The N-terminal domains of NLR immune receptors exhibit structural and functional similarities across divergent plant lineages.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors in plants. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade. Here, we describe an extended spectrum of NLR diversity across divergent plant lineages and demonstrate the structural and functional similarities of N-terminal domains that trigger immune responses. We show that the broadly distributed coiled-coil (CC) and toll/interleukin-1 receptor (TIR) domain families of non-flowering plants retain immune-related functions through trans-lineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to non-flowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable to motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the non-flowering liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death. Moreover, comparative transcriptomic analyses of CCMAEPL activity delineated a common CC-mediated immune program shared across evolutionarily divergent non-flowering and flowering plants. Collectively, our findings highlight the ancestral nature of NLR-mediated immunity during plant evolution that dates its origin to at least ∼500 million years ago.

Convergent evolution of water-conducting cells in Marchantia recruited the ZHOUPI gene promoting cell wall reinforcement and programmed cell death.

A key adaptation of plants to life on land is the formation of water-conducting cells (WCCs) for efficient long-distance water transport. Based on morphological analyses it is thought that WCCs have evolved independently on multiple occasions. For example, WCCs have been lost in all but a few lineages of bryophytes but, strikingly, within the liverworts a derived group, the complex thalloids, has evolved a novel externalized water-conducting tissue composed of reinforced, hollow cells termed pegged rhizoids. Here, we show that pegged rhizoid differentiation in Marchantia polymorpha is controlled by orthologs of the ZHOUPI and ICE bHLH transcription factors required for endosperm cell death in Arabidopsis seeds. By contrast, pegged rhizoid development was not affected by disruption of MpNAC5, the Marchantia ortholog of the VND genes that control WCC formation in flowering plants. We characterize the rapid, genetically controlled programmed cell death process that pegged rhizoids undergo to terminate cellular differentiation and identify a corresponding upregulation of conserved putative plant cell death effector genes. Lastly, we show that ectopic expression of MpZOU1 increases production of pegged rhizoids and enhances drought tolerance. Our results support that pegged rhizoids evolved independently of other WCCs. We suggest that elements of the genetic control of developmental cell death are conserved throughout land plants and that the ZHOUPI/ICE regulatory module has been independently recruited to promote cell wall modification and programmed cell death in liverwort rhizoids and in the endosperm of flowering plant seed.

Mucoromycotina 'fine root endophytes': a new molecular model for plant-fungal mutualisms?

The most studied plant-fungal symbioses to date are the interactions between plants and arbuscular mycorrhizal (AM) fungi of the Glomeromycotina clade. Advancements in phylogenetics and microbial community profiling have distinguished a group of symbiosis-forming fungi that resemble AM fungi as belonging instead to the Mucoromycotina. These enigmatic fungi are now known as Mucoromycotina 'fine root endophytes' and could provide a means to understand the origins of plant-fungal symbioses. Most of our knowledge of the mechanisms of fungal symbiosis comes from investigations using AM fungi. Here, we argue that inclusion of Mucoromycotina fine root endophytes in future studies will expand our understanding of the mechanisms, evolution, and ecology of plant-fungal symbioses.

Stiffness transitions in new walls post-cell division differ between Marchantia polymorpha gemmae and Arabidopsis thaliana leaves.

Plant morphogenesis is governed by the mechanics of the cell wall-a stiff and thin polymeric box that encloses the cells. The cell wall is a highly dynamic composite material. New cell walls are added during cell division. As the cells continue to grow, the properties of cell walls are modulated to undergo significant changes in shape and size without breakage. Spatial and temporal variations in cell wall mechanical properties have been observed. However, how they relate to cell division remains an outstanding question. Here, we combine time-lapse imaging with local mechanical measurements via atomic force microscopy to systematically map the cell wall's age and growth, with their stiffness. We make use of two systems, Marchantia polymorpha gemmae, and Arabidopsis thaliana leaves. We first characterize the growth and cell division of M. polymorpha gemmae. We then demonstrate that cell division in M. polymorpha gemmae results in the generation of a temporary stiffer and slower-growing new wall. In contrast, this transient phenomenon is absent in A. thaliana leaves. We provide evidence that this different temporal behavior has a direct impact on the local cell geometry via changes in the junction angle. These results are expected to pave the way for developing more realistic plant morphogenetic models and to advance the study into the impact of cell division on tissue growth.

EVO-MPMI: From fundamental science to practical applications.

In the unending coevolutionary dance between plants and microbes, each player impacts the evolution of the other. Here, we provide an overview of the burgeoning field of evolutionary molecular plant-microbe interactions (EVO-MPMI)-the study of mechanisms of plant-microbe interactions in the context of their evolutionary history-tracing its progression from foundational science to practical implementation. We present a snapshot of current research and delve into central concepts, such as conserved features and convergent evolution, as well as methodologies such as ancestral reconstruction. Moreover, we shed light on the practical applications of EVO-MPMI, particularly within the realm of disease control. Looking ahead, we discuss potential future trajectories for EVO-MPMI research, spotlighting the innovative tools and technologies propelling the discipline forward.

Antibodies for a bespoke plant immunity.

Plants do not have antibodies. However, in a recent Science article, Kourelis and Marchal et al. have demonstrated that plant immune receptors can be retrofitted with animal antibodies to provide plants potentially with hundreds and thousands of options to perceive attacking microbes. This is the dawn of bespoke plant immunity.

A pathogen effector FOLD diversified in symbiotic fungi.

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.

FIRE Mimics a 14-3-3-Binding Motif to Promote Phytophthora palmivora Infection.

The oomycete Phytophthora palmivora infects a wide range of tropical crops worldwide. Like other filamentous plant pathogens, it secretes effectors to colonize plant tissues. Here, we characterize FIRE, an RXLR effector that contains a canonical mode I 14-3-3 phospho-sensor-binding motif that is conserved in effectors of several Phytophthora species. FIRE is phosphorylated in planta and interacts with multiple 14-3-3 proteins. Binding is sensitive to the R18 14-3-3 inhibitor. FIRE promotes plant susceptibility and co-localizes with its target around haustoria. This work uncovers a new type of oomycete effector target mechanism. It demonstrates that substrate mimicry for 14-3-3 proteins is a cross-kingdom effector strategy used by both prokaryotic and eukaryotic plant pathogens to suppress host immunity. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Candidate effector proteins from the oomycetes Plasmopara viticola and Phytophthora parasitica share similar predicted structures and induce cell death in Nicotiana species.

Effector proteins secreted by plant pathogens are essential for infection. Cytoplasmic RXLR effectors from oomycetes are characterized by the presence of RXLR and EER motifs that are frequently linked to WY- and/or LWY-domains, folds that are exclusive to this effector family. A related family of secreted candidate effector proteins, carrying WY-domains and the EER motif but lacking the canonical RXLR motif, has recently been described in oomycetes and is mainly found in downy mildew pathogens. Plasmopara viticola is an obligate biotrophic oomycete causing grapevine downy mildew. Here we describe a conserved Pl. viticola secreted candidate non-RXLR effector protein with cell death-inducing activity in Nicotiana species. A similar RXLR effector candidate from the broad host range oomycete pathogen Phytophthora parasitica also induces cell death in Nicotiana. Through comparative tertiary structure modelling, we reveal that both proteins are predicted to carry WY- and LWY-domains. Our work supports the presence of LWY-domains in non-RXLR effectors and suggests that effector candidates with similar domain architecture may exert similar activities.

Nodulation and nitrogen fixation in Medicago truncatula strongly alters the abundance of its root microbiota and subtly affects its structure.

The plant common symbiosis signalling (SYM) pathway has shared function between interactions with rhizobia and arbuscular mycorrhizal fungi, the two most important symbiotic interactions between plants and microorganisms that are crucial in plant and agricultural yields. Here, we determine the role of the plant SYM pathway in the structure and abundance of the microbiota in the model legume Medicago truncatula and whether this is controlled by the nitrogen or phosphorus status of the plant. We show that SYM mutants (dmi3) differ substantially from the wild type (WT) in the absolute abundance of the root microbiota, especially under nitrogen limitation. Changes in the structure of the microbiota were less pronounced and depended on both plant genotype and nutrient status. Thus, the SYM pathway has a major impact on microbial abundance in M. truncatula and also subtly alters the composition of the microbiota.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Transcriptional activity and epigenetic regulation of transposable elements in the symbiotic fungus Rhizophagus irregularis.

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.

Deep learning-based quantification of arbuscular mycorrhizal fungi in plant roots.

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.

An oomycete effector subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface.

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here, we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labeled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a-mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization.

With its long filaments reaching deep inside its prey, the tiny fungi-like organism known as Phytophthora infestans has had a disproportionate impact on human history. Latching onto plants and feeding on their cells, it has caused large-scale starvation events such as the Irish or Highland potato famines. Many specialized proteins allow the parasite to accomplish its feat. For instance, PexRD54 helps P. infestans hijack a cellular process known as autophagy. Healthy cells use this 'self-eating' mechanism to break down invaders or to recycle their components, for example when they require specific nutrients. The process is set in motion by various pathways of molecular events that result in specific sac-like 'vesicles' filled with cargo being transported to specialized compartments for recycling. PexRD54 can take over this mechanism by activating one of the plant autophagy pathways, directing cells to form autophagic vesicles that Phytophthora could then possibly use to feed on or to destroy antimicrobial components. How or why this is the case remains poorly understood. To examine these questions, Pandey, Leary et al. used a combination of genetic and microscopy techniques and tracked how PexRD54 alters autophagy as P. infestans infects a tobacco-related plant. The results show that PexRD54 works by bridging two proteins: one is present on cellular vesicles filled with cargo, and the other on autophagic structures surrounding the parasite. This allows PexRD54 to direct the vesicles to the feeding sites of P. infestans so the parasite can potentially divert nutrients. Pandey, Leary et al. then went on to develop a molecule called the AIM peptide, which could block autophagy by mimicking part of PexRD54. These results help to better grasp how a key disease affects crops, potentially leading to new ways to protect plants without the use of pesticides. They also shed light on autophagy: ultimately, a deeper understanding of this fundamental biological process could allow the development of plants which can adapt to changing environments.

MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation.

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process.

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.

Plant evolution driven by interactions with symbiotic and pathogenic microbes.

During 450 million years of diversification on land, plants and microbes have evolved together. This is reflected in today's continuum of associations, ranging from parasitism to mutualism. Through phylogenetics, cell biology, and reverse genetics extending beyond flowering plants into bryophytes, scientists have started to unravel the genetic basis and evolutionary trajectories of plant-microbe associations. Protection against pathogens and support of beneficial, symbiotic, microorganisms are sustained by a blend of conserved and clade-specific plant mechanisms evolving at different speeds. We propose that symbiosis consistently emerges from the co-option of protection mechanisms and general cell biology principles. Exploring and harnessing the diversity of molecular mechanisms used in nonflowering plant-microbe interactions may extend the possibilities for engineering symbiosis-competent and pathogen-resilient crops.

Developmental Modulation of Root Cell Wall Architecture Confers Resistance to an Oomycete Pathogen.

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.

The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity.

Phytophthora palmivora is a destructive oomycete plant pathogen with a wide host range. So far, little is known about the factors governing its infection structure development and pathogenicity. From the culture filtrate of a P. palmivora strain isolated from papaya, we identified a secreted glycoprotein of 15 kDa, designated as Ppal15kDa, using liquid chromatography tandem mass spectrometry. Two gene variants, Ppal15kDaA and Ppal15kDaB were amplified from a P. palmivora papaya isolate. Transient expression of both variants in Nicotiana benthamiana by agroinfiltration enhanced P. palmivora infection. Six Ppal15kDa mutants with diverse mutations were generated via CRISPR/Cas9-mediated gene editing. All mutants were compromised in infectivity on N. benthamiana and papaya. Two mutants with all Ppal15kDa copies mutated almost completely lost pathogenicity. The pathogenicity of the other four containing at least one wild-type copy of Ppal15kDa was compromised at varying levels. The mutants were also affected in development as they produced smaller sporangia, shorter germ tubes, and fewer appressoria. The affected levels in development corresponded to the levels of reduction in pathogenicity, suggesting that Ppal15kDa plays an important role in normal development of P. palmivora infection structures. Consistent with its role in infection structure development and pathogenicity, Ppal15kDa was found to be highly induced during appressorium formation. In addition, Ppal15kDa homologs are broadly present in Phytophthora spp., but none were characterized. Altogether, this study identified a novel component involved in development and pathogenicity of P. palmivora and possibly other Phytophthora spp. known to contain a Ppal15kDa homolog.