Endophytic fungi related to the ash dieback causal agent encode signatures of pathogenicity on European ash.

Tree diseases constitute a significant threat to biodiversity worldwide. Pathogen discovery in natural habitats is of vital importance to understanding current and future threats and prioritising efforts towards developing disease management strategies. Ash dieback is a fungal disease of major conservational concern that is infecting common ash trees, Fraxinus excelsior, in Europe. The disease is caused by a non-native fungal pathogen, Hymenoscyphus fraxineus. Other dieback causing-species have not previously been identified in the genus Hymenoscyphus. Here, we discover the pathogenicity potential of two newly identified related species of Asian origin, H. koreanus and H. occultus, and one Europe-native related species, H. albidus. We sequence the genomes of all three Hymenoscyphus species and compare them to that of H. fraxineus. Phylogenetic analysis of core eukaryotic genes identified H. albidus and H. koreanus as sister species, whilst H. occultus diverged prior to these and H. fraxineus. All four Hymenoscyphus genomes are of comparable size (55-62 Mbp) and GC contents (42-44%) and encode for polymorphic secretomes. Surprisingly, 1133 predicted secreted proteins are shared between the ash dieback pathogen H. fraxineus and the three related Hymenoscyphus endophytes. Amongst shared secreted proteins are cell death-inducing effector candidates, such as necrosis, and ethylene-inducing peptide 1-like proteins, Nep1-like proteins, that are upregulated during in planta growth of all Hymenoscyphus species. Indeed, pathogenicity tests showed that all four related Hymenoscyphus species develop pathogenic growth on European ash stems, with native H. albidus being the least virulent. Our results identify the threat Hymenoscypohus species pose to the survival of European ash trees, and highlight the importance of promoting pathogen surveillance in environmental landscapes. Identifying new pathogens and including them in the screening for durable immunity of common ash trees is key to the long-term survival of ash in Europe.

Profile of the in silico secretome of the palm dieback pathogen, Fusarium oxysporum f. sp. albedinis, a fungus that puts natural oases at risk.

Understanding biotic changes that occur alongside climate change constitute a research priority of global significance. Here, we address a plant pathogen that poses a serious threat to life on natural oases, where climate change is already taking a toll and severely impacting human subsistence. Fusarium oxysporum f. sp. albedinis is a pathogen that causes dieback disease on date palms, a tree that provides several critical ecosystem services in natural oases; and consequently, of major importance in this vulnerable habitat. Here, we assess the current state of global pathogen spread, we annotate the genome of a sequenced pathogen strain isolated from the native range and we analyse its in silico secretome. The palm dieback pathogen secretes a large arsenal of effector candidates including a variety of toxins, a distinguished profile of secreted in xylem proteins (SIX) as well as an expanded protein family with an N-terminal conserved motif [SG]PC[KR]P that could be involved in interactions with host membranes. Using agrobiodiversity as a strategy to decrease pathogen infectivity, while providing short term resilient solutions, seems to be widely overcome by the pathogen. Hence, the urgent need for future mechanistic research on the palm dieback disease and a better understanding of pathogen genetic diversity.

The Multiple Facets of Plant-Fungal Interactions Revealed Through Plant and Fungal Secretomics.

The plant secretome is usually considered in the frame of proteomics, aiming at characterizing extracellular proteins, their biological roles and the mechanisms accounting for their secretion in the extracellular space. In this review, we aim to highlight recent results pertaining to secretion through the conventional and unconventional protein secretion pathways notably those involving plant exosomes or extracellular vesicles. Furthermore, plants are well known to actively secrete a large array of different molecules from polymers (e.g. extracellular RNA and DNA) to small compounds (e.g. ATP, phytochemicals, secondary metabolites, phytohormones). All of these play pivotal roles in plant-fungi (or oomycetes) interactions, both for beneficial (mycorrhizal fungi) and deleterious outcomes (pathogens) for the plant. For instance, recent work reveals that such secretion of small molecules by roots is of paramount importance to sculpt the rhizospheric microbiota. Our aim in this review is to extend the definition of the plant and fungal secretomes to a broader sense to better understand the functioning of the plant/microorganisms holobiont. Fundamental perspectives will be brought to light along with the novel tools that should support establishing an environment-friendly and sustainable agriculture.

The ash dieback invasion of Europe was founded by two genetically divergent individuals.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

Effector candidates in the secretome of Piriformospora indica, a ubiquitous plant-associated fungus.

One of the emerging systems in plant-microbe interaction is the study of proteins, referred to as effectors, secreted by microbes in order to modulate host cells function and structure and to promote microbial growth on plant tissue. Current knowledge on fungal effectors derives mainly from biotrophic and hemibiotrophic plant fungal pathogens that have a limited host range. Here, we focus on effectors of Piriformospora indica, a soil borne endophyte forming intimate associations with roots of a wide range of plant species. Complete genome sequencing provides an opportunity to investigate the role of effectors during the interaction of this mutualistic fungus with plants. We describe in silico analyses to predict effectors of P. indica and we explore effector features considered here to mine a high priority protein list for functional analysis.

Challenges and progress towards understanding the role of effectors in plant-fungal interactions.

Both mutualistic and biotrophic pathogenic fungi rely on living host plants for growth and reproduction and must modify host cell structure and function for successful infection. The deployment of a diverse set of secreted virulence determinants referred to as 'effectors', many of which are directly delivered into the host cell, is postulated to be the key to host infection. This review provides a snapshot of the current progress in fungal effector biology. Recent genome sequencing of rust and powdery mildew obligate biotrophs has provided insight into the repertoires of potential effectors of these highly specialised pathogens. Identification of the first host-translocated effectors from mutualistic fungi has revealed that these fungi also manipulate host cells through effectors. The biological activities of some fungal effectors are just beginning to be revealed, while much uncertainty still surrounds the mechanisms of transport into host cells.

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance.

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

Lipid binding activities of flax rust AvrM and AvrL567 effectors.

Effectors are pathogen-encoded proteins that are thought to facilitate infection by manipulation of host cells. Evidence showing that the effectors of some eukaryotic plant pathogens are able to interact directly with cytoplasmic host proteins indicates that translocation of these proteins into host cells is an important part of infection. Recently, we showed that the flax rust effectors AvrM and AvrL567 are able to internalize into plant cells in the absence of the pathogen. Further, N-terminal sequences that were sufficient for uptake were identified for both these proteins. In light of the possibility that the internalization of fungal and oomycete effectors may require binding to specific phospholipids, the lipid binding activities of AvrM and AvrL567 mutants with different abilities to enter cells were tested. While AvrL567 was not found to bind to phospholipids, AvrM bound strongly to phosphatidyl inositol, phosphatidyl inositol monophosphates and phosphatidyl serine. However, a fragment of AvrM sufficient to direct uptake of a fusion protein into plant cells did not bind to these phospholipids. Thus, our results do not support the role of specific binding of AvrM and AvrL567 to phospholipids for uptake into the plant cytoplasm.

Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen.

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.

Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance.

Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.

Recent progress in discovery and functional analysis of effector proteins of fungal and oomycete plant pathogens.

Plant-pathogen interactions involve processes of pathogen offence, host defence and pathogen counter-attack that are commonly played out using molecules secreted by hosts and pathogens. Secreted pathogen molecules involved in these events, referred to as 'effectors', function either in the plant extracellular space (apoplast) or inside of plant cells after translocation from the pathogen. These molecules have evolved as virulence factors that can be detected by polymorphic host resistance proteins. Advances are being made in the identification and in understanding the evolution of effectors and of host uptake signals used by eukaryotic effectors to enter host cells.

In the trenches of plant pathogen recognition: Role of NB-LRR proteins.

As in nearly every discipline of plant biology, new insights are constantly changing our understanding of plant immunity. It is now clear that plant immunity is controlled by two layers of inducible responses: basal responses triggered by conserved microbial features and specific responses triggered by gene-for-gene recognition of pathogen effector proteins by host resistance (R) proteins. The nucleotide-binding domain leucine-rich repeat (NB-LRR) class of R proteins plays a major role in the combat against a wide range of plant pathogens. The variation that has been generated and is maintained within these conserved proteins has diversified their specificity, subcellular localisations, activation and recognition mechanisms, allowing them to specifically adapt to different plant-pathogen interaction systems. This review addresses recent advances in the molecular role of NB-LRR proteins in pathogen recognition and activation of plant defence responses.

Identification of a protein from rust fungi transferred from haustoria into infected plant cells.

The formation of haustoria is one of the hallmarks of the interaction of obligate biotrophic fungi with their host plants. In addition to their role in nutrient uptake, it is hypothesized that haustoria are actively involved in establishing and maintaining the biotrophic relationship. We have identified a 24.3-kDa protein that exhibited a very unusual allocation. Rust transferred protein 1 from Uromyces fabae (Uf-RTP1p) was not only detected in the host parasite interface, the extrahaustorial matrix, but also inside infected plant cells by immunofluorescence and electron microscopy. Uf-RTP1p does not exhibit any similarity to sequences currently listed in the public databases. However, we identified a homolog of Uf-RTP1p in the related rust fungus Uromyces striatus (Us-RTP1p). The localization of Uf-RTP1p and Us-RTP1p inside infected plant cells was confirmed, using four independently raised polyclonal antibodies. Depending on the developmental stage of haustoria, Uf-RTP1p was found in increasing amounts in host cells, including the host nucleus. Putative nuclear localization signals (NLS) were found in the predicted RTP1p sequences. However, functional efficiency could only be verified for the Uf-RTP1p NLS by means of green fluorescent protein fusions in transformed tobacco protoplasts. Western blot analysis indicated that Uf-RTP1p and Us-RTP1p most likely enter the host cell as N-glycosylated proteins. However, the mechanism by which they cross the extrahaustorial membrane and accumulate in the host cytoplasm is unknown. The localization of RTP1p suggests that it might play an important role in the maintenance of the biotrophic interaction.