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Summary: Pathogenic fungi use secreted effector proteins to suppress immunity and support their infection, but effectors have also been reported from fungi that engage in nutritional symbioses with plants. Sequence‐based effector comparisons between pathogens and symbiotic arbuscular mycorrhizal (AM) fungi are hampered by the huge diversity of effector sequences even within closely related microbes. To find sequence‐divergent but structurally similar effectors shared between symbiotic and pathogenic fungi, we compared secreted protein structure models of the AM fungus Rhizophagus irregularis to known pathogen effectors. We identified proteins with structural similarity to known Fusarium oxysporum f. sp. lycopersici dual domain (FOLD) effectors, which occur in low numbers in several fungal pathogens. Contrastingly, FOLD genes from AM fungi (MycFOLDs) are found in enlarged and diversified gene families with higher levels of positive selection in their C‐terminal domains. Our structure model comparison suggests that MycFOLDs are similar to carbohydrate‐binding motifs. Different MycFOLD genes are expressed during colonisation of different hosts and MycFOLD‐17 transcripts accumulate in plant intracellular arbuscules. The exclusive presence of MycFOLDs across unrelated plant‐colonising fungi, their inducible expression, lineage‐specific sequence diversification and transcripts in arbuscules suggest that FOLD proteins act as effectors during plant colonisation of symbiotic and pathogenic fungi.

Arbuscular mycorrhizal (AM) fungi form mutualistic relationships with most land plant species. AM fungi have long been considered as ancient asexuals. Long-term clonal evolution would be remarkable for a eukaryotic lineage and suggests the importance of alternative mechanisms to promote genetic variability facilitating adaptation. Here, we assessed the potential of transposable elements for generating such genomic diversity. The dynamic expression of TEs during Rhizophagus irregularis spore development suggests ongoing TE activity. We find Mutator-like elements located near genes belonging to highly expanded gene families. Whole-genome epigenomic profiling of R. irregularis provides direct evidence of DNA methylation and small RNA production occurring at TE loci. Our results support a model in which TE activity shapes the genome, while DNA methylation and small RNA–mediated silencing keep their overproliferation in check. We propose that a well-controlled TE activity directly contributes to genome evolution in AM fungi.

Soil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the abundance of hyphal structures in mutant roots rely on staining and human scoring involving simple yet repetitive tasks which are prone to variation between experimenters. We developed Automatic Mycorrhiza Finder (AMFinder) which allows for automatic computer vision-based identification and quantification of AM fungal colonisation and intraradical hyphal structures on ink-stained root images using convolutional neural networks. AMFinder delivered high-confidence predictions on image datasets of roots of multiple plant hosts (Nicotiana benthamiana, Medicago truncatula, Lotus japonicus, Oryza sativa) and captured the altered colonisation in ram1-1, str, and smax1 mutants. A streamlined protocol for sample preparation and imaging allowed us to quantify mycobionts from the genera Rhizophagus, Claroideoglomus, Rhizoglomus and Funneliformis via flatbed scanning or digital microscopy, including dynamic increases in colonisation in whole root systems over time. AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses. AMFinder can be accessed at https://github.com/SchornacklabSLCU/amfinder.

Arbuscular mycorrhizal (AM) fungi form mutualistic relationships with most land plant species and have long been considered as ancient asexuals. Long-term clonal evolution would be remarkable for a eukaryotic lineage and suggests the importance of alternative mechanisms to promote genetic variability facilitating adaptation. Here, we assessed the potential of transposable elements (TEs) for generating genomic diversity. The dynamic expression of TEs during Rhizophagus irregularis spore development suggests ongoing TE activity. We find Mutator-like elements located near genes belonging to highly expanded gene families. Characterising the epigenomic status of R. irregularis provides evidence of DNA methylation and small RNA production occurring at TE loci. Our results support a potential role for TEs in shaping the genome, and roles for DNA methylation and small RNA-mediated silencing in regulating TEs. A well-controlled balance between TE activity and repression may therefore contribute to genome evolution in AM fungi.

SummarySoil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the relative abundance of intraradical hyphal structures in mutant roots rely on staining and human scoring involving simple, yet repetitive tasks prone to variations between experimenters.We developed the software AMFinder which allows for automatic computer vision-based identification and quantification of AM fungal colonisation and intraradical hyphal structures on ink-stained root images using convolutional neural networks.AMFinder delivered high-confidence predictions on image datasets of colonised roots ofMedicago truncatula,Lotus japonicus,Oryza sativaandNicotiana benthamianaobtained via flatbed scanning or digital microscopy enabling reproducible and transparent data analysis. A streamlined protocol for sample preparation and imaging allowed us to quantify dynamic increases in colonisation in whole root systems over time.AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses.AMFinder can be accessed here:https://github.com/SchornacklabSLCU/amfinder.git

Summary The cell wall is the primary interface between plant cells and their immediate environment and must balance multiple functionalities, including the regulation of growth, the entry of beneficial microbes, and protection against pathogens. Here, we demonstrate how API, a SCAR2 protein component of the SCAR/WAVE complex, controls the root cell wall architecture important for pathogenic oomycete and symbiotic bacterial interactions in legumes. A mutation in API results in root resistance to the pathogen Phytophthora palmivora and colonization defects by symbiotic rhizobia. Although api mutant plants do not exhibit significant overall growth and development defects, their root cells display delayed actin and endomembrane trafficking dynamics and selectively secrete less of the cell wall polysaccharide xyloglucan. Changes associated with a loss of API establish a cell wall architecture with altered biochemical properties that hinder P. palmivora infection progress. Thus, developmental stage-dependent modifications of the cell wall, driven by SCAR/WAVE, are important in balancing cell wall developmental functions and microbial invasion., Graphical Abstract, Highlights • The SCAR protein API controls actin and endomembrane trafficking dynamics • SCAR proteins of several plant species can support symbiosis and pathogen infection • A mutation in API affects specific biochemical properties of plant cell walls • An altered wall architecture results in root resistance to Phytophthora palmivora, Subtly altered plant cell walls can be decisive for disease. Gavrin et al. show that the Medicago SCAR/WAVE complex protein API controls actin cytoskeleton dynamics in roots. Local changes associated with a loss of API establish a cell wall architecture with altered biochemical properties that hinder infection progress by an oomycete pathogen.

Background Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce. Results We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features. Conclusions These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0379-1) contains supplementary material, which is available to authorized users.

Polyenergid fungi and oomycetes are phylogenetically distant but structurally similar. To address whether they share similar nuclear dynamics we carried out time-lapse imaging of fluorescently labelledPhytophthora palmivoranuclei. Nuclei underwent coordinated bi-directional movements during plant infection. Within hyphal networks growingin plantaor in axenic culture nuclei are either dragged passively with the cytoplasm or actively become rerouted toward nuclei-depleted hyphal sections and often display a very stretched shape. Benomyl-induced depolymerization of microtubules reduced active movements and the occurrence of stretched nuclei. A centrosome protein localized at the leading end of stretched nuclei, suggesting that like fungi astral microtubule-guided movements contribute to nuclear distribution within oomycete hyphae. The remarkable hydrodynamic shape adaptations ofPhytophthoranuclei contrast those in fungi and likely enable them to migrate over longer distances. Therefore, our work summarises mechanisms which enable a near equal nuclear distribution in an oomycete. We provide a basis for computational modelling of hydrodynamic nuclear deformation within branched tubular networks.

International audience; Plasmopara viticola is a biotrophic oomycete pathogen causing grapevine downy mildew. We characterized the repertoire of P. viticola effector proteins which may be translocated into plants to support the disease. We found several secreted proteins that contain canonical dEER motifs and conserved WY-domains but lack the characteristic RXLR motif reported previously from oomycete effectors. We cloned four candidates and showed that one of them, Pv33, induces plant cell death in grapevine and Nicotiana species. This activity is dependent on the nuclear localization of the protein. Sequence similar effectors were present in seven European, but in none of the tested American isolates. Together our work contributes a new type of conserved P. viticola effector candidates.

Despite their fungal morphology, oomycetes constitute a distinct group of protists related to brown algae and diatoms. Many oomycetes are pathogens and cause diseases of plants, insects, mammals, and humans. Extensive efforts have been made to understand the molecular basis of oomycete infection, but durable protection against these pathogens is yet to be achieved. We use a plant-pathogenic oomycete to decipher a key physiological aspect of oomycete growth and infection. We show that oomycete nuclei travel actively and over long distances within hyphae and during infection. Such movements require microtubules anchored on the centrosome. Nuclei hydrodynamically adapt their shape to travel in or against the flow. In contrast, fungi lack a centrosome and have much less flexible nuclei. Our findings provide a basis for modeling of flexible nuclear shapes in branched hyphal networks and may help in finding hard-to-evade targets to develop specific antioomycete strategies and achieve durable crop disease protection., Multinucleate fungi and oomycetes are phylogenetically distant but structurally similar. To address whether they share similar nuclear dynamics, we carried out time-lapse imaging of fluorescently labeled Phytophthora palmivora nuclei. Nuclei underwent coordinated bidirectional movements during plant infection. Within hyphal networks growing in planta or in axenic culture, nuclei either are dragged passively with the cytoplasm or actively become rerouted toward nucleus-depleted hyphal sections and often display a very stretched shape. Benomyl-induced depolymerization of microtubules reduced active movements and the occurrence of stretched nuclei. A centrosome protein localized at the leading end of stretched nuclei, suggesting that, as in fungi, astral microtubule-guided movements contribute to nuclear distribution within oomycete hyphae. The remarkable hydrodynamic shape adaptations of Phytophthora nuclei contrast with those in fungi and likely enable them to migrate over longer distances. Therefore, our work summarizes mechanisms which enable a near-equal nuclear distribution in an oomycete. We provide a basis for computational modeling of hydrodynamic nuclear deformation within branched tubular networks.

Graphical abstract, Highlights • Phytopathogen effectors converge on similar sets of host proteins in angiosperms. • Effectors may target host proteins and processes present across the green plant lineage. • Bryophyte model plants are promising systems to investigate effector–target relationships. Plant-associated microbes secrete effector proteins that subvert host cellular machinery to facilitate the colonization of plant tissues and cells. Accumulating data suggests that independently evolved effectors from bacterial, fungal, and oomycete pathogens may converge on a similar set of host proteins in certain angiosperm models, however, whether this concept is relevant throughout the green plant lineage is unknown. Here, we explore the idea that pathogen effector molecules target host proteins present across evolutionarily distant land plant lineages to promote disease. We discuss that host proteins targeted by phytopathogens or integrated into angiosperm immune receptors are likely found across green plant genomes, from early diverging non-vascular lineages (bryophytes) to flowering plants (angiosperms). This would suggest that independently evolved pathogens might manipulate their hosts by targeting `vulnerability’ hubs that are present across land plants. Future work focusing on accessible early divergent land plant model systems may therefore provide an insightful evolutionary backdrop for effector–target research.

Plant-pathogen interactions can result in dramatic visual changes in the host, such as galls, phyllody, pseudoflowers, and altered root-system architecture, indicating that the invading microbe has perturbed normal plant growth and development. These effects occur on a cellular level but range up to the organ scale, and they commonly involve attenuation of hormone homeostasis and deployment of effector proteins with varying activities to modify host cell processes. This review focuses on the cellular-reprogramming mechanisms of filamentous and bacterial plant pathogens that exhibit a biotrophic lifestyle for part, if not all, of their lifecycle in association with the host. We also highlight strategies for exploiting our growing knowledge of microbial host reprogramming to study plant processes other than immunity and to explore alternative strategies for durable plant resistance.

Rubber tree (Hevea brasiliensis Muell. Arg) is an important economic crop in Thailand. Leaf fall and black stripe diseases caused by the aggressive oomycete pathogen Phytophthora palmivora, cause deleterious damage on rubber tree growth leading to decrease of latex production. To gain insights into the molecular function of H. brasiliensis subtilisin-like serine proteases, the HbSPA, HbSPB, and HbSPC genes were transiently expressed in Nicotiana benthamiana via agroinfiltration. A functional protease encoded by HbSPA was successfully expressed in the apoplast of N. benthamiana leaves. Transient expression of HbSPA in N. benthamiana leaves enhanced resistance to P. palmivora, suggesting that HbSPA plays an important role in plant defense. P. palmivora Kazal-like extracellular protease inhibitor 10 (PpEPI10), an apoplastic effector, has been implicated in pathogenicity through the suppression of H. brasiliensis protease. Semi-quantitative RT-PCR revealed that the PpEPI10 gene was significantly up-regulated during colonization of rubber tree by P. palmivora. Concurrently, the HbSPA gene was highly expressed during infection. To investigate a possible interaction between HbSPA and PpEPI10, the recombinant PpEPI10 protein (rPpEPI10) was expressed in Escherichia coli and purified using affinity chromatography. In-gel zymogram and co-immunoprecipitation (co-IP) assays demonstrated that rPpEPI10 specifically inhibited and interacted with HbSPA. The targeting of HbSPA by PpEPI10 revealed a defense-counterdefense mechanism, which is mediated by plant protease and pathogen protease inhibitor, in H. brasiliensis-P. palmivora interactions.

BackgroundPlant-pathogenic oomycetes are responsible for economically important losses on crops worldwide. Phytophthora palmivora, a broad-host-range tropical relative of the potato late blight pathogen, causes rotting diseases in many important tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus.Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped understanding of how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce.ResultsWe employed the model plant N. benthamiana to study the P. palmivora root infections at the cellular and molecular level. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features.ConclusionsThese results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.

Plant roots are host to a multitude of filamentous microorganisms. Among these, arbuscular mycorrhizal fungi provide benefits to plants, while pathogens trigger diseases resulting in significant crop yield losses. It is therefore imperative to study processes which allow plants to discriminate detrimental and beneficial interactions in order to protect crops from diseases while retaining the ability for sustainable bio-fertilisation strategies. Accumulating evidence suggests that some symbiosis processes also affect plant-pathogen interactions. A large part of this overlap likely constitutes plant developmental processes. Moreover, microbes utilise effector proteins to interfere with plant development. Here we list relevant recent findings on how plant-microbe interactions intersect with plant development and highlight future research leads.

Background Oomycetes are a group of filamentous microorganisms that includes both animal and plant pathogens and causes major agricultural losses. Phytophthora species can infect most crops and plants from natural ecosystems. Despite their tremendous economic and ecologic importance, few effective methods exist for limiting the damage caused by these species. New solutions are required, and their development will require improvements in our understanding of the molecular events governing infection by these pathogens. In this study, we characterized the genetic program activated during penetration of the plant by the soil-borne pathogen Phytophthora parasitica. Results Using all the P. parasitica sequences available in public databases, we generated a custom oligo-array and performed a transcriptomic analysis of the early events of Arabidopsis thaliana infection. We characterized biological stages, ranging from the appressorium-mediated penetration of the pathogen into the roots to the occurrence of first dead cells in the plant. We identified a series of sequences that were transiently modulated during host penetration. Surprisingly, we observed an overall down regulation of genes encoding proteins involved in lipid and sugar metabolism, and an upregulation of functions controlling the transport of amino acids. We also showed that different groups of genes were expressed by P. parasitica during host penetration and the subsequent necrotrophic phase. Differential expression patterns were particularly marked for cell wall-degrading enzymes and other proteins involved in pathogenicity, including RXLR effectors. By transforming P. parasitica with a transcriptional fusion with GFP, we showed that an RXLR-ecoding gene was expressed in the appressorium and infectious hyphae during infection of the first plant cell. Conclusion We have characterized the genetic program activated during the initial invasion of plant cells by P. parasitica. We showed that a specific set of proteins, including effectors, was mobilized for penetration and to facilitate infection. Our detection of the expression of an RXLR encoding gene by the appressorium and infection hyphae highlights a role of this structure in the manipulation of the host cells. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-538) contains supplementary material, which is available to authorized users.

International audience; Pathogenic oomycetes have evolved RXLR effectors to thwart plant defense mechanisms and invade host tissues. We analysed the function of one of these effectors (Penetration-Specific Effector 1 (PSE1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete Phytophthora parasitica. Expression of PSE1 protein in tobacco (Nicotiana tabacum and Nicotiana benthamiana) leaves and in Arabidopsis thaliana plants was used to assess the role of this effector in plant physiology and in interactions with pathogens. A pharmacological approach and marker lines were used to charcterize the A. thaliana phenotypes. Expression of PSE1 in A. thaliana led to developmental perturbations associated with low concentrations of auxin at the root apex. This modification of auxin content was associated with an altered distribution of the PIN4 and PIN7 auxin efflux carriers. The PSE1 protein facilitated plant infection: it suppressed plant cell death activated by Pseudomonas syringae avirulence gene AvrPto and Phytophthora cryptogea elicitin cryptogein in tobacco and exacerbated disease symptoms upon inoculation of transgenic A. thaliana plantlets with P. parasitica in an auxin-dependant manner. We propose that P.parasitica secretes the PSE1 protein during the penetration process to favour the infection by locally modulating the auxin content. These results support the hypothesis that effectors from plant pathogens may act on a limited set of targets, including hormones.

Root-knot nematodes (RKN) are obligate biotrophic parasites that settle close to the vascular tissues in roots, where they induce the differentiation of specialized feeding cells and maintain a compatible interaction for 3 to 8 weeks. Transcriptome analyses of the plant response to parasitic infection have shown that plant defenses are strictly controlled during the interaction. This suggests that, similar to other pathogens, RKN secrete effectors that suppress host defenses. We show here that Mi-CRT, a calreticulin (CRT) secreted by the nematode into the apoplasm of infected tissues, plays an important role in infection success, because Mi-CRT knockdown by RNA interference affected the ability of the nematodes to infect plants. Stably transformed Arabidopsis thaliana plants producing the secreted form of Mi-CRT were more susceptible to nematode infection than wild-type plants. They were also more susceptible to infection with another root pathogen, the oomycete Phytophthora parasitica. Mi-CRT overexpression in A. thaliana suppressed the induction of defense marker genes and callose deposition after treatment with the pathogen-associated molecular pattern elf18. Our results show that Mi-CRT secreted in the apoplasm by the nematode has a role in the suppression of plant basal defenses during the interaction.