The receptor-like kinase ARK controls symbiotic balance across land plants.

The mutualistic arbuscular mycorrhizal (AM) symbiosis arose in land plants more than 450 million years ago and is still widely found in all major land plant lineages. Despite its broad taxonomic distribution, little is known about the molecular components underpinning symbiosis outside of flowering plants. The ARBUSCULAR RECEPTOR-LIKE KINASE (ARK) is required for sustaining AM symbiosis in distantly related angiosperms. Here, we demonstrate that ARK has an equivalent role in symbiosis maintenance in the bryophyte Marchantia paleacea and is part of a broad AM genetic program conserved among land plants. In addition, our comparative transcriptome analysis identified evolutionarily conserved expression patterns for several genes in the core symbiotic program required for presymbiotic signaling, intracellular colonization, and nutrient exchange. This study provides insights into the molecular pathways that consistently associate with AM symbiosis across land plants and identifies an ancestral role for ARK in governing symbiotic balance.

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts.

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying ß-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

Phytophthora palmivora establishes tissue-specific intracellular infection structures in the earliest divergent land plant lineage.

The expansion of plants onto land was a formative event that brought forth profound changes to the earth's geochemistry and biota. Filamentous eukaryotic microbes developed the ability to colonize plant tissues early during the evolution of land plants, as demonstrated by intimate, symbiosis-like associations in >400 million-year-old fossils. However, the degree to which filamentous microbes establish pathogenic interactions with early divergent land plants is unclear. Here, we demonstrate that the broad host-range oomycete pathogen Phytophthora palmivora colonizes liverworts, the earliest divergent land plant lineage. We show that P. palmivora establishes a complex tissue-specific interaction with Marchantia polymorpha, where it completes a full infection cycle within air chambers of the dorsal photosynthetic layer. Remarkably, P. palmivora invaginates M. polymorpha cells with haustoria-like structures that accumulate host cellular trafficking machinery and the membrane syntaxin MpSYP13B, but not the related MpSYP13A. Our results indicate that the intracellular accommodation of filamentous microbes is an ancient plant trait that is successfully exploited by pathogens like P. palmivora.

Isolation of Natural Fungal Pathogens from Marchantia polymorpha Reveals Antagonism between Salicylic Acid and Jasmonate during Liverwort-Fungus Interactions.

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts.

A novel experimental system using the liverwort Marchantia polymorpha and its fungal endophytes reveals diverse and context-dependent effects.

Fungal symbioses are ubiquitous in plants, but their effects have mostly been studied in seed plants. This study aimed to assess the diversity of fungal endophyte effects in a bryophyte and identify factors contributing to the variability of outcomes in these interactions. Fungal endophyte cultures and axenic liverwort clones were isolated from wild populations of the liverwort, Marchantia polymorpha. These collections were combined in a gnotobiotic system to test the effects of fungal isolates on the growth rates of hosts under laboratory conditions. Under the experimental conditions, fungi isolated from M. polymorpha ranged from aggressively pathogenic to strongly growth-promoting, but the majority of isolates caused no detectable change in host growth. Growth promotion by selected fungi depended on nutrient concentrations and was inhibited by coinoculation with multiple fungi. The M. polymorpha endophyte system expands the resources for this model liverwort. The experiments presented here demonstrate a wealth of diversity in fungal interactions even in a host reported to lack standard mycorrhizal symbiosis. In addition, they show that some known pathogens of vascular plants live in M. polymorpha and can confer benefits to this nonvascular host. This highlights the importance of studying endophyte effects across the plant tree of life.

AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L.

The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.

Lipid exchanges drove the evolution of mutualism during plant terrestrialization.

Symbiosis with arbuscular mycorrhizal fungi (AMF) improves plant nutrition in most land plants, and its contribution to the colonization of land by plants has been hypothesized. Here, we identify a conserved transcriptomic response to AMF among land plants, including the activation of lipid metabolism. Using gain of function, we show the transfer of lipids from the liverwort Marchantia paleacea to AMF and its direct regulation by the transcription factor WRINKLED (WRI). Arbuscules, the nutrient-exchange structures, were not formed in loss-of-function wri mutants in M. paleacea, leading to aborted mutualism. Our results show the orthology of the symbiotic transfer of lipids across land plants and demonstrate that mutualism with arbuscular mycorrhizal fungi was present in the most recent ancestor of land plants 450 million years ago.

An Evolutionarily Ancient Immune System Governs the Interactions between Pseudomonas syringae and an Early-Diverging Land Plant Lineage.

Evolutionary molecular plant-microbe interactions (EvoMPMI) is an emerging field bridging the gap between molecular phytopathology and evolutionary studies. EvoMPMI research is currently challenging due to the scarcity of pathogenic model systems in early-diverging land plants. Liverworts are among the earliest diverging land-plant lineages, and Marchantia polymorpha has emerged as a liverwort model for evolutionary studies. However, bacterial pathogens of Marchantia have not yet been discovered, and the molecular mechanisms controlling plant-pathogen interactions in this early-diverging land plant remain unknown. Here, we describe a robust experimental plant-bacterial pathosystem for EvoMPMI studies and discover that an ancient immune system governs plant-microbe interactions between M. polymorpha and the hemi-biotrophic pathogenic bacteria Pseudomonas syringae. We show that P. syringae pv tomato (Pto) DC3000, causal agent of tomato bacterial speck disease, colonizes M. polymorpha and activates typical hallmarks of plant innate immunity. Virulence of Pto DC3000 on M. polymorpha relies on effector activities inside liverwort cells, including conserved AvrPto and AvrPtoB functions. Host specificity analyses uncovered pathogenic differences among P. syringae strains, suggesting that M. polymorpha-P. syringae interactions are controlled by the genetic backgrounds of both host and pathogen. Finally, we show that ancient phytohormone defensive networks govern M. polymorpha-P. syringae interactions. Altogether, our results demonstrate that the basic structure of the plant immune system of extant angiosperms is evolutionarily ancient and conserved in early-diverging land plants. This basic immune system may have been instrumental for land colonization by the common ancestor of land plants.

Conserved Biochemical Defenses Underpin Host Responses to Oomycete Infection in an Early-Divergent Land Plant Lineage.

The expansion of plants onto land necessitated the evolution of robust defense strategies to protect against a wide array of microbial invaders. Whereas host responses to microbial colonization are extensively explored in evolutionarily young land plant lineages such as angiosperms, we know relatively little about plant-pathogen interactions in early-diverging land plants thought to better represent the ancestral state. Here, we define the transcriptional and proteomic response of the early-divergent liverwort Marchantia polymorpha to infection with the oomycete pathogen Phytophthora palmivora. We uncover a robust molecular response to oomycete colonization in Marchantia that consists of conserved land plant gene families. Direct macroevolutionary comparisons of host infection responses in Marchantia and the model angiosperm Nicotiana benthamiana further reveal a shared set of orthologous microbe-responsive genes that include members of the phenylpropanoid metabolic pathway. In addition, we identify a role for the Marchantia R2R3-MYB transcription factor MpMyb14 in activating phenylpropanoid (flavonoid) biosynthesis during oomycete infection. Mpmyb14 mutants infected with P. palmivora fail to activate phenylpropanoid biosynthesis gene expression and display enhanced disease susceptibility compared to wild-type plants. Conversely, the ectopic induction of MpMyb14 led to the accumulation of anthocyanin-like pigments and dramatically enhanced liverwort resistance to P. palmivora infection. Collectively, our results demonstrate that the Marchantia response to oomycete infection displays evolutionarily conserved features indicative of an ancestral pathogen deterrence strategy centered on phenylpropanoid-mediated biochemical defenses.

Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology.

Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.

Marchantia liverworts as a proxy to plants' basal microbiomes.

Microbiomes influence plant establishment, development, nutrient acquisition, pathogen defense, and health. Plant microbiomes are shaped by interactions between the microbes and a selection process of host plants that distinguishes between pathogens, commensals, symbionts and transient bacteria. In this work, we explore the microbiomes through massive sequencing of the 16S rRNA genes of microbiomes two Marchantia species of liverworts. We compared microbiomes from M. polymorpha and M. paleacea plants collected in the wild relative to their soils substrates and from plants grown in vitro that were established from gemmae obtained from the same populations of wild plants. Our experimental setup allowed identification of microbes found in both native and in vitro Marchantia species. The main OTUs (97% identity) in Marchantia microbiomes were assigned to the following genera: Methylobacterium, Rhizobium, Paenibacillus, Lysobacter, Pirellula, Steroidobacter, and Bryobacter. The assigned genera correspond to bacteria capable of plant-growth promotion, complex exudate degradation, nitrogen fixation, methylotrophs, and disease-suppressive bacteria, all hosted in the relatively simple anatomy of the plant. Based on their long evolutionary history Marchantia is a promising model to study not only long-term relationships between plants and their microbes but also the transgenerational contribution of microbiomes to plant development and their response to environmental changes.

Targeted destruction of fungal structures of Erysiphe trifoliorum on flat leaf surfaces of Marchantia polymorpha.

In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet-like leaf tissues) and gemmae (small, flat, sheet-like leaf tissues that propagate asexually via bud-like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP-02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP-4N) and examined their germination and subsequent appressorium formation under a high-fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP-4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP-4N conidia inoculated onto M. polymorpha on both leaf surfaces. No differences in destruction of the KRCP-4N fungal structures between thalli and gemmae were observed. At 4 h post-inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6 h post-inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP-02 conidia were not destroyed and formed normal, well-lobed appressoria on the surface of M. polymorpha gemmae.

The liverwort Marchantia foliacea forms a specialized symbiosis with arbuscular mycorrhizal fungi in the genus Glomus.

Microscopic evidence suggests that fungi forming endosymbioses with liverworts in the Marchantiales are arbuscular mycorrhizal (AM) fungi from the Glomeromycota. Polymerase chain reaction amplification of ribosomal sequences confirmed that endophytes of the New Zealand liverwort, Marchantia foliacea, were members of the genus Glomus. Endophytes from two Glomus rDNA phylotypes were repeatedly isolated from geographically separated liverwort samples. Multiple phylotypes were present in the same liverwort patch. The colonizing Glomus species exhibited substantial internal transcribed spacer sequence variation within phylotypes. This work suggests that certain liverwort species may serve as a model for studying DNA sequence variation in colonizing AM phylotypes and specificity in AM-host relationships.