Ryohei Thomas Nakano

Establishment of the apoplastic root barrier known as the Casparian strip occurs early in root development. In legumes, this area overlaps with nitrogen-fixing nodule formation, which raises the possibility that nodulation and barrier formation are connected. Nodules also contain Casparian strips, yet, in this case, their role is unknown. We established mutants with defective barriers in Lotus japonicus . This revealed that effective apoplastic blockage in the endodermis is important for root-to-shoot signals underlying nodulation. Our findings further revealed that in nodules, the genetic machinery for Casparian strip formation is shared with roots. Apoplastic blockage controls the metabolic source-sink status required for nitrogen fixation. This identifies Casparian strips as a model system to study spatially constrained symbiotic plant-microbe relationships.

Roots are composed of concentric tissue layers that embrace the centrally localized vasculature. Of these layers, particularly the endodermis stands out as it contains barriers that facilitate selective uptake across the plasma membrane. In mature root regions, endodermal cells undergo additional differentiation and become coated with suberin, a hydrophobic polymer that blocks membrane transport and seals off the inner root parts. Intriguingly, individual cells adjacent to the water-conducting xylem remain unsuberized. These are termed “passage cells”, based on the assumption that they facilitate radial vascular access in a zone which is otherwise impenetrable. The identity of passage cells remain unknown, but their existence suggests that distinct identities and developmental trajectories exist within the second differentiation of the endodermis. In this study, we investigate this in the model plant Arabidopsis thaliana . Our work identifies a genetic regulator that controls pole-specific endodermal differentiation and tissue-forming divisions connected to passage cells. Through a number of analyses, we provide spatiotemporal insights into suberization, establish a framework for radial organization of the endodermis and highlight putative function(s) of passage cells. Combined our findings illustrate how multi-dimensional developmental proceses integrate with environmental inputs in order to provide distinct cellular functions within root tissues.

This article is a Commentary on Walsh et al. (2024), 243: 1951–1965.

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 multi-modal 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.

The molecular framework underlying apoplastic root barrier formation has been unveiled in the model species Arabidopsis thaliana where establishment of Casparian strips occurs at an early stage of root development. In legumes, this region overlaps with the area where nitrogen-fixing bacteria can induce nodule formation, termed the susceptible zone. Moreover, while nodules themselves also contain an endodermis spanning their vascular bundles, it is current unknown if Casparian strips serve as a filter for transport across this specialized organ. Here we establish barrier mutants in the symbiosis model Lotus japonicus. We find that the while genetic network controlling Casparian strip formation is conserved in this legume species, formation of functional barriers is crucial for establishment of N-fixing nodules. By probing this in detail, we establish a model where the Casparian strip, via its linked Schengen pathway, converge with long distance N signaling and systemic regulation of nodulation. Moreover, this also reveal that the genetic system for barrier establishment in the root endodermis is shared in nodule vascular endodermis and required for nodule function. Combined, our findings uncover a novel role of apoplastic root barriers and establishes a mutant collection suitable to probe the role of root barriers in symbiotic plant-microbe relationships.

Summary: 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.

DNA amplicon-based microbiota profiling currently relies on polymorphisms in microbial marker genes to estimate species diversity and abundance. However, this technique cannot resolve genetic differences among microbial individuals of the same species. We report here the development of modular bacterial tags (MoBacTags) encoding DNA barcodes. These tags facilitate tracking of near-isogenic bacterial commensals in synthetic communities (SynComs), which allow assessment of the contributions of individual bacterial genes to root microbiota establishment in Arabidopsis thaliana . Chromosomally-integrated DNA barcodes are co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We generated MoBacTag-labeled strains of wild-type Pseudomonas capeferrum WCS358 and of pqqF and cyoB mutants with known defects in gluconic acid-mediated host immunosuppression and validated reduced root colonization of both mutants in a 15-member synthetic microbiota. We detected a reduced SynCom load on roots in the presence of the WCS358:pqqF mutant, but not WCS358:cyoB, revealing distinct pqqF and cyoB activities in a community context. Using MoBacTags, we also show that WCS358 pqqF mutant-specific colonization and community establishment is not trans -complemented by wild-type WCS358. Given that gluconic acid production in P. capeferrum is indirectly abolished in the pqqF mutant by disruption of pyrroloquinoline quinone (PQQ) biosynthesis, we propose that drastic changes in the root-associated community result from depletion of the cofactor PQQ, which might serve as a common good during root microbiota establishment. Our proof-of-principle experiments illustrate how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in the plant microbiota.

Indole glucosinolates (IGs) are tryptophan (Trp)-derived sulfur-containing specialized metabolites that play a crucial role in plant-microbe interactions in plants of the order Brassicales, including Arabidopsis thaliana . Despite the growing body of evidence implicating IG biosynthetic pathways in root-microbiota interactions, how myrosinases, the enzymes that convert inert IGs into bioactive intermediate/terminal products, contribute to this process remains unknown. Here, we describe the roles of the PYK10 and BGLU21 myrosinases in root-microbiota assembly partly via metabolites secreted from roots into the rhizosphere. PYK10 and BGLU21 localize to the endoplasmic reticulum (ER) body, an ER-derived organelle observed in plants of the family Brassicaceae. We investigated the root microbiota structure of mutants defective in the Trp metabolic ( cyp79b2b3 and myb34/51/122 ) and ER body ( nai1 and pyk10bglu21 ) pathways and found that these factors together contribute to the assembly of root microbiota. Microbial community composition in soils as well as in bacterial synthetic communities (SynComs) treated with root exudates axenically collected from pyk10bglu21 and cyp79b2b3 differed significantly from those treated with exudates derived from wild-type plants, pointing to a direct role of root-exuded compounds. We also show that growth of the pyk10bglu21 and cyp79b2b3 mutants was severely inhibited by fungal endophytes isolated from healthy A. thaliana plants. Overall, our findings demonstrate that root ER body-resident myrosinases influencing the secretion of Trp-derived specialized metabolites represent a lineage-specific innovation that evolved in Brassicaceae to regulate root microbiota structure. Significance: ER bodies were first identified in roots of Brassicaceae plants more than 50 years ago, but their physiological functions have remained uncharacterized. A series of previous studies have suggested their possible role in root-microbe interactions. Here, we provide clear experimental evidence showing a role for ER bodies in root-microbiota interactions, which overlaps with that of root-exuded Trp-derived metabolites. Our findings delineate a plant lineage-specific innovation involving intracellular compartments and metabolic enzymes that evolved to regulate plant-microbe interactions at the root-soil interface.

Significance: Understanding how host–microbe homeostasis is controlled and maintained in plant roots is key to enhance plant productivity. However, the factors that contribute to the maintenance of this equilibrium between plant roots and their multikingdom microbial communities remain largely unknown. Here, we observed a link between fungal load in roots and plant health, and we showed that modulation of fungal abundance is tightly controlled by a two-layer regulatory circuit involving the host innate immune system on one hand and bacterial root commensals on another hand. Our results shed a light into how host–microbe and microbe–microbe interactions act in concert to prevent dysbiosis in Arabidopsis thaliana roots, thereby promoting plant health and maintaining growth-promoting activities of multikingdom microbial commensals.