance, 105, 55 and 129 cDNA clones were identified showing significant changes in steady-state transcript levels, across the two tissues, in response to HL, HT and HL + HT, respectively. A significant number of these transcripts were found to be specific to each stress. Comparing gene expression profiles between leaves and immature seeds revealed that 89, 113 and 186 cDNA clones can be considered as differentially expressed in response to HL, HT and HL + HT, respectively. More than half of the cDNA clones showing significant differences between embryo and leaf tissues in response to HL + HT were specific to this stress. Significant differences between leaves and seeds shared by all three stress treatments were observed for only eight genes.
Taken together, these results indicate that vegetative and reproductive tissues employ different transcriptome responses to these stress treatments. Careful examination of the putative functions of these genes revealed novel and specific responses. The potential roles of many of the differentially expressed genes in stress tolerance are mentioned and discussed.
Annals of Botany 08/2008; 102(1):127-40. · 3.45 Impact Factor