The root-knot nematode effector MiMSP32 targets host 12-oxophytodienoate reductase 2 to regulate plant susceptibility.

To establish persistent infections in host plants, herbivorous invaders, such as root-knot nematodes, must rely on effectors for suppressing damage-induced jasmonate-dependent host defenses. However, at present, the effector mechanisms targeting the biosynthesis of biologically active jasmonates to avoid adverse host responses are unknown. Using yeast two-hybrid, in planta co-immunoprecipitation, and mutant analyses, we identified 12-oxophytodienoate reductase 2 (OPR2) as an important host target of the stylet-secreted effector MiMSP32 of the root-knot nematode Meloidogyne incognita. MiMSP32 has no informative sequence similarities with other functionally annotated genes but was selected for the discovery of novel effector mechanisms based on evidence of positive, diversifying selection. OPR2 catalyzes the conversion of a derivative of 12-oxophytodienoate to jasmonic acid (JA) and operates parallel to 12-oxophytodienoate reductase 3 (OPR3), which controls the main pathway in the biosynthesis of jasmonates. We show that MiMSP32 targets OPR2 to promote parasitism of M. incognita in host plants independent of OPR3-mediated JA biosynthesis. Artificially manipulating the conversion of the 12-oxophytodienoate by OPRs increases susceptibility to multiple unrelated plant invaders. Our study is the first to shed light on a novel effector mechanism targeting this process to regulate the susceptibility of host plants.

GLYCINE-RICH RNA-BINDING PROTEIN 7 potentiates effector-triggered immunity through an RNA recognition motif.

The activity of intracellular plant nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors is fine-tuned by interactions between the receptors and their partners. Identifying NB-LRR interacting proteins is therefore crucial to advance our understanding of how these receptors function. A co-immunoprecipitation/mass spectrometry screening was performed in Nicotiana benthamiana to identify host proteins associated with the resistance protein Gpa2, a CC-NB-LRR immune receptor conferring resistance against the potato cyst nematode Globodera pallida. A combination of biochemical, cellular, and functional assays was used to assess the role of a candidate interactor in defense. A N. benthamiana homolog of the GLYCINE-RICH RNA-BINDING PROTEIN7 (NbGRP7) protein was prioritized as a Gpa2-interacting protein for further investigations. NbGRP7 also associates in planta with the homologous Rx1 receptor, which confers immunity to Potato Virus X. We show that NbGRP7 positively regulates extreme resistance by Rx1 and cell death by Gpa2. Mutating the NbGRP7 RNA recognition motif (RRM) compromises its role in Rx1-mediated defense. Strikingly, ectopic NbGRP7 expression is likely to impact the steady-state levels of Rx1, which relies on an intact RRM. Our findings illustrate that NbGRP7 is a pro-immune component in effector-triggered immunity by regulating Gpa2/Rx1 function at a posttranscriptional level.

Sequence Exchange between Homologous NB-LRR Genes Converts Virus Resistance into Nematode Resistance, and Vice Versa.

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1CN/Gpa2L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.

Dual disease resistance mediated by the immune receptor Cf-2 in tomato requires a common virulence target of a fungus and a nematode.

Plants lack the seemingly unlimited receptor diversity of a somatic adaptive immune system as found in vertebrates and rely on only a relatively small set of innate immune receptors to resist a myriad of pathogens. Here, we show that disease-resistant tomato plants use an efficient mechanism to leverage the limited nonself recognition capacity of their innate immune system. We found that the extracellular plant immune receptor protein Cf-2 of the red currant tomato (Solanum pimpinellifolium) has acquired dual resistance specificity by sensing perturbations in a common virulence target of two independently evolved effectors of a fungus and a nematode. The Cf-2 protein, originally identified as a monospecific immune receptor for the leaf mold fungus Cladosporium fulvum, also mediates disease resistance to the root parasitic nematode Globodera rostochiensis pathotype Ro1-Mierenbos. The Cf-2-mediated dual resistance is triggered by effector-induced perturbations of the apoplastic Rcr3(pim) protein of S. pimpinellifolium. Binding of the venom allergen-like effector protein Gr-VAP1 of G. rostochiensis to Rcr3(pim) perturbs the active site of this papain-like cysteine protease. In the absence of the Cf-2 receptor, Rcr3(pim) increases the susceptibility of tomato plants to G. rostochiensis, thus showing its role as a virulence target of these nematodes. Furthermore, both nematode infection and transient expression of Gr-VAP1 in tomato plants harboring Cf-2 and Rcr3(pim) trigger a defense-related programmed cell death in plant cells. Our data demonstrate that monitoring host proteins targeted by multiple pathogens broadens the spectrum of disease resistances mediated by single plant immune receptors.

Bunyaviral N Proteins Localize at RNA Processing Bodies and Stress Granules: The Enigma of Cytoplasmic Sources of Capped RNA for Cap Snatching.

Most cytoplasmic-replicating negative-strand RNA viruses (NSVs) initiate genome transcription by cap snatching. The source of host mRNAs from which the cytoplasmic NSVs snatch capped-RNA leader sequences has remained elusive. Earlier reports have pointed towards cytoplasmic-RNA processing bodies (P body, PB), although several questions have remained unsolved. Here, the nucleocapsid (N) protein of plant- and animal-infecting members of the order Bunyavirales, in casu Tomato spotted wilt virus (TSWV), Rice stripe virus (RSV), Sin nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Schmallenberg virus (SBV) have been expressed and localized in cells of their respective plant and animal hosts. All N proteins localized to PBs as well as stress granules (SGs), but extensively to docking stages of PB and SG. TSWV and RSV N proteins also co-localized with Ran GTPase-activating protein 2 (RanGAP2), a nucleo-cytoplasmic shuttling factor, in the perinuclear region, and partly in the nucleus when co-expressed with its WPP domain containing a nuclear-localization signal. Upon silencing of PB and SG components individually or concomitantly, replication levels of a TSWV minireplicon, as measured by the expression of a GFP reporter gene, ranged from a 30% reduction to a four-fold increase. Upon the silencing of RanGAP homologs in planta, replication of the TSWV minireplicon was reduced by 75%. During in vivo cap-donor competition experiments, TSWV used transcripts destined to PB and SG, but also functional transcripts engaged in translation. Altogether, the results implicate a more complex situation in which, besides PB, additional cytoplasmic sources are used during transcription/cap snatching of cytoplasmic-replicating and segmented NSVs.

Plant degradation: a nematode expansin acting on plants.

Expansin proteins, which have so far been identified only in plants, rapidly induce extension of plant cell walls by weakening the non-covalent interactions that help to maintain their integrity. Here we show that an animal, the plant-parasitic roundworm Globodera rostochiensis, can also produce a functional expansin, which it uses to loosen cell walls when invading its host plant. As this nematode is known to be able to disrupt covalent bonds in plant cell walls, its accompanying ability to loosen non-covalent bonds challenges the prevailing view that animals are genetically poorly equipped to degrade plant cell walls.

The effector GpRbp-1 of Globodera pallida targets a nuclear HECT E3 ubiquitin ligase to modulate gene expression in the host.

Plant-parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp-1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp-1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6-AP C-Terminus (HECT)-type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3-5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3-5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT-type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp-1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.

A secreted SPRY domain-containing protein (SPRYSEC) from the plant-parasitic nematode Globodera rostochiensis interacts with a CC-NB-LRR protein from a susceptible tomato.

Esophageal gland secretions from nematodes are believed to include effectors that play important roles in plant parasitism. We have identified a novel gene family encoding secreted proteins specifically expressed in the dorsal esophageal gland of Globodera rostochiensis early in the parasitic cycle, and which contain the B30.2/SPRY domain. The secondary structure of these proteins, named the secreted SPRY domain-containing proteins (SPRYSEC), includes highly conserved regions folding into beta-strands interspersed with loops varying in sequence and in length. Mapping sequence diversity onto a three-dimensional structure model of the SPRYSEC indicated that most of the variability is in the extended loops that shape the so-called surface A in the SPRY domains. Seven of nine amino acid sites subjected to diversifying selection in the SPRYSEC are also at this surface. In both yeast-two-hybrid screening using a library from a susceptible tomato and in an in vitro pull-down assay, one of the SPRYSEC interacted with the leucine-rich repeat (LRR) region of a novel coiled-coil nucleotide-binding LRR protein, which is highly similar to members of the SW5 resistance gene cluster. Given that the tomato cultivar used is susceptible to nematode infection, this SPRYSEC could be an evolutionary intermediate that binds to a classical immune receptor but does not yet, or no longer, triggers a resistance response. Alternatively, this SPRYSEC may bind to the immune receptor to downregulate its activity.

A high-resolution map of the Grp1 locus on chromosome V of potato harbouring broad-spectrum resistance to the cyst nematode species Globodera pallida and Globodera rostochiensis.

The Grp1 locus confers broad-spectrum resistance to the potato cyst nematode species Globodera pallida and Globodera rostochiensis and is located in the GP21-GP179 interval on the short arm of chromosome V of potato. A high-resolution map has been developed using the diploid mapping population RHAM026, comprising 1,536 genotypes. The flanking markers GP21 and GP179 have been used to screen the 1,536 genotypes for recombination events. Interval mapping of the resistances to G. pallida Pa2 and G. rostochiensis Ro5 resulted in two nearly identical LOD graphs with the highest LOD score just north of marker TG432. Detailed analysis of the 44 recombinant genotypes showed that G. pallida and G. rostochiensis resistance could not be separated and map to the same location between marker SPUD838 and TG432. It is suggested that the quantitative resistance to both nematode species at the Grp1 locus is mediated by one or more tightly linked R genes that might belong to the NBS-LRR class.

Identification and characterization of the most abundant cellulases in stylet secretions from Globodera rostochiensis.

Plant-parasitic cyst nematodes secrete cell wall modifying proteins during their invasion of host plants. In this study, we used a monoclonal antibody to immunopurify and to sequence the N terminus of the most abundant cellulases in stylet secretions of preparasitic juveniles of Globodera rostochiensis. The N-terminal amino acid sequence perfectly matched the sequence of an expressed sequence tag of two nearly identical genes, named Gr-eng3 and Gr-eng4, which show relatively low similarity with the previously identified Gr-eng1 and Gr-eng2 (i.e., 62% similarity and 42% identity). The recombinantly produced proteins from Gr-eng3 and Gr-eng4 demonstrated specific activity on carboxymethylcellulose, indicating that these genes encode active cellulases. To date, the cellulases in cyst nematodes are comprised of three possible domain structure variants with different types of ancillary domains at the C terminus of the glycosyl hydrolase family 5 (GHF5) domain. We used Bayesian inference to show that the phylogeny of the GHF5 domain based on currently available data suggest that the extant nematode cellulases arose through reshuffling of the GHF5 domain with different types of ancillary domains as relatively independent units. Knocking-down Gr-eng3 and Gr-eng4 using RNA interference resulted in a reduction of nematode infectivity by 57%. Our observations show that the reduced infectivity of the nematodes can be attributed to poor penetration of the host's root system at the onset of parasitism.

A symbiont-independent endo-1,4-beta-xylanase from the plant-parasitic nematode Meloidogyne incognita.

Substituted xylan polymers constitute a major part of the hemicellulose fraction of plant cell walls, especially in monocotyledons. Endo-1,4-beta-xylanases (EC 3.2.1.8) are capable of hydrolyzing substituted xylan polymers into fragments of random size. Many herbivorous animals have evolved intimate relationships with endosymbionts to exploit their enzyme complexes for the degradation of xylan. Here, we report the first finding of a functional endo-1,4-beta-xylanase gene from an animal. The gene (Mi-xyl1) was found in the obligate plant-parasitic root-knot nematode Meloidogyne incognita, and encodes a protein that is classified as a member of glycosyl hydrolase family 5. The expression of Mi-xyl1 is localized in the subventral esophageal gland cells of the nematode. Previous studies have shown that M. incognita has the ability to degrade cellulose and pectic polysaccharides in plant cell walls independent of endosymbionts. Including our current data on Mi-xyl1, we show that the endogenous enzyme complex in root-knot nematode secretions targets essentially all major cell wall carbohydrates to facilitate a stealthy intercellular migration in the host plant.

The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence.

BACKGROUND: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. RESULTS: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. CONCLUSIONS: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.

Origin, distribution and 3D-modeling of Gr-EXPB1, an expansin from the potato cyst nematode Globodera rostochiensis.

Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modeling predicted that Gr-EXPB1 domain 1 (D1) has a flat beta-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.

Structural and functional characterization of a novel, host penetration-related pectate lyase from the potato cyst nematode Globodera rostochiensis.

SUMMARY The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene (Gr-pel2, 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40-42% identity and 58-60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca(2+) is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes.

Feeding cell development by cyst and root-knot nematodes involves a similar early, local and transient activation of a specific auxin-inducible promoter element.

SUMMARY To study the role of the phytohormone auxin in nematode feeding cell induction and early development, the transcriptional regulation of the artificial auxin-responsive promoter element DR5 was monitored in Arabidopsis thaliana roots infected with the cyst nematode Heterodera schachtii or the root-knot nematode Meloidogyne incognita. For both nematode species, a specific and strong activation of DR5::gusA was observed inside the initial feeding cells at 18 h post inoculation, pointing to an increase in the perceived auxin concentration. This high expression was maintained until 3-5 days post inoculation and subsequently the GUS staining was reduced. Cyst and root-knot nematodes are distantly related and the feeding sites they induce are highly dissimilar. In this respect, the similarities between the two nematode-induced DR5 activation patterns in A. thaliana roots are remarkable. A transient and local increase in auxin perception could be due to an accumulation or to an increased sensitivity. Based on previously published data, a local auxin accumulation seems to be the more probable explanation. The observed early and localized increase of the perceived IAA concentration in the initial feeding structure underlines that this phytohormone could be an important clue in feeding cell induction by plant parasitic nematodes.