Transcription factor WOX11 modulates tolerance to cyst nematodes via adventitious lateral root formation.

The transcription factor WUSCHEL-RELATED HOMEOBOX 11 (WOX11) in Arabidopsis (Arabidopsis thaliana) initiates the formation of adventitious lateral roots upon mechanical injury in primary roots. Root-invading nematodes also induce de novo root organogenesis leading to excessive root branching, but it is not known if this symptom of disease involves mediation by WOX11 and if it benefits the plant. Here, we show with targeted transcriptional repression and reporter gene analyses in Arabidopsis that the beet cyst nematode Heterodera schachtii activates WOX11-mediated adventitious lateral rooting from primary roots close to infection sites. The activation of WOX11 in nematode-infected roots occurs downstream of jasmonic acid-dependent damage signaling via ETHYLENE RESPONSE FACTOR109, linking adventitious lateral root formation to nematode damage to host tissues. By measuring different root system components, we found that WOX11-mediated formation of adventitious lateral roots compensates for nematode-induced inhibition of primary root growth. Our observations further demonstrate that WOX11-mediated rooting reduces the impact of nematode infections on aboveground plant development and growth. Altogether, we conclude that the transcriptional regulation by WOX11 modulates root system plasticity under biotic stress, which is one of the key mechanisms underlying the tolerance of Arabidopsis to cyst nematode infections.

From root to shoot: quantifying nematode tolerance in Arabidopsis thaliana by high-throughput phenotyping of plant development.

Nematode migration, feeding site formation, withdrawal of plant assimilates, and activation of plant defence responses have a significant impact on plant growth and development. Plants display intraspecific variation in tolerance limits for root-feeding nematodes. Although disease tolerance has been recognized as a distinct trait in biotic interactions of mainly crops, we lack mechanistic insights. Progress is hampered by difficulties in quantification and laborious screening methods. We turned to the model plant Arabidopsis thaliana, since it offers extensive resources to study the molecular and cellular mechanisms underlying nematode-plant interactions. Through imaging of tolerance-related parameters, the green canopy area was identified as an accessible and robust measure for assessing damage due to cyst nematode infection. Subsequently, a high-throughput phenotyping platform simultaneously measuring the green canopy area growth of 960 A. thaliana plants was developed. This platform can accurately measure cyst nematode and root-knot nematode tolerance limits in A. thaliana through classical modelling approaches. Furthermore, real-time monitoring provided data for a novel view of tolerance, identifying a compensatory growth response. These findings show that our phenotyping platform will enable a new mechanistic understanding of tolerance to below-ground biotic stress.

Comparative genomics among cyst nematodes reveals distinct evolutionary histories among effector families and an irregular distribution of effector-associated promoter motifs.

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are essential for PCN control. However, the poor delineation of G. pallida pathotypes has hampered the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs (venom allergen-like proteins) diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCNs after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression ("DOG boxes") were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with the expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Resequencing of PCN populations with different virulence characteristics will allow for the linking of these characteristics to the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

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.

Root architecture plasticity in response to endoparasitic cyst nematodes is mediated by damage signaling.

Plant root architecture plasticity in response to biotic stresses has not been thoroughly investigated. Infection by endoparasitic cyst nematodes induces root architectural changes that involve the formation of secondary roots at infection sites. However, the molecular mechanisms regulating secondary root formation in response to cyst nematode infection remain largely unknown. We first assessed whether secondary roots form in a nematode density-dependent manner by challenging wild-type Arabidopsis plants with increasing numbers of cyst nematodes (Heterodera schachtii). Next, using jasmonate-related reporter lines and knockout mutants, we tested whether tissue damage by nematodes triggers jasmonate-dependent secondary root formation. Finally, we verified whether damage-induced secondary root formation depends on local auxin biosynthesis at nematode infection sites. Intracellular host invasion by H. schachtii triggers a transient local increase in jasmonates, which activates the expression of ERF109 in a COI1-dependent manner. Knockout mutations in COI1 and ERF109 disrupt the nematode density-dependent increase in secondary roots observed in wild-type plants. Furthermore, ERF109 regulates secondary root formation upon H. schachtii infection via local auxin biosynthesis. Host invasion by H. schachtii triggers secondary root formation via the damage-induced jasmonate-dependent ERF109 pathway. This points at a novel mechanism underlying plant root plasticity in response to biotic stress.

The Arabidopsis transcription factor TCP9 modulates root architectural plasticity, reactive oxygen species-mediated processes, and tolerance to cyst nematode infections.

Infections by root-feeding nematodes have profound effects on root system architecture and consequently shoot growth of host plants. Plants harbor intraspecific variation in their growth responses to belowground biotic stresses by nematodes, but the underlying mechanisms are not well understood. Here, we show that the transcription factor TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR-9 (TCP9) modulates root system architectural plasticity in Arabidopsis thaliana in response to infections by the endoparasitic cyst nematode Heterodera schachtii. Young seedlings of tcp9 knock-out mutants display a significantly weaker primary root growth inhibition response to cyst nematodes than wild-type Arabidopsis. In older plants, tcp9 reduces the impact of nematode infections on the emergence and growth of secondary roots. Importantly, the altered growth responses by tcp9 are most likely not caused by less biotic stress on the root system, because TCP9 does not affect the number of infections, nematode development, and size of the nematode-induced feeding structures. RNA-sequencing of nematode-infected roots of the tcp9 mutants revealed differential regulation of enzymes involved in reactive oxygen species (ROS) homeostasis and responses to oxidative stress. We also found that root and shoot growth of tcp9 mutants is less sensitive to exogenous hydrogen peroxide and that ROS accumulation in nematode infection sites in these mutants is reduced. Altogether, these observations demonstrate that TCP9 modulates the root system architectural plasticity to nematode infections via ROS-mediated processes. Our study further points at a novel regulatory mechanism contributing to the tolerance of plants to root-feeding nematodes by mitigating the impact of belowground biotic stresses.

Comparative Transcriptome Analysis Reveals the Specific Activation of Defense Pathways Against Globodera pallida in Gpa2 Resistant Potato Roots.

Cyst nematodes are considered a dominant threat to yield for a wide range of major food crops. Current control strategies are mainly dependent on crop rotation and the use of resistant cultivars. Various crops exhibit single dominant resistance (R) genes that are able to activate effective host-specific resistance to certain cyst nematode species and/or populations. An example is the potato R gene Gpa2, which confers resistance against the potato cyst nematode (PCN), Globodera pallida population D383. Activation of Gpa2 results in a delayed resistance response, which is characterized by a layer of necrotic cells formed around the developing nematode feeding structure. However, knowledge about the Gpa2-induced defense pathways is still lacking. Here, we uncover the transcriptional changes and gene expression network induced upon Gpa2 activation in potato roots infected with G. pallida. To this end, in vitro-grown Gpa2-resistant potato roots were infected with the avirulent population D383 and virulent population Rookmaker. Infected root segments were harvested at 3 and 6 dpi and sent for RNA sequencing. Comparative transcriptomics revealed a total of 1,743 differentially expressed genes (DEGs) upon nematode infection, of which 559 DEGs were specifically regulated in response to D383 infection. D383-specific DEGs associated with Gpa2-mediated defense mainly relates to calcium-binding activity, salicylic acid (SA) biosynthesis, and systemic acquired resistance (SAR). These data reveal that cyst nematode resistance in potato roots depends on conserved downstream signaling pathways involved in plant immunity, which are also known to contribute to R genes-mediated resistance against other pathogens with different lifestyles.

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.

Comparative genomics of two inbred lines of the potato cyst nematode Globodera rostochiensis reveals disparate effector family-specific diversification patterns.

BACKGROUND: Potato cyst nematodes belong to the most harmful pathogens in potato, and durable management of these parasites largely depends on host-plant resistances. These resistances are pathotype specific. The current Globodera rostochiensis pathotype scheme that defines five pathotypes (Ro1 - Ro5) is both fundamentally and practically of limited value. Hence, resistant potato varieties are used worldwide in a poorly informed manner. RESULTS: We generated two novel reference genomes of G. rostochiensis inbred lines derived from a Ro1 and a Ro5 population. These genome sequences comprise 173 and 189 scaffolds respectively, marking a ≈ 24-fold reduction in fragmentation as compared to the current reference genome. We provide copy number variations for 19 effector families. Four dorsal gland effector families were investigated in more detail. SPRYSECs, known to be implicated in plant defence suppression, constitute by far the most diversified family studied herein with 60 and 99 variants in Ro1 and Ro5 distributed over 18 and 26 scaffolds. In contrast, CLEs, effectors involved in feeding site induction, show strong physical clustering. The 10 and 16 variants cluster on respectively 2 and 1 scaffolds. Given that pathotypes are defined by their effectoromes, we pinpoint the disparate nature of the contributing effector families in terms of sequence diversification and loss and gain of variants. CONCLUSIONS: Two novel reference genomes allow for nearly complete inventories of effector diversification and physical organisation within and between pathotypes. Combined with insights we provide on effector family-specific diversification patterns, this constitutes a basis for an effectorome-based virulence scheme for this notorious pathogen.

Toward genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles.

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

The TIR-NB-LRR pair DSC1 and WRKY19 contributes to basal immunity of Arabidopsis to the root-knot nematode Meloidogyne incognita.

BACKGROUND: Root-knot nematodes transform vascular host cells into permanent feeding structures to withdraw nutrients from the host plant. Ecotypes of Arabidopsis thaliana can display large quantitative variation in susceptibility to the root-knot nematode Meloidogyne incognita, which is thought to be independent of dominant major resistance genes. However, in an earlier genome-wide association study of the interaction between Arabidopsis and M. incognita we identified a quantitative trait locus harboring homologs of dominant resistance genes but with minor effect on susceptibility to the M. incognita population tested. RESULTS: Here, we report on the characterization of two of these genes encoding the TIR-NB-LRR immune receptor DSC1 (DOMINANT SUPPRESSOR OF Camta 3 NUMBER 1) and the TIR-NB-LRR-WRKY-MAPx protein WRKY19 in nematode-infected Arabidopsis roots. Nematode infection studies and whole transcriptome analyses using the Arabidopsis mutants showed that DSC1 and WRKY19 co-regulate susceptibility of Arabidopsis to M. incognita. CONCLUSION: Given the head-to-head orientation of DSC1 and WRKY19 in the Arabidopsis genome our data suggests that both genes may function as a TIR-NB-LRR immune receptor pair. Unlike other TIR-NB-LRR pairs involved in dominant disease resistance in plants, DSC1 and WRKY19 most likely regulate basal levels of immunity to root-knot nematodes.

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.

Systems Genetics for Evolutionary Studies.

Systems genetics combines high-throughput genomic data with genetic analysis. In this chapter, we review and discuss application of systems genetics in the context of evolutionary studies, in which high-throughput molecular technologies are being combined with quantitative trait locus (QTL) analysis in segregating populations.The recent explosion of high-throughput data-measuring thousands of RNAs, proteins, and metabolites, using deep sequencing, mass spectrometry, chromatin, methyl-DNA immunoprecipitation, etc.-allows the dissection of causes of genetic variation underlying quantitative phenotypes of all types. To deal with the sheer amount of data, powerful statistical tools are needed to analyze multidimensional relationships and to extract valuable information and new modes and mechanisms of changes both within and between species. In the context of evolutionary computational biology, a well-designed experiment and the right population can help dissect complex traits likely to be under selection using proven statistical methods for associating phenotypic variation with chromosomal locations.Recent evolutionary expression QTL (eQTL) studies focus on gene expression adaptations, mapping the gene expression landscape, and, tentatively, define networks of transcripts and proteins that are jointly modulated sets of eQTL networks. Here, we discuss the possibility of introducing an evolutionary "prior" in the form of gene families displaying evidence of positive selection, and using that prior in the context of an eQTL experiment for elucidating host-pathogen protein-protein interactions.Here we review one exemplar evolutionairy eQTL experiment and discuss experimental design, choice of platforms, analysis methods, scope, and interpretation of results. In brief we highlight how eQTL are defined; how they are used to assemble interacting and causally connected networks of RNAs, proteins, and metabolites; and how some QTLs can be efficiently converted to reasonably well-defined sequence variants.

Scalable Workflows and Reproducible Data Analysis for Genomics.

Biological, clinical, and pharmacological research now often involves analyses of genomes, transcriptomes, proteomes, and interactomes, within and between individuals and across species. Due to large volumes, the analysis and integration of data generated by such high-throughput technologies have become computationally intensive, and analysis can no longer happen on a typical desktop computer.In this chapter we show how to describe and execute the same analysis using a number of workflow systems and how these follow different approaches to tackle execution and reproducibility issues. We show how any researcher can create a reusable and reproducible bioinformatics pipeline that can be deployed and run anywhere. We show how to create a scalable, reusable, and shareable workflow using four different workflow engines: the Common Workflow Language (CWL), Guix Workflow Language (GWL), Snakemake, and Nextflow. Each of which can be run in parallel.We show how to bundle a number of tools used in evolutionary biology by using Debian, GNU Guix, and Bioconda software distributions, along with the use of container systems, such as Docker, GNU Guix, and Singularity. Together these distributions represent the overall majority of software packages relevant for biology, including PAML, Muscle, MAFFT, MrBayes, and BLAST. By bundling software in lightweight containers, they can be deployed on a desktop, in the cloud, and, increasingly, on compute clusters.By bundling software through these public software distributions, and by creating reproducible and shareable pipelines using these workflow engines, not only do bioinformaticians have to spend less time reinventing the wheel but also do we get closer to the ideal of making science reproducible. The examples in this chapter allow a quick comparison of different solutions.

A Jasmonate Signaling Network Activates Root Stem Cells and Promotes Regeneration.

Plants are sessile and have to cope with environmentally induced damage through modification of growth and defense pathways. How tissue regeneration is triggered in such responses and whether this involves stem cell activation is an open question. The stress hormone jasmonate (JA) plays well-established roles in wounding and defense responses. JA also affects growth, which is hitherto interpreted as a trade-off between growth and defense. Here, we describe a molecular network triggered by wound-induced JA that promotes stem cell activation and regeneration. JA regulates organizer cell activity in the root stem cell niche through the RBR-SCR network and stress response protein ERF115. Moreover, JA-induced ERF109 transcription stimulates CYCD6;1 expression, functions upstream of ERF115, and promotes regeneration. Soil penetration and response to nematode herbivory induce and require this JA-mediated regeneration response. Therefore, the JA tissue damage response pathway induces stem cell activation and regeneration and activates growth after environmental stress.

Mediator of tolerance to abiotic stress ERF6 regulates susceptibility of Arabidopsis to Meloidogyne incognita.

Root-knot nematodes transform vascular host cells into permanent feeding structures to selectively withdraw their nutrients from host plants during the course of several weeks. The susceptibility of host plants to root-knot nematode infections is thought to be a complex trait involving many genetic loci. However, genome-wide association (GWA) analysis has so far revealed only four quantitative trait loci (QTLs) linked to the reproductive success of the root-knot nematode Meloidogyne incognita in Arabidopsis thaliana, which suggests that the genetic architecture underlying host susceptibility could be much simpler than previously thought. Here, we report that, by using a relaxed stringency approach in a GWA analysis, we could identify 15 additional loci linked to quantitative variation in the reproductive success of M. incognita in Arabidopsis. To test the robustness of our analysis, we functionally characterized six genes located in a QTL with the lowest acceptable statistical support and smallest effect size. This led us to identify ETHYLENE RESPONSE FACTOR 6 (ERF6) as a novel susceptibility gene for M. incognita in Arabidopsis. ERF6 functions as a transcriptional activator and suppressor of genes in response to various abiotic stresses independent of ethylene signalling. However, whole-transcriptome analysis of nematode-infected roots of the Arabidopsis erf6-1 knockout mutant line showed that allelic variation at this locus may regulate the conversion of aminocyclopropane-1-carboxylate (ACC) into ethylene by altering the expression of 1-aminocyclopropane-1-carboxylate oxidase 3 (ACO3). Our data further suggest that tolerance to abiotic stress mediated by ERF6 forms a novel layer of control in the susceptibility of Arabidopsis to M. incognita.

Secreted venom allergen-like proteins of helminths: Conserved modulators of host responses in animals and plants.

Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops.

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

Granulocyte-macrophage colony-stimulating factor negatively regulates early IL-10-mediated responses.

AIM: Treatment of inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. IL-10 is a cytokine with therapeutic potential, but until now has not been as successful as previously anticipated. A reason for this may be that IL-10 responsiveness depends on the environment of the inflamed tissue. In this study we investigated whether GM-CSF is able to influence IL-10-mediated responses. METHODOLOGY: Dendritic cells and macrophages were differentiated from mouse bone marrow and treated or depleted from GM-CSF prior to analyze their response to IL-10. Activity was assessed by measuring cytokine expression upon lipopolysaccharide stimulation, IL-10-induced signaling and down-stream gene expression. CONCLUSION: This study describes that GM-CSF negatively regulates IL-10-mediated responses.