A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging ß-glucan decasaccharide to subvert immune responses.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) ß-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved ß-1,3;1,6-glucan decasaccharide (ß-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight ß-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by ß-1,3-endoglucanases due to the presence of three ß-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of ß-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.

Plant species-specific recognition of long and short ß-1,3-linked glucans is mediated by different receptor systems.

Plants survey their environment for the presence of potentially harmful or beneficial microbes. During colonization, cell surface receptors perceive microbe-derived or modified-self ligands and initiate appropriate responses. The recognition of fungal chitin oligomers and the subsequent activation of plant immunity are well described. In contrast, the mechanisms underlying ß-glucan recognition and signaling activation remain largely unexplored. Here, we systematically tested immune responses towards different ß-glucan structures and show that responses vary between plant species. While leaves of the monocots Hordeum vulgare and Brachypodium distachyon can recognize longer (laminarin) and shorter (laminarihexaose) ß-1,3-glucans with responses of varying intensity, duration and timing, leaves of the dicot Nicotiana benthamiana activate immunity in response to long ß-1,3-glucans, whereas Arabidopsis thaliana and Capsella rubella perceive short ß-1,3-glucans. Hydrolysis of the ß-1,6 side-branches of laminarin demonstrated that not the glycosidic decoration but rather the degree of polymerization plays a pivotal role in the recognition of long-chain ß-glucans. Moreover, in contrast to the recognition of short ß-1,3-glucans in A. thaliana, perception of long ß-1,3-glucans in N. benthamiana and rice is independent of CERK1, indicating that ß-glucan recognition may be mediated by multiple ß-glucan receptor systems.

Unraveling the sugar code: the role of microbial extracellular glycans in plant-microbe interactions.

To defend against microbial invaders but also to establish symbiotic programs, plants need to detect the presence of microbes through the perception of molecular signatures characteristic of a whole class of microbes. Among these molecular signatures, extracellular glycans represent a structurally complex and diverse group of biomolecules that has a pivotal role in the molecular dialog between plants and microbes. Secreted glycans and glycoconjugates such as symbiotic lipochitooligosaccharides or immunosuppressive cyclic ß-glucans act as microbial messengers that prepare the ground for host colonization. On the other hand, microbial cell surface glycans are important indicators of microbial presence. They are conserved structures normally exposed and thus accessible for plant hydrolytic enzymes and cell surface receptor proteins. While the immunogenic potential of bacterial cell surface glycoconjugates such as lipopolysaccharides and peptidoglycan has been intensively studied in the past years, perception of cell surface glycans from filamentous microbes such as fungi or oomycetes is still largely unexplored. To date, only few studies have focused on the role of fungal-derived cell surface glycans other than chitin, highlighting a knowledge gap that needs to be addressed. The objective of this review is to give an overview on the biological functions and perception of microbial extracellular glycans, primarily focusing on their recognition and their contribution to plant-microbe interactions.

Serendipita indica E5'NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization.

Extracellular adenosine 5'-triphosphate (eATP) is an essential signaling molecule that mediates different cellular processes through its interaction with membrane-associated receptor proteins in animals and plants. eATP regulates plant growth, development, and responses to biotic and abiotic stresses. Its accumulation in the apoplast induces ROS production and cytoplasmic calcium increase mediating a defense response to invading microbes. We show here that perception of extracellular nucleotides, such as eATP, is important in plant-fungus interactions and that during colonization by the beneficial root endophyte Serendipita indica eATP accumulates in the apoplast at early symbiotic stages. Using liquid chromatography-tandem mass spectrometry, and cytological and functional analysis, we show that S. indica secrets SiE5'NT, an enzymatically active ecto-5'-nucleotidase capable of hydrolyzing nucleotides in the apoplast. Arabidopsis thaliana lines producing extracellular SiE5'NT are significantly better colonized, have reduced eATP levels, and altered responses to biotic stresses, indicating that SiE5'NT functions as a compatibility factor. Our data suggest that extracellular bioactive nucleotides and their perception play an important role in fungus-root interactions and that fungal-derived enzymes can modify apoplastic metabolites to promote fungal accommodation.

The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import.

BACKGROUND: The mitochondrial intermembrane space (IMS) is home to proteins fulfilling numerous essential cellular processes, particularly in metabolism and mitochondrial function. All IMS proteins are nuclear encoded and synthesized in the cytosol and must therefore be correctly targeted and transported to the IMS, either through mitochondrial targeting sequences or conserved cysteines and the mitochondrial disulfide relay system. The mitochondrial oxidoreductase MIA40, which catalyzes disulfide formation in the IMS, is imported by the combined action of the protein AIFM1 and MIA40 itself. Here, we characterized the function of the conserved highly negatively charged C-terminal region of human MIA40. RESULTS: We demonstrate that the C-terminal region is critical during posttranslational mitochondrial import of MIA40, but is dispensable for MIA40 redox function in vitro and in intact cells. The C-terminal negatively charged region of MIA40 slowed import into mitochondria, which occurred with a half-time as slow as 90 min. During this time, the MIA40 precursor persisted in the cytosol in an unfolded state, and the C-terminal negatively charged region served in protecting MIA40 from proteasomal degradation. This stabilizing property of the MIA40 C-terminal region could also be conferred to a different mitochondrial precursor protein, COX19. CONCLUSIONS: Our data suggest that the MIA40 precursor contains the stabilizing information to allow for postranslational import of sufficient amounts of MIA40 for full functionality of the essential disulfide relay. We thereby provide for the first time mechanistic insights into the determinants controlling cytosolic surveillance of IMS precursor proteins.

A secreted fungal histidine- and alanine-rich protein regulates metal ion homeostasis and oxidative stress.

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S. indica, and inhibits H2 O2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization.

The RxLR Motif of the Host Targeting Effector AVR3a of Phytophthora infestans Is Cleaved before Secretion.

When plant-pathogenic oomycetes infect their hosts, they employ a large arsenal of effector proteins to establish a successful infection. Some effector proteins are secreted and are destined to be translocated and function inside host cells. The largest group of translocated proteins from oomycetes is the RxLR effectors, defined by their conserved N-terminal Arg-Xaa-Leu-Arg (RxLR) motif. However, the precise role of this motif in the host cell translocation process is unclear. Here, detailed biochemical studies of the RxLR effector AVR3a from the potato pathogen Phytophthora infestans are presented. Mass spectrometric analysis revealed that the RxLR sequence of native AVR3a is cleaved off prior to secretion by the pathogen and the N terminus of the mature effector was found likely to be acetylated. High-resolution NMR structure analysis of AVR3a indicates that the RxLR motif is well accessible to potential processing enzymes. Processing and modification of AVR3a is to some extent similar to events occurring with the export element (PEXEL) found in malaria effector proteins from Plasmodium falciparum These findings imply a role for the RxLR motif in the secretion of AVR3a by the pathogen, rather than a direct role in the host cell entry process itself.

FGB1 and WSC3 are in planta-induced ß-glucan-binding fungal lectins with different functions.

In the root endophyte Serendipita indica, several lectin-like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in ß-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long-chain ß1-3-glucan but has no affinity for shorter ß1-3- or ß1-6-linked glucose oligomers. Comparative analysis with the previously identified ß-glucan-binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require ß1-6-linked glucose for efficient binding to branched ß1-3-glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two ß-glucan-binding lectins. Our results highlight the importance of the ß-glucan cell wall component in plant-fungus interactions and the potential of ß-glucan-binding lectins as specific detection tools for fungi in vivo.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate-dependent manner.

The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate-modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host-microbe interactions.

Avirulence protein 3a (AVR3a) from the potato pathogen Phytophthora infestans forms homodimers through its predicted translocation region and does not specifically bind phospholipids.

The mechanism of translocation of RxLR effectors from plant pathogenic oomycetes into the cytoplasm of their host is currently the object of intense research activity and debate. Here, we report the biochemical and thermodynamic characterization of the Phytophthora infestans effector AVR3a in vitro. We show that the amino acids surrounding the RxLR leader mediate homodimerization of the protein. Dimerization was considerably attenuated by a localized mutation within the RxLR motif that was previously described to prevent translocation of the protein into host. Importantly, we confirm that the reported phospholipid-binding properties of AVR3a are mediated by its C-terminal effector domain, not its RxLR leader. However, we show that the observed phospholipid interaction is attributable to a weak association with denatured protein molecules and is therefore most likely physiologically irrelevant.

In vitro translocation experiments with RxLR-reporter fusion proteins of Avr1b from Phytophthora sojae and AVR3a from Phytophthora infestans fail to demonstrate specific autonomous uptake in plant and animal cells.

Plant-pathogenic oomycetes have a large set of secreted effectors that can be translocated into their host cells during infection. One group of these effectors are the RxLR effectors for which it has been shown, in a few cases, that the RxLR motif is important for their translocation. It has been suggested that the RxLR-leader sequences alone are enough to translocate the respective effectors into eukaryotic cells through binding to surface-exposed phosphoinositol-3-phosphate. These conclusions were primary based on translocation experiments conducted with recombinant fusion proteins whereby the RxLR leader of RxLR effectors (i.e., Avr1b from Phytophthora sojae) were fused to the green fluorescent protein reporter-protein. However, we failed to observe specific cellular uptake for a comparable fusion protein where the RxLR leader of the P. infestans AVR3a was fused to monomeric red fluorescent protein. Therefore, we reexamined the ability of the reported P. sojae AVR1b RxLR leader to enter eukaryotic cells. Different relevant experiments were performed in three independent laboratories, using fluorescent reporter fusion constructs of AVR3a and Avr1b proteins in a side-by-side comparative study on plant tissue and human and animal cells. We report that we were unable to obtain conclusive evidence for specific RxLR-mediated translocation.

A GH81-type ß-glucan-binding protein enhances colonization by mutualistic fungi in barley.

Cell walls are important interfaces of plant-fungal interactions, acting as robust physical and chemical barriers against invaders. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other's cell walls. These enzymes release various sugar oligomers into the apoplast, some of which activate host immunity via surface receptors. Recent characterization of cell walls from plant-colonizing fungi has emphasized the abundance of ß-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated ß-glucan laminarin. Thereby, we identified a plant glycoside hydrolase family 81-type glucan-binding protein (GBP) as a ß-glucan interactor. Mutation of GBP1 and its only paralog, GBP2, in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of colonization was accompanied by enhanced responses at the host cell wall, including an extension of callose-containing cell wall appositions. Moreover, GBP mutation in barley also reduced fungal biomass in roots by the hemibiotrophic pathogen Bipolaris sorokiniana and inhibited the penetration success of the obligate biotrophic leaf pathogen Blumeria hordei. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi-a role that has potentially been appropriated by barley-adapted pathogens.

The fungal root endophyte Serendipita vermifera displays inter-kingdom synergistic beneficial effects with the microbiota in Arabidopsis thaliana and barley.

Plant root-associated bacteria can confer protection against pathogen infection. By contrast, the beneficial effects of root endophytic fungi and their synergistic interactions with bacteria remain poorly defined. We demonstrate that the combined action of a fungal root endophyte from a widespread taxon with core bacterial microbiota members provides synergistic protection against an aggressive soil-borne pathogen in Arabidopsis thaliana and barley. We additionally reveal early inter-kingdom growth promotion benefits which are host and microbiota composition dependent. Using RNA-sequencing, we show that these beneficial activities are not associated with extensive host transcriptional reprogramming but rather with the modulation of expression of microbial effectors and carbohydrate-active enzymes.

Towards understanding the virulence functions of RXLR effectors of the oomycete plant pathogen Phytophthora infestans.

Plant pathogens establish infection by secretion of effector proteins that may be delivered inside host cells to manipulate innate immunity. It is increasingly apparent that the ubiquitin proteasome system (UPS) contributes significantly to the regulation of plant defences and, as such, is a target for pathogen effectors. Bacterial effectors delivered by the type III and IV secretion systems have been shown to interact with components of the host UPS. Some of these effectors possess functional domains that are conserved in UPS enzymes, whilst others contain novel domains with ubiquitination activities. Relatively little is known about effector activities in eukaryotic microbial plant pathogens. Nevertheless, effectors from oomycetes that contain an RXLR motif for translocation to the inside of plant cells have been shown to suppress host defences. Annotation of the genome of one such oomycete, the potato late blight pathogen Phytophthora infestans, and protein-protein interaction assays to discover host proteins targeted by the RXLR effector AVR3a, have revealed that this eukaryotic plant pathogen also has the potential to manipulate host plant UPS functions.

Cell entry of a host-targeting protein of oomycetes requires gp96.

The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants.

The fungal-specific ß-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants.

ß-glucans are well-known modulators of the immune system in mammals but little is known about ß-glucan triggered immunity in planta. Here we show by isothermal titration calorimetry, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy that the FGB1 gene from the root endophyte Piriformospora indica encodes for a secreted fungal-specific ß-glucan-binding lectin with dual function. This lectin has the potential to both alter fungal cell wall composition and properties, and to efficiently suppress ß-glucan-triggered immunity in different plant hosts, such as Arabidopsis, barley and Nicotiana benthamiana. Our results hint at the existence of fungal effectors that deregulate innate sensing of ß-glucan in plants.

Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding.

Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export.