Arabidopsis HIPP27 is a host susceptibility gene for the beet cyst nematode Heterodera schachtii

Sedentary plant‐parasitic cyst nematodes are obligate biotrophs that infect the roots of their host plant. Their parasitism is based on the modification of root cells to form a hypermetabolic syncytium from which the nematodes draw their nutrients. The aim of this study was to identify nematode susceptibility genes in Arabidopsis thaliana and to characterize their roles in supporting the parasitism of Heterodera schachtii. By selecting genes that were most strongly upregulated in response to cyst nematode infection, we identified HIPP27 (HEAVY METAL‐ASSOCIATED ISOPRENYLATED PLANT PROTEIN 27) as a host susceptibility factor required for beet cyst nematode infection and development. Detailed expression analysis revealed that HIPP27 is a cytoplasmic protein and that HIPP27 is strongly expressed in leaves, young roots and nematode‐induced syncytia. Loss‐of‐function Arabidopsis hipp27 mutants exhibited severely reduced susceptibility to H. schachtii and abnormal starch accumulation in syncytial and peridermal plastids. Our results suggest that HIPP27 is a susceptibility gene in Arabidopsis whose loss of function reduces plant susceptibility to cyst nematode infection without increasing the susceptibility to other pathogens or negatively affecting the plant phenotype.     

Sequence divergences between cyst nematode effector protein orthologs may contribute to host specificity

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 five-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.Key words: Heterodera schachtii, arabidopsis, 10A06 effector protein, spermidine synthase 2Cyst nematodes are sedentary pathogens of roots of many economically important crop plants and induce the formation of specialized feeding cells, so-called syncytia, that provide the nematodes with nourishment. The infection process is mediated through secretion of an array of nematode effector proteins inside plant tissues and cells. One of these effector proteins is 10A06, which was initially identified from a gland cell cDNA library from H. glycines, the soybean cyst nematode.¹ The 927 bp full-length H. glycines Hg-10A06 cDNA (GenBank Accession {"type":"entrez-nucleotide","attrs":{"text":"AF502391","term_id":"30315231","term_text":"AF502391"}}AF502391) encoded a predicted protein of 308 amino acids with an N-terminal signal peptide of 17 amino acids for secretion. Recently, we identified the orthologous 10A06 sequence from the sugar beet cyst nematode H. schachtii (Hs-10A06), which is able to infect the model plant Arabidopsis thaliana. The Hs-10A06 cDNA (GenBank Accession {"type":"entrez-nucleotide","attrs":{"text":"GQ373256","term_id":"255528984","term_text":"GQ373256"}}GQ373256) contained an open reading frame of 858 bp encoding a 285-amino acid protein with an N-terminal signal peptide for secretion.² Sequence alignment of H. glycines and H. schachtii 10A06 proteins revealed a strong homology between both orthologues with 86% identity and 87% similarity. The largest difference between the two proteins is the lack of a stretch of 23 amino acids in Hs-10A06. Additionally, a region of 15 amino acid residues located between amino acid 167 and 181 exhibited a high degree of divergence between both proteins. Constitutive expression of Hs-10A06 in Arabidopsis affected plant morphology and increased susceptibility to H. schachtii.² We uncovered in yeast two-hybrid assays that the Hs-10A06 protein interacts with Arabidopsis SPDS2, a key enzyme involved in polyamine biosynthesis, to mediate susceptibility. Here, we assessed the effects of ectopic Hg-10A06 expression in the non-host Arabidopsis on plant morphology and nematode susceptibility. Moreover, we assayed whether Hg-10A06 also is able to interact with SPDS2 from Arabidopsis.     

Cellulose Binding Protein from the Parasitic Nematode Heterodera schachtii Interacts with Arabidopsis Pectin Methylesterase: Cooperative Cell Wall Modification during Parasitism

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.     

Manipulation of two α‐endo‐β‐1,4‐glucanase genes,AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots

Plant endo‐β‐1,4‐glucanases (EGases) include cell wall‐modifying enzymes that are involved in nematode‐induced growth of syncytia (feeding structures) in nematode‐infected roots. EGases in the α‐ and β‐subfamilies contain signal peptides and are secreted, whereas those in the γ‐subfamily have a membrane‐anchoring domain and are not secreted. The Arabidopsis α‐EGase At1g48930, designated as AtCel6, is known to be down‐regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α‐EGase, AtCel2, is up‐regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ‐EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α‐EGases are likely to be more useful than membrane‐bound γ‐EGases in the development of an SCN‐resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis–H. schachtii interaction as a model for the soybean–H. glycines pathosystem.     

Dactylella oviparasitica parasitism of the sugar beet cyst nematode observed in trixenic culture plates

The nematophagous fungus Dactylella oviparasitica is considered the primary cause of a sugar beet cyst nematode (Heterodera schachtii) population suppression in a field at the Agricultural Operations, University of California, Riverside. Parasitism of H. schachtii by the ascomycete D. oviparasitica was studied using both Arabidopsis thaliana (type Landsberg erecta) and cabbage as host plants in gnotobiotic agar culture. Suitability of Arabidopsis as a host for H. schachtii was confirmed using seedlings grown with the nematode in axenic sand culture. Both developing males and females of H. schachtii broke through the Arabidopsis root surface during late juvenile stages and both were susceptible to D. oviparasitica parasitism. In contrast to Arabidopsis, developing juvenile males remained in nearly all observed cases enclosed within the cabbage root tissues while the larger body expansion of the female juveniles caused the root cortex to split; consequently only the latter ones were accessible to the fungus. In the presence of D. oviparasitica, the number of females with eggs was reduced by more than 95% and the number of eggs per female by almost 60% as compared to females developing on plates without the fungus. Viable eggs were not susceptible to parasitism while more than 90% of heat- or cold-killed eggs were rendered susceptible. These observations suggest that parasitism of developing juveniles may be the essential mode of action in the population suppression of H. schachtii.     

Divergent expression of cytokinin biosynthesis,signaling and catabolism genes underlying differences in feeding sites induced by cyst and root‐knot nematodes

Cyst and root‐knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root‐knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.     

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.     

Apoplastic Venom Allergen-like Proteins of Cyst Nematodes Modulate the Activation of Basal Plant Innate Immunity by Cell Surface Receptors

Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize venom allergen-like proteins to suppress the activation of defenses by immunogenic breakdown products in damaged host tissue.     

A novel Meloidogyne enterolobii effector MeTCTP promotes parasitism by suppressing programmed cell death in host plants

Meloidogyne enterolobii is one of the most important plant‐parasitic nematodes that can overcome the Mi‐1 resistance gene and damage many economically important crops. Translationally controlled tumour protein (TCTP) is a multifunctional protein that exists in various eukaryotes and plays an important role in parasitism. In this study, a novel M. enterolobii TCTP effector, named MeTCTP, was identified and functionally characterized. MeTCTP was specifically expressed within the dorsal gland and was up‐regulated during M. enterolobii parasitism. Transient expression of MeTCTP in protoplasts from tomato roots showed that MeTCTP was localized in the cytoplasm of the host cells. Transgenic Arabidopsis thaliana plants overexpressing MeTCTP were more susceptible to M. enterolobii infection than wild‐type plants in a dose‐dependent manner. By contrast, in planta RNA interference (RNAi) targeting MeTCTP suppressed the expression of MeTCTP in infecting nematodes and attenuated their parasitism. Furthermore, MeTCTP could suppress programmed cell death triggered by the pro‐apoptotic protein BAX. These results demonstrate that MeTCTP is a novel plant‐parasitic nematode effector that promotes parasitism, probably by suppressing programmed cell death in host plants.     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.     

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 endoparasitesthat use parasitism proteins injected through the stylet intohost tissues to successfully parasitize plants. These secretoryproteins likely are essential for parasitism as they are involvedin a variety of parasitic events leading to the establishmentof specialized feeding cells required by the nematode to obtainnourishment. With the advent of RNA interference (RNAi) technologyand 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 silencingof cyst nematode genes so far has had only limited success butsimilarly should disrupt the parasitic cycle and render thehost plant resistant. Additional in planta RNAi data for cystnematodes are being provided by targeting four parasitism genesthrough host-induced RNAi gene silencing in transgenic Arabidopsisthaliana, which is a host for the sugar beet cyst nematode Heteroderaschachtii. Here it is reported that mRNA abundances of targetednematode genes were specifically reduced in nematodes feedingon plants expressing corresponding RNAi constructs. Furthermore,this host-induced RNAi of all four nematode parasitism genesled to a reduction in the number of mature nematode females.Although no complete resistance was observed, the reductionof developing females ranged from 23% to 64% in different RNAilines. These observations demonstrate the relevance of the targetedparasitism genes during the nematode life cycle and, potentiallymore importantly, suggest that a viable level of resistancein crop plants may be accomplished in the future using thistechnology against cyst nematodes. Key words: beet cyst nematode (BCN), soybean cyst nematode (SCN), host induced, in planta RNAi, resistance, RNAi, transgenic Received 19 August 2008; Revised 25 October 2008 Accepted 27 October 2008     

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development

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.     

Parasitism of Heterodera schachtii and Meloidogyne javanica by Hirsutella rhossiliensis in Microplots over Two Growing Seasons

Numbers of cyst and root-knot nematodes and percentage parasitism by the nematophagous fungus Hirsutella rhossiliensis were quantified in microplots over 2 years. The microplots contained either sugarbeets in loam infested with Heterodera schachtii or tomatoes in sand infested with Meloidogyne javanica. The fungus was added to half of the microplots for each crop. Although H. rhossiliensis established in both microplot soils, the percentage of nematodes parasitized did not increase with nematode density and nematode numbers were not affected by the fungus. The results indicate that long-term interactions between populations of the fungus and cyst or root-knot nematodes will not result in biological control.