Chromosomal barcodes for simultaneous tracking of near-isogenic bacterial strains in plant microbiota.

DNA-amplicon-based microbiota profiling can estimate species diversity and abundance but cannot resolve genetic differences within individuals of the same species. Here we report the development of modular bacterial tags (MoBacTags) encoding DNA barcodes that enable tracking of near-isogenic bacterial commensals in an array of complex microbiome communities. Chromosomally integrated DNA barcodes are then co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We use this approach to assess the contributions of individual bacterial genes to Arabidopsis thaliana root microbiota establishment with synthetic communities that include MoBacTag-labelled strains of Pseudomonas capeferrum. Results show reduced root colonization for certain mutant strains with defects in gluconic-acid-mediated host immunosuppression, which would not be detected with traditional amplicon sequencing. Our work illustrates how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in the plant microbiota.

Long-Term Consequences of PTI Activation and Its Manipulation by Root-Associated Microbiota.

In nature, plants are constantly colonized by a massive diversity of microbes engaged in mutualistic, pathogenic or commensal relationships with the host. Molecular patterns present in these microbes activate pattern-triggered immunity (PTI), which detects microbes in the apoplast or at the tissue surface. Whether and how PTI distinguishes among soil-borne pathogens, opportunistic pathogens, and commensal microbes within the soil microbiota remains unclear. PTI is a multimodal series of molecular events initiated by pattern perception, such as Ca2+ influx, reactive oxygen burst, and extensive transcriptional and metabolic reprogramming. These short-term responses may manifest within minutes to hours, while the long-term consequences of chronic PTI activation persist for days to weeks. Chronic activation of PTI is detrimental to plant growth, so plants need to coordinate growth and defense depending on the surrounding biotic and abiotic environments. Recent studies have demonstrated that root-associated commensal microbes can activate or suppress immune responses to variable extents, clearly pointing to the role of PTI in root-microbiota interactions. However, the molecular mechanisms by which root commensals interfere with root immunity and root immunity modulates microbial behavior remain largely elusive. Here, with a focus on the difference between short-term and long-term PTI responses, we summarize what is known about microbial interference with host PTI, especially in the context of root microbiota. We emphasize some missing pieces that remain to be characterized to promote the ultimate understanding of the role of plant immunity in root-microbiota interactions.

ER body-resident myrosinases and tryptophan specialized metabolism modulate root microbiota assembly.

Endoplasmic reticulum (ER) bodies are ER-derived structures that contain a large amount of PYK10 myrosinase, which hydrolyzes tryptophan (Trp)-derived indole glucosinolates (IGs). Given the well-described role of IGs in root-microbe interactions, we hypothesized that ER bodies in roots are important for interaction with soil-borne microbes at the root-soil interface. We used mutants impaired in ER bodies (nai1), ER body-resident myrosinases (pyk10bglu21), IG biosynthesis (myb34/51/122), and Trp specialized metabolism (cyp79b2b3) to profile their root microbiota community in natural soil, evaluate the impact of axenically collected root exudates on soil or synthetic microbial communities, and test their response to fungal endophytes in a mono-association setup. Tested mutants exhibited altered bacterial and fungal communities in rhizoplane and endosphere, respectively. Natural soils and bacterial synthetic communities treated with mutant root exudates exhibited distinctive microbial profiles from those treated with wild-type (WT) exudates. Most tested endophytes severely restricted the growth of cyp79b2b3, a part of which also impaired the growth of pyk10bglu21. Our results suggest that root ER bodies and their resident myrosinases modulate the profile of root-secreted metabolites and thereby influence root-microbiota interactions.

Tryptophan metabolism and bacterial commensals prevent fungal dysbiosis in Arabidopsis roots.

In nature, roots of healthy plants are colonized by multikingdom microbial communities that include bacteria, fungi, and oomycetes. A key question is how plants control the assembly of these diverse microbes in roots to maintain host-microbe homeostasis and health. Using microbiota reconstitution experiments with a set of immunocompromised Arabidopsis thaliana mutants and a multikingdom synthetic microbial community (SynCom) representative of the natural A. thaliana root microbiota, we observed that microbiota-mediated plant growth promotion was abolished in most of the tested immunocompromised mutants. Notably, more than 40% of between-genotype variation in these microbiota-induced growth differences was explained by fungal but not bacterial or oomycete load in roots. Extensive fungal overgrowth in roots and altered plant growth was evident at both vegetative and reproductive stages for a mutant impaired in the production of tryptophan-derived, specialized metabolites (cyp79b2/b3). Microbiota manipulation experiments with single- and multikingdom microbial SynComs further demonstrated that 1) the presence of fungi in the multikingdom SynCom was the direct cause of the dysbiotic phenotype in the cyp79b2/b3 mutant and 2) bacterial commensals and host tryptophan metabolism are both necessary to control fungal load, thereby promoting A. thaliana growth and survival. Our results indicate that protective activities of bacterial root commensals are as critical as the host tryptophan metabolic pathway in preventing fungal dysbiosis in the A. thaliana root endosphere.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

Tobacco Root Endophytic Arthrobacter Harbors Genomic Features Enabling the Catabolism of Host-Specific Plant Specialized Metabolites.

Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.

Discovery of a Family of Mixed Lineage Kinase Domain-like Proteins in Plants and Their Role in Innate Immune Signaling.

HeLo domain-containing mixed lineage kinase domain-like protein (MLKL), a pseudokinase, mediates necroptotic cell death in animals. Here, we report the discovery of a conserved protein family across seed plants that structurally resembles vertebrate MLKL. The Arabidopsis genome encodes three MLKLs (AtMLKLs) with overlapping functions in disease resistance mediated by Toll-interleukin 1-receptor domain intracellular immune receptors (TNLs). The HeLo domain of AtMLKLs confers cell death activity but is dispensable for immunity. Cryo-EM structures reveal a tetrameric configuration, in which the HeLo domain is buried, suggestive of an auto-repressed complex. The mobility of AtMLKL1 along microtubules is reduced by chitin, a fungal immunity-triggering molecule. An AtMLKL1 phosphomimetic variant exhibiting reduced mobility enhances immunity. Coupled with the predicted presence of HeLo domains in plant helper NLRs, our data reveal the importance of HeLo domain proteins for TNL-dependent immunity and argue for a cell death-independent immune mechanism mediated by MLKLs.

Characterization of rhizome transcriptome and identification of a rhizomatous ER body in the clonal plant Cardamine leucantha.

The rhizome is a plant organ that develops from a shoot apical meristem but penetrates into belowground environments. To characterize the gene expression profile of rhizomes, we compared the rhizome transcriptome with those of the leaves, shoots and roots of a rhizomatous Brassicaceae plant, Cardamine leucantha. Overall, rhizome transcriptomes were characterized by the absence of genes that show rhizome-specific expression and expression profiles intermediate between those of shoots and roots. Our results suggest that both endogenous developmental factors and external environmental factors are important for controlling the rhizome transcriptome. Genes that showed relatively high expression in the rhizome compared to shoots and roots included those related to belowground defense, control of reactive oxygen species and cell elongation under dark conditions. A comparison of transcriptomes further allowed us to identify the presence of an ER body, a defense-related belowground organelle, in epidermal cells of the C. leucantha rhizome, which is the first report of ER bodies in rhizome tissue.

Apoplastic Fluid Preparation from Arabidopsis thaliana Leaves Upon Interaction with a Nonadapted Powdery Mildew Pathogen.

Proteins in the extracellular space (apoplast) play a crucial role at the interface between plant cells and their proximal environment. Consequently, it is not surprising that plants actively control the apoplastic proteomic profile in response to biotic and abiotic cues. Comparative quantitative proteomics of plant apoplastic fluids is therefore of general interest in plant physiology. We here describe an efficient method to isolate apoplastic fluids from Arabidopsis thaliana leaves inoculated with a nonadapted powdery mildew pathogen.

A Golgi-Released Subpopulation of the Trans-Golgi Network Mediates Protein Secretion in Arabidopsis.

Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis (Arabidopsis thaliana), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N-ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant-fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components.

Modular Traits of the Rhizobiales Root Microbiota and Their Evolutionary Relationship with Symbiotic Rhizobia.

Rhizobia are a paraphyletic group of soil-borne bacteria that induce nodule organogenesis in legume roots and fix atmospheric nitrogen for plant growth. In non-leguminous plants, species from the Rhizobiales order define a core lineage of the plant microbiota, suggesting additional functional interactions with plant hosts. In this work, genome analyses of 1,314 Rhizobiales isolates along with amplicon studies of the root microbiota reveal the evolutionary history of nitrogen-fixing symbiosis in this bacterial order. Key symbiosis genes were acquired multiple times, and the most recent common ancestor could colonize roots of a broad host range. In addition, root growth promotion is a characteristic trait of Rhizobiales in Arabidopsis thaliana, whereas interference with plant immunity constitutes a separate, strain-specific phenotype of root commensal Alphaproteobacteria. Additional studies with a tripartite gnotobiotic plant system reveal that these traits operate in a modular fashion and thus might be relevant to microbial homeostasis in healthy roots.

Glutathione Transferase U13 Functions in Pathogen-Triggered Glucosinolate Metabolism.

Glutathione (GSH) and indole glucosinolates (IGs) exert key functions in the immune system of the model plant Arabidopsis (Arabidopsis thaliana). Appropriate GSH levels are important for execution of both pre- and postinvasive disease resistance mechanisms to invasive pathogens, whereas an intact PENETRATION2 (PEN2)-pathway for IG metabolism is essential for preinvasive resistance in this species. Earlier indirect evidence suggested that the latter pathway involves conjugation of GSH with unstable products of IG metabolism and further processing of the resulting adducts to biologically active molecules. Here we describe the identification of Glutathione-S-Transferase class-tau member 13 (GSTU13) as an indispensable component of the PEN2 immune pathway for IG metabolism. gstu13 mutant plants are defective in the pathogen-triggered biosynthesis of end products of the PEN2 pathway, including 4-O-ß-d-glucosyl-indol-3-yl formamide, indole-3-ylmethyl amine, and raphanusamic acid. In line with this metabolic defect, lack of functional GSTU13 results in enhanced disease susceptibility toward several fungal pathogens including Erysiphe pisi, Colletotrichum gloeosporioides, and Plectosphaerella cucumerina Seedlings of gstu13 plants fail also to deposit the (1,3)-ß-glucan cell wall polymer, callose, after recognition of the bacterial flg22 epitope. We show that GSTU13 mediates specifically the role of GSH in IG metabolism without noticeable impact on other immune functions of this tripeptide. We postulate that GSTU13 connects GSH with the pathogen-triggered PEN2 pathway for IG metabolism to deliver metabolites that may have numerous functions in the innate immune system of Arabidopsis.

Root Endophyte Colletotrichum tofieldiae Confers Plant Fitness Benefits that Are Phosphate Status Dependent.

A staggering diversity of endophytic fungi associate with healthy plants in nature, but it is usually unclear whether these represent stochastic encounters or provide host fitness benefits. Although most characterized species of the fungal genus Colletotrichum are destructive pathogens, we show here that C. tofieldiae (Ct) is an endemic endophyte in natural Arabidopsis thaliana populations in central Spain. Colonization by Ct initiates in roots but can also spread systemically into shoots. Ct transfers the macronutrient phosphorus to shoots, promotes plant growth, and increases fertility only under phosphorus-deficient conditions, a nutrient status that might have facilitated the transition from pathogenic to beneficial lifestyles. The host's phosphate starvation response (PSR) system controls Ct root colonization and is needed for plant growth promotion (PGP). PGP also requires PEN2-dependent indole glucosinolate metabolism, a component of innate immune responses, indicating a functional link between innate immunity and the PSR system during beneficial interactions with Ct.

ERMO3/MVP1/GOLD36 is involved in a cell type-specific mechanism for maintaining ER morphology in Arabidopsis thaliana.

The endoplasmic reticulum (ER) has a unique, network-like morphology. The ER structures are composed of tubules, cisternae, and three-way junctions. This morphology is highly conserved among eukaryotes, but the molecular mechanism that maintains ER morphology has not yet been elucidated. In addition, certain Brassicaceae plants develop a unique ER-derived organelle called the ER body. This organelle accumulates large amounts of PYK10, a ß-glucosidase, but its physiological functions are still obscure. We aimed to identify a novel factor required for maintaining the morphology of the ER, including ER bodies, and employed a forward-genetic approach using transgenic Arabidopsis thaliana (GFP-h) with fluorescently-labeled ER. We isolated and investigated a mutant (designated endoplasmic reticulum morphology3, ermo3) with huge aggregates and abnormal punctate structures of ER. ERMO3 encodes a GDSL-lipase/esterase family protein, also known as MVP1. Here, we showed that, although ERMO3/MVP1/GOLD36 was expressed ubiquitously, the morphological defects of ermo3 were specifically seen in a certain type of cells where ER bodies developed. Coimmunoprecipitation analysis combined with mass spectrometry revealed that ERMO3/MVP1/GOLD36 interacts with the PYK10 complex, a huge protein complex that is thought to be important for ER body-related defense systems. We also found that the depletion of transcription factor NAI1, a master regulator for ER body formation, suppressed the formation of ER-aggregates in ermo3 cells, suggesting that NAI1 expression plays an important role in the abnormal aggregation of ER. Our results suggest that ERMO3/MVP1/GOLD36 is required for preventing ER and other organelles from abnormal aggregation and for maintaining proper ER morphology in a coordinated manner with NAI1.

GNOM-LIKE1/ERMO1 and SEC24a/ERMO2 are required for maintenance of endoplasmic reticulum morphology in Arabidopsis thaliana.

The endoplasmic reticulum (ER) is composed of tubules, sheets, and three-way junctions, resulting in a highly conserved polygonal network in all eukaryotes. The molecular mechanisms responsible for the organization of these structures are obscure. To identify novel factors responsible for ER morphology, we employed a forward genetic approach using a transgenic Arabidopsis thaliana plant (GFP-h) with fluorescently labeled ER. We isolated two mutants with defects in ER morphology and designated them endoplasmic reticulum morphology1 (ermo1) and ermo2. The cells of both mutants developed a number of ER-derived spherical bodies, approximately 1 microm in diameter, in addition to the typical polygonal network of ER. The spherical bodies were distributed throughout the ermo1 cells, while they formed a large aggregate in ermo2 cells. We identified the responsible gene for ermo1 to be GNOM-LIKE1 (GNL1) and the gene for ermo2 to be SEC24a. Homologs of both GNL1 and SEC24a are involved in membrane trafficking between the ER and Golgi in yeast and animal cells. Our findings, however, suggest that GNL1/ERMO1 and SEC24a/ERMO2 have a novel function in ER morphology in higher plants.

Quantitative analysis of ER body morphology in an Arabidopsis mutant.

Although fluorescence microscopy screening has proven useful in the identification of genes involved in plant organelle biogenesis and integrity, the quantitative and statistical study of the geometric phenotype is highly limited. This situation could generate unconscious bias in the understanding and presentation of a mutant phenotype. Therefore, we have developed an automated quantification system for green fluorescent protein (GFP) images, which enabled us to easily obtain quantitative data on ER bodies (an endoplasmic reticulum-derived organelle). We isolated an ER body morphology mutant of Arabidopsis thaliana, leb-1 (long ER body). The leb-1 mutant had significantly fewer and larger ER bodies than the wild-type. An amino acid substitution of Cys29 with tyrosine (C29Y) on PYK10, a major component protein of ER bodies, was found in leb-1. Non-reducing SDS-PAGE revealed that the electrophoretic mobility of PYK10 in the leb-1 mutant was clearly different from that in the wild type. This difference suggests that the C29Y amino acid substitution caused a tertiary structural change of the PYK10 protein. While the bglu21-1 and pyk10-1 single mutations slightly affected the number and morphology of the ER bodies, a bglu21-1 pyk10-1 double mutant had fewer and larger ER bodies than the wild type. The quantitative ER body phenotypes of leb-1 were similar to those of bglu21-1 pyk10-1 and bglu21-1 leb-1, suggesting that the leb-1 mutation allele acts dominantly to the BGLU21 wild-type allele. The leb-1 type PYK10 protein, which has an abnormal structure, may competitively inhibit interactions between the wild-type BGLU21/PYK10 protein and an unknown partner.