Discovery of Novel Effector Protein Candidates Produced in the Dorsal Gland of Adult Female Root-Knot Nematodes.

Root-knot nematodes (RKN) (Meloidogyne spp.) represent one of the most damaging groups of plant-parasitic nematodes. They secrete effector proteins through a protrusible stylet to manipulate host cells for their benefit. Stylet-secreted effector proteins are produced within specialized secretory esophageal gland cells, one dorsal gland (DG) and two subventral glands (SvG), whose activity differ throughout the nematode life cycle. Previous gland transcriptomic profiling studies identified dozens of candidate RKN effectors but were focused on the juvenile stages of the nematode, when the SvGs are most active. We developed a new approach to enrich for the active DGs of M. incognita adult female RKN for RNA and protein extraction. Female heads were manually cut from the body, and a combination of sonication and vortexing was used to dislodge contents inside the heads. DG-enriched fractions were collected by filtering, using cell strainers. Comparative transcriptome profiling of pre-parasitic second-stage juveniles, female heads, and DG-enriched samples was conducted using RNA sequencing. Application of an established effector mining pipeline led to the identification of 83 candidate effector genes upregulated in DG-enriched samples of adult females that code for proteins with a predicted signal peptide but lack transmembrane domains or homology to proteins in the free-living nematode Caenorhabditis elegans. In situ hybridization resulted in the identification of 14 new DG-specific candidate effectors expressed in adult females. Taken together, we have identified novel candidate Meloidogyne effector genes that may have essential roles during later stages of parasitism. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phytonematode peptide effectors exploit a host post-translational trafficking mechanism to the ER using a novel translocation signal.

Cyst nematodes induce a multicellular feeding site within roots called a syncytium. It remains unknown how root cells are primed for incorporation into the developing syncytium. Furthermore, it is unclear how CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide effectors secreted into the cytoplasm of the initial feeding cell could have an effect on plant cells so distant from where the nematode is feeding as the syncytium expands. Here we describe a novel translocation signal within nematode CLE effectors that is recognized by plant cell secretory machinery to redirect these peptides from the cytoplasm to the apoplast of plant cells. We show that the translocation signal is functionally conserved across CLE effectors identified in nematode species spanning three genera and multiple plant species, operative across plant cell types, and can traffic other unrelated small peptides from the cytoplasm to the apoplast of host cells via a previously unknown post-translational mechanism of endoplasmic reticulum (ER) translocation. Our results uncover a mechanism of effector trafficking that is unprecedented in any plant pathogen to date, andthey illustrate how phytonematodes can deliver effector proteins into host cells and then hijack plant cellular processes for their export back out of the cell to function as external signaling molecules to distant cells.

The cyst nematode effector protein 10A07 targets and recruits host posttranslational machinery to mediate its nuclear trafficking and to promote parasitism in Arabidopsis.

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.

Eighteen New Candidate Effectors of the Phytonematode Heterodera glycines Produced Specifically in the Secretory Esophageal Gland Cells During Parasitism.

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 60 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 expressed sequence tags. 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 18 new H. glycines esophageal gland-cell-specific candidate effectors. Of these candidate effectors, 11 sequences were pioneers without similarities to known proteins while 7 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.

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita.

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 toward 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 the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated 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 M. 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 on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest.

Members of the Meloidogyne avirulence protein family contain multiple plant ligand-like motifs.

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.

Nematode effector proteins: an emerging paradigm of parasitism.

Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda.

The 8D05 parasitism gene of Meloidogyne incognita is required for successful infection of host roots.

Parasitism genes encode effector proteins that are secreted through the stylet of root-knot nematodes to dramatically modify selected plant cells into giant-cells for feeding. The Mi8D05 parasitism gene previously identified was confirmed to encode a novel protein of 382 amino acids that had only one database homolog identified on contig 2374 within the Meloidogyne hapla genome. Mi8D05 expression peaked in M. incognita parasitic second-stage juveniles within host roots and its encoded protein was limited to the subventral esophageal gland cells that produce proteins secreted from the stylet. Constitutive expression of Mi8D05 in transformed Arabidopsis thaliana plants induced accelerated shoot growth and early flowering but had no visible effects on root growth. Independent lines of transgenic Arabidopsis that expressed a double-stranded RNA complementary to Mi8D05 in host-derived RNA interference (RNAi) tests had up to 90% reduction in infection by M. incognita compared with wild-type control plants, suggesting that Mi8D05 plays a critical role in parasitism by the root-knot nematode. Yeast two-hybrid experiments confirmed the specific interaction of the Mi8D05 protein with plant aquaporin tonoplast intrinsic protein 2 (TIP2) and provided evidence that the Mi8D05 effector may help regulate solute and water transport within giant-cells to promote the parasitic interaction.

Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE.

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ESR (CLE)-like effector proteins. These proteins have been shown to act as ligand mimics of plant CLE peptides and are required for successful nematode infection; however, the receptors for nematode CLE-like peptides have not been identified. Here we demonstrate that CLV2 and CORYNE (CRN), members of the receptor kinase family, are required for nematode CLE signaling. Exogenous peptide assays and overexpression of nematode CLEs in Arabidopsis demonstrated that CLV2 and CRN are required for perception of nematode CLEs. In addition, promoter-reporter assays showed that both receptors are expressed in nematode-induced syncytia. Lastly, infection assays with receptor mutants revealed a decrease in both nematode infection and syncytium size. Taken together, our results indicate that perception of nematode CLEs by CLV2 and CRN is not only required for successful nematode infection but is also involved in the formation and/or maintenance of nematode-induced syncytia.

The interaction of the novel 30C02 cyst nematode effector protein with a plant ß-1,3-endoglucanase may suppress host defence to promote parasitism.

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 ß-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 ß-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.

A novel sugar beet cyst nematode effector 2D01 targets the Arabidopsis HAESA receptor-like kinase.

Plant-parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185-amino-acid secreted protein, was previously shown to be up-regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana-H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface-associated leucine-rich repeat (LRR) receptor-like kinase (RLK) involved in signalling the activation of cell wall-remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA-LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01-AtHAE interaction may play an important role in sugar beet cyst nematode parasitism.

Arabidopsis spermidine synthase is targeted by an effector protein of the cyst nematode Heterodera schachtii.

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.

The plant apoplasm is an important recipient compartment for nematode secreted proteins.

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode.

Parasitism proteins in nematode-plant interactions.

The current battery of candidate parasitism proteins secreted by nematodes to modify plant tissues for parasitism includes cell-wall-modifying enzymes of potential prokaryotic origin, multiple regulators of host cell cycle and metabolism, proteins that can localize to the plant cell nucleus, potential suppressors of host defense, mimics of plant molecules, and a relatively large cadre of predicted novel nematode parasitism proteins. Phenotypic effects of expressing nematode parasitism proteins in transformed plant tissues, protein-protein interaction assays, and RNA-mediated interference (RNAi) analyses are currently providing exciting evidence of the biological role of candidate nematode secreted parasitism proteins and identifying potential novel means of developing transgenic resistance to nematodes in crops.

A nematode effector protein similar to annexins in host plants.

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.

Targeted suppression of soybean BAG6-induced cell death in yeast by soybean cyst nematode effectors.

While numerous effectors that suppress plant immunity have been identified from bacteria, fungi, and oomycete pathogens, relatively little is known for nematode effectors. Several dozen effectors have been reported from the soybean cyst nematode (SCN). Previous studies suggest that a hypersensitive response-like programmed cell death is triggered at nematode feeding sites in soybean during an incompatible interaction. However, virulent SCN populations overcome this incompatibility using unknown mechanisms. A soybean BAG6 (Bcl-2 associated anthanogene 6) gene previously reported by us to be highly up-regulated in degenerating feeding sites induced by SCN in a resistant soybean line was attenuated in response to a virulent SCN population. We show that GmBAG6-1 induces cell death in yeast like its Arabidopsis homolog AtBAG6 and also in soybean. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean defence responses during infection. Thus, we used a yeast viability assay to screen SCN effector candidates for their ability to specifically suppress GmBAG6-1-induced cell death. We identified several effectors that strongly suppressed cell death mediated by GmBAG6-1. Two effectors identified as suppressors showed direct interaction with GmBAG6-1 in yeast, suggesting that one mechanism of cell death suppression may occur through an interaction with this host protein.

Sequence mining and transcript profiling to explore cyst nematode parasitism.

BACKGROUND: Cyst nematodes are devastating plant parasites that become sedentary within plant roots and induce the transformation of normal plant cells into elaborate feeding cells with the help of secreted effectors, the parasitism proteins. These proteins are the translation products of parasitism genes and are secreted molecular tools that allow cyst nematodes to infect plants. RESULTS: We present here the expression patterns of all previously described parasitism genes of the soybean cyst nematode, Heterodera glycines, in all major life stages except the adult male. These insights were gained by analyzing our gene expression dataset from experiments using the Affymetrix Soybean Genome Array GeneChip, which contains probeset sequences for 6,860 genes derived from preparasitic and parasitic H. glycines life stages. Targeting the identification of additional H. glycines parasitism-associated genes, we isolated 633 genes encoding secretory proteins using algorithms to predict secretory signal peptides. Furthermore, because some of the known H. glycines parasitism proteins have strongest similarity to proteins of plants and microbes, we searched for predicted protein sequences that showed their highest similarities to plant or microbial proteins and identified 156 H. glycines genes, some of which also contained a signal peptide. Analyses of the expression profiles of these genes allowed the formulation of hypotheses about potential roles in parasitism. This is the first study combining sequence analyses of a substantial EST dataset with microarray expression data of all major life stages (except adult males) for the identification and characterization of putative parasitism-associated proteins in any parasitic nematode. CONCLUSION: We have established an expression atlas for all known H. glycines parasitism genes. Furthermore, in an effort to identify additional H. glycines genes with putative functions in parasitism, we have reduced the currently known 6,860 H. glycines genes to a pool of 788 most promising candidate genes (including known parasitism genes) and documented their expression profiles. Using our approach to pre-select genes likely involved in parasitism now allows detailed functional analyses in a manner not feasible for larger numbers of genes. The generation of the candidate pool described here is an important enabling advance because it will significantly facilitate the unraveling of fascinating plant-animal interactions and deliver knowledge that can be transferred to other pathogen-host systems. Ultimately, the exploration of true parasitism genes verified from the gene pool delineated here will identify weaknesses in the nematode life cycle that can be exploited by novel anti-nematode efforts.

Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success.

Cyst nematodes are highly evolved sedentary plant endoparasites that use parasitism proteins injected through the stylet into host tissues to successfully parasitize plants. These secretory proteins likely are essential for parasitism as they are involved in a variety of parasitic events leading to the establishment of specialized feeding cells required by the nematode to obtain nourishment. With the advent of RNA interference (RNAi) technology and the demonstration of host-induced gene silencing in parasites, a new strategy to control pests and pathogens has become available, particularly in root-knot nematodes. Plant host-induced silencing of cyst nematode genes so far has had only limited success but similarly should disrupt the parasitic cycle and render the host plant resistant. Additional in planta RNAi data for cyst nematodes are being provided by targeting four parasitism genes through host-induced RNAi gene silencing in transgenic Arabidopsis thaliana, which is a host for the sugar beet cyst nematode Heterodera schachtii. Here it is reported that mRNA abundances of targeted nematode genes were specifically reduced in nematodes feeding on plants expressing corresponding RNAi constructs. Furthermore, this host-induced RNAi of all four nematode parasitism genes led to a reduction in the number of mature nematode females. Although no complete resistance was observed, the reduction of developing females ranged from 23% to 64% in different RNAi lines. These observations demonstrate the relevance of the targeted parasitism genes during the nematode life cycle and, potentially more importantly, suggest that a viable level of resistance in crop plants may be accomplished in the future using this technology against cyst nematodes.

Active uptake of cyst nematode parasitism proteins into the plant cell nucleus.

Cyst nematodes produce parasitism proteins that contain putative nuclear localisation signals (NLSs) and, therefore, are predicted to be imported into the nucleus of the host plant cell. The in planta localisation patterns of eight soybean cyst nematode (Heterodera glycines) parasitism proteins with putative NLSs were determined by producing these proteins as translational fusions with the GFP and GUS reporter proteins. Two parasitism proteins were found to be imported into the nuclei of onion epidermal cells as well as Arabidopsis protoplasts. One of these two parasitism proteins was further transported into the nucleoli. Mutations introduced into the NLS domains of these two proteins abolished nuclear import and caused a cytoplasmic accumulation. Furthermore, we observed active nuclear uptake for three additional parasitism proteins, however, only when these proteins were synthesised as truncated forms. Two of these proteins were further transported into nucleoli. We hypothesise that nuclear uptake and nucleolar localisation are important mechanisms for H. glycines to modulate the nuclear biology of parasitised cells of its host plant.

A root-knot nematode secretory peptide functions as a ligand for a plant transcription factor.

Parasitism genes expressed in the esophageal gland cells of root-knot nematodes encode proteins that are secreted into host root cells to transform the recipient cells into enlarged multinucleate feeding cells called giant-cells. Expression of a root-knot nematode parasitism gene which encodes a novel 13-amino-acid secretory peptide in plant tissues stimulated root growth. Two SCARECROW-like transcription factors of the GRAS protein family were identified as the putative targets for this bioactive nematode peptide in yeast two-hybrid analyses and confirmed by in vitro and in vivo coimmunoprecipitations. This discovery is the first demonstration of a direct interaction of a nematode-secreted parasitism peptide with a plant-regulatory protein, which may represent an early signaling event in the root-knot nematode-host interaction.