Exploiting breakdown in nonhost effector-target interactions to boost host disease resistance.

Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis, enhanced pathogen infection when expressed in host plants (Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P. infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P. infestans and H. arabidopsidis. Few P. infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H. arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P. infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P. infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease.

The Plant Sesquiterpene Nootkatone Efficiently Reduces Heterodera schachtii Parasitism by Activating Plant Defense.

Plant parasitic nematodes, including the beet cyst nematode Heterodera schachtii, constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents. Here, we evaluated the impact of eight plant terpenoids on the H. schachtii parasitism of Arabidopsis thaliana. None of the metabolites affected the plant development (5 or 10 ppm). Nootkatone decreased the number of adult nematodes on A. thaliana to 50%, with the female nematodes being smaller compared to the control. In contrast, three other terpenoids increased the parasitism and/or female size. We discovered that nootkatone considerably decreased the number of nematodes that penetrated A. thaliana roots, but neither affected the nematode viability or attraction to plant roots, nor triggered the production of plant reactive oxygen species or changed the plant's sesquiterpene profile. However, we demonstrated that nootkatone led to a significant upregulation of defense-related genes involved in salicylic and jasmonic acid pathways. Our results indicate that nootkatone is a promising candidate to be developed into a novel plant protection agent acting as a stimulator of plant immunity against parasitic 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.

Nicotinamide Inhibits Aphid Fecundity and Impacts Survival.

Nicotinamide (NAM) alters behavior in C. elegans and Drosophila, serving as an agonist of TRPV channels affecting sensory neurons and mimicking the mode of action of insecticides used to control phloem-feeding insects. The impact of NAM on green peach aphid (Myzus persicae) behaviors was assessed in artificial diet assays and foliar applications to Arabidopsis plants. Aphids feeding on artificial diets supplemented with NAM impaired stylet movement causing feeding interruptions and ultimately starvation and death. Aphid feeding behaviors were negatively impacted on NAM sprayed plants at concentrations as low as 2.5 mM leading to increased mortality. In choice assays with NAM sprayed leaves aphids showed clear preference for untreated control leaves. NAM is an intermediate in the NAD salvage pathway that should accumulate in nicotinamidase (nic) mutants. LC-MS analysis showed NAM accumulates 60-fold in nic-1-1 Arabidopsis mutants as compared with Col-0. Aphid reproductive potential was significantly decreased on nic-1-1 mutant plants, resulting in a smaller colony size and arrested population development. The results support the hypothesis that dietary NAM causes behavioral changes in aphids, including altered feeding, reduced reproduction, and increased mortality. NAM is thought to bind to TRPV channels causing overstimulation of sensory neurons in the aphid feeding apparatus.

Cell Wall Modifications in Giant Cells Induced by the Plant Parasitic Nematode Meloidogyne incognita in Wild-Type (Col-0) and the fra2 Arabidopsis thaliana Katanin Mutant.

Meloidogyne incognita is a root knot nematode (RKN) species which is among the most notoriously unmanageable crop pests with a wide host range. It inhabits plants and induces unique feeding site structures within host roots, known as giant cells (GCs). The cell walls of the GCs undergo the process of both thickening and loosening to allow expansion and finally support nutrient uptake by the nematode. In this study, a comparative in situ analysis of cell wall polysaccharides in the GCs of wild-type Col-0 and the microtubule-defective fra2 katanin mutant, both infected with M. incognita has been carried out. The fra2 mutant had an increased infection rate. Moreover, fra2 roots exhibited a differential pectin and hemicellulose distribution when compared to Col-0 probably mirroring the fra2 root developmental defects. Features of fra2 GC walls include the presence of high-esterified pectic homogalacturonan and pectic arabinan, possibly to compensate for the reduced levels of callose, which was omnipresent in GCs of Col-0. Katanin severing of microtubules seems important in plant defense against M. incognita, with the nematode, however, to be nonchalant about this "katanin deficiency" and eventually induce the necessary GC cell wall modifications to establish a feeding site.

Risk versus reward: host dependent parasite mortality rates and phenotypes in the facultative generalist Triphysaria versicolor.

BACKGROUND: Parasitic plants engage in a complex molecular dialog with potential host plants to identify a host and overcome host defenses to initiate development of the parasitic feeding organ, the haustorium, invade host tissues, and withdraw water and nutrients. While one of two critical signaling events in the parasitic plant life cycle (germination via stimulant chemicals) has been relatively well-studied, the signaling event that triggers haustorium formation remains elusive. Elucidation of this poorly understood molecular dialogue will shed light on plant-plant communication, parasitic plant physiology, and the evolution of parasitism in plants. RESULTS: Here we present an experimental framework that develops easily quantifiable contrasts for the facultative generalist parasitic plant, Triphysaria, as it feeds across a broad range of diverse flowering plants. The contrasts, including variable parasite growth form and mortality when grown with different hosts, suggest a dynamic and host-dependent molecular dialogue between the parasite and host. Finally, by comparing transcriptome datasets from attached versus unattached parasites we gain insight into some of the physiological processes that are altered during parasitic behavior including shifts in photosynthesis-related and stress response genes. CONCLUSIONS: This work sheds light on Triphysaria's parasitic life habit and is an important step towards understanding the mechanisms of haustorium initiation factor perception, a unique form of plant-plant communication.

Soybean Resistance Locus Rhg1 Confers Resistance to Multiple Cyst Nematodes in Diverse Plant Species.

Cyst nematodes consistently threaten agricultural production, causing billions of dollars in losses globally. The Rhg1 (resistance to Heterodera glycines 1) locus of soybean (Glycine max) is the most popular resistance source used against soybean cyst nematodes (H. glycines). Rhg1 is a complex locus that has multiple repeats of an ≈30-kilobase segment carrying three genes that contribute to resistance. We investigated whether soybean Rhg1 could function in different plant families, conferring resistance to their respective cyst nematode parasites. Transgenic Arabidopsis thaliana and potato (Solanum tuberosum) plants expressing the three soybean Rhg1 genes were generated. The recipient Brassicaceae and Solanaceae plant species exhibited elevated resistance to H. schachtii and Globodera rostochiensis and to G. pallida, respectively. However, some negative consequences including reduced root growth and tuber biomass were observed upon Rhg1 expression in heterologous species. One of the genes at Rhg1 encodes a toxic version of an alpha-SNAP protein that has been demonstrated to interfere with vesicle trafficking. Using a transient expression assay for Nicotiana benthamiana, native Arabidopsis and potato alpha-SNAPs (soluble NSF [N-ethylamine sensitive factor] attachment protein) were found to compensate for the toxicity of soybean Rhg1 alpha-SNAP proteins. Hence, future manipulation of the balance between Rhg1 alpha-SNAP and the endogenous wild-type alpha-SNAPs (as well as the recently discovered soybean NSF-RAN07) may mitigate impacts of Rhg1 on plant productivity. The multispecies efficacy of soybean Rhg1 demonstrates that the encoded mechanisms can function across plant and cyst nematode species and offers a possible avenue for engineered resistance in diverse crop species.

Pectin Methylesterases Modulate Plant Homogalacturonan Status in Defenses against the Aphid Myzus persicae.

Because they suck phloem sap and act as vectors for phytopathogenic viruses, aphids pose a threat to crop yields worldwide. Pectic homogalacturonan (HG) has been described as a defensive element for plants during infections with phytopathogens. However, its role during aphid infestation remains unexplored. Using immunofluorescence assays and biochemical approaches, the HG methylesterification status and associated modifying enzymes during the early stage of Arabidopsis (Arabidopsis thaliana) infestation with the green peach aphid (Myzus persicae) were analyzed. Additionally, the influence of pectin methylesterase (PME) activity on aphid settling and feeding behavior was evaluated by free choice assays and the Electrical Penetration Graph technique, respectively. Our results revealed that HG status and HG-modifying enzymes are significantly altered during the early stage of the plant-aphid interaction. Aphid infestation induced a significant increase in total PME activity and methanol emissions, concomitant with a decrease in the degree of HG methylesterification. Conversely, inhibition of PME activity led to a significant decrease in the settling and feeding preference of aphids. Furthermore, we demonstrate that the PME inhibitor AtPMEI13 has a defensive role during aphid infestation, since pmei13 mutants are significantly more susceptible to M. persicae in terms of settling preference, phloem access, and phloem sap drainage.

Re-targeting of a plant defense protease by a cyst nematode effector.

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector-coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two-hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain-like cysteine protease (PLCP) 'Responsive to Dehydration 21A' (RD21A), which has been shown to function in the plant defense response. Activity-based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re-localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two-hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector-mediated re-localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense-inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.

Disruption of the Pathogenicity and Sex Ratio of the Beet Cyst Nematode Heterodera schachtii by Host-Delivered RNA Interference.

The beet cyst nematode (BCN) Heterodera schachtii causes serious damage and yield losses in numerous important crops worldwide. This study examines the efficacy of three types of transgenic Arabidopsis RNA interference (RNAi) lines to decrease the biological activity of this devastating nematode. The first RNAi construct (E1E2-RNAi) targets two nematode endoglucanase genes, which are involved in BCN pathogenicity, the second construct (MSP-RNAi) contains a fragment corresponding to the major sperm protein transcript necessary for BCN development and reproduction, and the third construct (E1E2MSP-RNAi) comprises all three target fragments. Transcript expression profiles of the target genes in all biological stages of the nematode were determined for the initial inoculated population and the resulting progeny. Bioassay data under indoor aseptic cultivation indicated that feeding on these RNAi lines did not affect pathogenic activity and reproductive capacity of the initial population, whereas inoculating the progeny into new transgenic plants corresponding with the lines from which they were recovered reduced the nematode penetration and the number of eggs per cyst. In addition, the male/female ratio increased more than the double, and the effects of RNAi continued in the second generation of the nematodes, because the progeny derived from E1E2-RNAi and E1E2MSP-RNAi lines showed an impaired ability to infect wild-type plants.

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to Mpersicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner.

A cyst nematode effector binds to diverse plant proteins, increases nematode susceptibility and affects root morphology.

Cyst nematodes are plant-parasitic roundworms that are of significance in many cropping systems around the world. Cyst nematode infection is facilitated by effector proteins secreted from the nematode into the plant host. The cDNAs of the 25A01-like effector family are novel sequences that were isolated from the oesophageal gland cells of the soybean cyst nematode (Heterodera glycines). To aid functional characterization, we identified an orthologous member of this protein family (Hs25A01) from the closely related sugar beet cyst nematode H. schachtii, which infects Arabidopsis. Constitutive expression of the Hs25A01 CDS in Arabidopsis plants caused a small increase in root length, accompanied by up to a 22% increase in susceptibility to H. schachtii. A plant-expressed RNA interference (RNAi) construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii. These data document that Hs25A01 has physiological functions in planta and a role in cyst nematode parasitism. In vivo and in vitro binding assays confirmed the specific interactions of Hs25A01 with an Arabidopsis F-box-containing protein, a chalcone synthase and the translation initiation factor eIF-2 ß subunit (eIF-2bs), making these proteins probable candidates for involvement in the observed changes in plant growth and parasitism. A role of eIF-2bs in the mediation of Hs25A01 virulence function is further supported by the observation that two independent eIF-2bs Arabidopsis knock-out lines were significantly more susceptible to H. schachtii.

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.

GroEL from the endosymbiont Buchnera aphidicola betrays the aphid by triggering plant defense.

Aphids are sap-feeding plant pests and harbor the endosymbiont Buchnera aphidicola, which is essential for their fecundity and survival. During plant penetration and feeding, aphids secrete saliva that contains proteins predicted to alter plant defenses and metabolism. Plants recognize microbe-associated molecular patterns and induce pattern-triggered immunity (PTI). No aphid-associated molecular pattern has yet been identified. By mass spectrometry, we identified in saliva from potato aphids (Macrosiphum euphorbiae) 105 proteins, some of which originated from Buchnera, including the chaperonin GroEL. Because GroEL is a widely conserved bacterial protein with an essential function, we tested its role in PTI. Applying or infiltrating GroEL onto Arabidopsis (Arabidopsis thaliana) leaves induced oxidative burst and expression of PTI early marker genes. These GroEL-induced defense responses required the known coreceptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1. In addition, in transgenic Arabidopsis plants, inducible expression of groEL activated PTI marker gene expression. Moreover, Arabidopsis plants expressing groEL displayed reduced fecundity of the green peach aphid (Myzus persicae), indicating enhanced resistance against aphids. Furthermore, delivery of GroEL into tomato (Solanum lycopersicum) or Arabidopsis through Pseudomonas fluorescens, engineered to express the type III secretion system, also reduced potato aphid and green peach aphid fecundity, respectively. Collectively our data indicate that GroEL is a molecular pattern that triggers PTI.

Overexpression of the transcription factor RAP2.6 leads to enhanced callose deposition in syncytia and enhanced resistance against the beet cyst nematode Heterodera schachtii in Arabidopsis roots.

BACKGROUND: Cyst nematodes invade the roots of their host plants as second stage juveniles and induce a syncytium which is their source of nutrients throughout their life. A transcriptome analysis of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots has shown that gene expression in the syncytium is different from that of the root with thousands of genes upregulated or downregulated. Among the downregulated genes are many which code for defense-related proteins. One gene which is strongly downregulated codes for the ethylene response transcription factor RAP2.6. The genome of Arabidopsis contains 122 ERF transcription factor genes which are involved in a variety of developmental and stress responses. RESULTS: Expression of RAP2.6 was studied with RT-PCR and a promoter::GUS line. During normal growth conditions the gene was expressed especially in roots and stems. It was inducible by Pseudomonas syringae but downregulated in syncytia from a very early time point on. Overexpression of the gene enhanced the resistance against H. schachtii which was seen by a lower number of nematodes developing on these plants as well as smaller syncytia and smaller female nematodes. A T-DNA mutant had a reduced RAP2.6 transcript level but this did not further increase the susceptibility against H. schachtii. Neither overexpression lines nor mutants had an effect on P. syringae. Overexpression of RAP2.6 led to an elevated expression of JA-responsive genes during early time points after infection by H. schachtii. Syncytia developing on overexpression lines showed enhanced deposition of callose. CONCLUSIONS: Our results showed that H. schachtii infection is accompanied by a downregulation of RAP2.6. It seems likely that the nematodes use effectors to actively downregulate the expression of this and other defense-related genes to avoid resistance responses of the host plant. Enhanced resistance of RAP2.6 overexpression lines seemed to be due to enhanced callose deposition at syncytia which might interfere with nutrient import into syncytia.

Synergistic interaction of CLAVATA1, CLAVATA2, and RECEPTOR-LIKE PROTEIN KINASE 2 in cyst nematode parasitism of Arabidopsis.

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (CLE)-like effector proteins. These proteins act as ligand mimics of plant CLE peptides and are required for successful nematode infection. Previously, we showed that the CLV2/CORYNE (CRN) heterodimer receptor complex is required for nematode CLE signaling. However, there was only a partial reduction in nematode infection when this signaling was disrupted, indicating that there might be additional nematode CLE receptors. In this study, we demonstrate that CLV1 and RECEPTOR-LIKE PROTEIN KINASE 2/TOADSTOOL2 (RPK2), two additional receptors that can transmit the CLV3 signal independent of CLV2/CRN for shoot apical meristem maintenance, also play a role in nematode CLE perception. Localization studies showed that both receptors are expressed in nematode-induced syncytia. Infection assays with clv1 and rpk2 single mutants revealed a decrease in both nematode infection and syncytium size. Significantly, further reduction in nematode infection was observed when rpk2 was combined with clv1 and clv2 mutants. Taken together, our results indicate that parallel signaling pathways involving CLV1, CLV2, and RPK2 are important for nematode parasitism.

The cell walls of syncytia formed by Heterodera schachtii in Arabidopsis thaliana are abundant in methyl-esterified pectin.

Plant-parasitic cyst nematodes form a specialized feeding site, termed a syncytium, in the roots of host plants. Monoclonal antibodies to defined glycans, in addition to a cellulose-binding module, were used to characterize the cell walls of a functioning syncytia in situ. Cell walls of syncytia were found to contain cellulose, xyloglucan and mannan. Analysis of the pectin network revealed syncytial cell walls are abundant in homogalacturonan, which was heavily methyl-esterified. Arabinan was also detected and the results suggest the cell walls of syncytia are highly flexible.

Syncytia formed by adult female Heterodera schachtii in Arabidopsis thaliana roots have a distinct cell wall molecular architecture.

• Plant-parasitic cyst nematodes form a feeding site, termed a syncytium, through which the nematode obtains nutrients from the host plant to support nematode development. The structural features of cell walls of syncytial cells have yet to be elucidated. • Monoclonal antibodies to defined glycans and a cellulose-binding module were used to determine the cell wall architectures of syncytial and surrounding cells in the roots of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii. • Fluorescence imaging revealed that the cell walls of syncytia contain cellulose and the hemicelluloses xyloglucan and heteromannan. Heavily methyl-esterified pectic homogalacturonan and arabinan are abundant in syncytial cell walls; galactan could not be detected. This is suggestive of highly flexible syncytial cell walls. • This work provides important information on the structural architecture of the cell walls of this novel cell type and reveals factors that enable the feeding site to perform its functional requirements to support nematode development.