NCR343 is required to maintain the viability of differentiated bacteroids in nodule cells in Medicago truncatula.

Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen-fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen-reducing activities remains elusive. Here, through mutant screen, map-based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA-Seq assay shows that many stress-responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response-related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.

A signal peptide peptidase is required for ER-symbiosome proximal association and protein secretion.

During legume-rhizobia symbiosis, differentiation of the symbiosome (engulfed intracellular rhizobia) is necessary for successful nitrogen fixation. To control symbiosome differentiation, host cell subcellular components, e.g., ER (endoplasmic reticulum), must adapt robustly to ensure large-scale host protein secretion to the new organelle. However, the key components controlling the adaption of ER in nodule cells remain elusive. We report that Medicago BID1, a nodule-specific signal peptide peptidase (SPP), is central to ER structural dynamics and host protein secretion. In bid1, symbiosome differentiation is blocked. BID1 localizes specifically to the ER membrane and expresses exclusively in nodule cells with symbiosomes. In the wild type ER forms proximal association structures with symbiosomes, but not in bid1. Consequently, in bid1 excessive ER stress responses are induced and ER-to-symbiosome protein secretion is impaired. In summary, a nodule-specific SPP is necessary for ER-symbiosome proximal association, host protein secretion, and symbiosome differentiation.

Endophytic Streptomyces hygroscopicus OsiSh-2-Mediated Balancing between Growth and Disease Resistance in Host Rice.

Plants fine-tune the growth-defense trade-off to survive when facing pathogens. Meanwhile, plant-associated microbes, such as the endophytes inside plant tissues, can benefit plant growth and stress resilience. However, the mechanisms for the beneficial microbes to increase stress resistance with little yield penalty in host plants remain poorly understood. In the present study, we report that endophytic Streptomyces hygroscopicus OsiSh-2 can form a sophisticated interaction with host rice, maintaining cellular homeostasis under pathogen-infection stress, and optimize plant growth and disease resistance in rice. Four-year field trials consistently showed that OsiSh-2 could boost host resistance to rice blast pathogen Magnaporthe oryzae while still maintaining a high yield. The integration of the proteomic, physiological, and transcriptional profiling analysis revealed that OsiSh-2 induced rice defense priming and controlled the expression of energy-consuming defense-related proteins, thus increasing the defense capability with the minimized costs of plant immunity. Meanwhile, OsiSh-2 improved the chloroplast development and optimally maintained the expression of proteins related to plant growth under pathogen stress, thus promoting the crop yield. Our results provided a representative example of an endophyte-mediated modulation of disease resistance and fitness in the host plant. The multilayer effects of OsiSh-2 implicate a promising future of using endophytic actinobacteria for disease control and crop yield promotion. IMPORTANCE Under disease stress, activation of defense response in plants often comes with the cost of a reduction in growth and yield, which is referred as the growth-defense trade-off. The microorganisms which can be recruited by plants to mitigate the growth-defense trade-off are of great value in crop breeding. Here, we reported a rice endophytic actinomycetes Streptomyces hygroscopicus OsiSh-2, which can improve host performances on resistance to rice blast while still sustaining high yield in the 4-year field trials. The proteomic, physiological, and transcriptional profiling data offer insights into the molecular basis underlying the balancing between defense and growth in OsiSh-2-rice symbiont. The findings provide an example for the endophyte-mediated modulation of growth-defense trade-offs in plants and indicated the promising application of endophytic actinobacterial strains in agriculture to breed "microbe-optimized crops."

Malectin/Malectin-like domain-containing proteins: A repertoire of cell surface molecules with broad functional potential.

Cell walls are at the front line of interactions between walled-organisms and their environment. They support cell expansion, ensure cell integrity and, for multicellular organisms such as plants, they provide cell adherence, support cell shape morphogenesis and mediate cell-cell communication. Wall-sensing, detecting perturbations in the wall and signaling the cell to respond accordingly, is crucial for growth and survival. In recent years, plant signaling research has suggested that a large family of receptor-like kinases (RLKs) could function as wall sensors partly because their extracellular domains show homology with malectin, a diglucose binding protein from the endoplasmic reticulum of animal cells. Studies of several malectin/malectin-like (M/ML) domain-containing RLKs (M/MLD-RLKs) from the model plant Arabidopsis thaliana have revealed an impressive array of biological roles, controlling growth, reproduction and stress responses, processes that in various ways rely on or affect the cell wall. Malectin homologous sequences are widespread across biological kingdoms, but plants have uniquely evolved a highly expanded family of proteins with ML domains embedded within various protein contexts. Here, we present an overview on proteins with malectin homologous sequences in different kingdoms, discuss the chromosomal organization of Arabidopsis M/MLD-RLKs and the phylogenetic relationship between these proteins from several model and crop species. We also discuss briefly the molecular networks that enable the diverse biological roles served by M/MLD-RLKs studied thus far.

Medicago Plants Control Nodulation by Regulating Proteolysis of the Receptor-Like Kinase DMI2.

Plants use receptor-like kinases to monitor environmental changes and transduce signals into plant cells. The Medicago truncatula (hereafter Mtruncatula) DOES NOT MAKE INFECTIONS2 (DMI2) protein functions as a coreceptor of rhizobial signals to initiate nodule development and rhizobial infection during nitrogen-fixing symbiosis, but the mechanisms regulating DMI2 protein level and folding are still unknown. Here, we report that DMI2 protein abundance changes during nitrogen-fixing symbiosis. DMI2 accumulates in the nodules and is induced by rhizobia treatment through a posttranscriptional process. However, DMI2 induction is independent of the perception of Nod factor, a group of lipochitooligosaccharides secreted by rhizobia. The stability of the DMI2 protein is controlled by the proteasome pathway: in rhizobia-free environments, DMI2 is degraded by the proteasome, but during rhizobial infection, DMI2 is protected from the proteasome, resulting in protein accumulation. Furthermore, proteasome inhibitor-promoted accumulation of DMI2 protein in Medicago roots induces the expression of two early nodulation marker genes, supporting the hypothesis that DMI2 accumulation activates downstream symbiosis signaling. The extracellular region of DMI2 contains two malectin-like domains (MLDs) and a leucine-rich repeat. When conserved amino acids in the MLDs are mutated, DMI2 fails to restore nodule development in dmi2 mutants, and point-mutated MLD proteins are degraded constitutively, suggesting that the MLD may be vital for the accumulation of DMI2. Our findings suggest that legumes control nodule development through modulating the protein level of DMI2, revealing a layer of regulation in the interaction between plants and rhizobia in nitrogen-fixing symbiosis.

Microsymbiont discrimination mediated by a host-secreted peptide in Medicago truncatula.

The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix-) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.

Nodule cysteine-rich peptides maintain a working balance during nitrogen-fixing symbiosis.

The nitrogen-fixing symbiosis between legumes and rhizobia is highly relevant to human society and global ecology. One recent breakthrough in understanding the molecular interplay between the plant and the prokaryotic partner is that, at least in certain legumes, the host deploys a number of antimicrobial peptides, called nodule cysteine-rich (NCR) peptides, to control the outcome of this symbiosis. Under this plant dominance, the bacteria are subject to the sub-lethal toxicity of these antimicrobial peptides, resulting in limited reproductive potential. However, recent genetic studies have added unexpected twists to this mechanism: certain NCR peptides are essential for the bacteria to adapt to the intracellular environment needed for a successful symbiosis, and the absence of these peptides can break down the mutualism. Meanwhile, some rhizobial strains have evolved a peptidase to specifically degrade these antimicrobial peptides, allowing the bacteria to escape host control. These findings challenge the preconceptions about 'antimicrobial' peptides, supporting the notion that their role in biotic interactions extends beyond toxicity to the microbial partners.

Arabidopsis glycosylphosphatidylinositol-anchored protein LLG1 associates with and modulates FLS2 to regulate innate immunity.

Plants detect and respond to pathogen invasion with membrane-localized pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and activate downstream immune responses. Here we report that Arabidopsis thaliana LORELEI-LIKE GPI-ANCHORED PROTEIN 1 (LLG1), a coreceptor of the receptor-like kinase FERONIA, regulates PRR signaling. In a forward genetic screen for suppressors of enhanced disease resistance 1 (edr1), we identified the point mutation llg1-3, which suppresses edr1 disease resistance but does not affect plant growth and development. The llg1 mutants show enhanced susceptibility to various virulent pathogens, indicating that LLG1 has an important role in plant immunity. LLG1 constitutively associates with the PAMP receptor FLAGELLIN SENSING 2 (FLS2) and the elongation factor-Tu receptor, and forms a complex with BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 in a ligand-dependent manner, indicating that LLG1 functions as a key component of PAMP-recognition immune complexes. Moreover, LLG1 contributes to accumulation and ligand-induced degradation of FLS2, and is required for downstream innate immunity responses, including ligand-induced phosphorylation of BOTRYTIS-INDUCED KINASE 1 and production of reactive oxygen species. Taken together, our findings reveal that LLG1 associates with PAMP receptors and modulates their function to regulate disease responses. As LLG1 functions as a coreceptor of FERONIA and plays central roles in plant growth and development, our findings indicate that LLG1 participates in separate pathways, and may suggest a potential connection between development and innate immunity in plants.

A symbiotic SNARE protein generated by alternative termination of transcription.

Many microbes interact with their hosts across a membrane interface, which is often distinct from existing membranes. Understanding how this interface acquires its identity has significant implications. In the symbiosis between legumes and rhizobia, the symbiosome encases the intracellular bacteria and receives host secretory proteins important for bacterial development. We show that the Medicago truncatula SYNTAXIN 132 (SYP132) gene undergoes alternative cleavage and polyadenylation during transcription, giving rise to two target-membrane soluble NSF attachment protein receptor (t-SNARE) isoforms. One of these isoforms, SYP132A, is induced during the symbiosis, is able to localize to the peribacteroid membrane, and is required for the maturation of symbiosomes into functional forms. The second isoform, SYP132C, has important functions unrelated to symbiosis. The SYP132A sequence is broadly found in flowering plants that form arbuscular mycorrhizal symbiosis, an ancestral mutualism between soil fungi and most land plants. SYP132A silencing severely inhibited arbuscule colonization, indicating that SYP132A is an ancient factor specifying plant-microbe interfaces.

The THO/TREX complex functions in disease resistance in Arabidopsis.

Powdery mildew pathogens are biotrophic fungi that infect large number of plant species. EDR1 (ENHANCED DISEASE RESISTANCE 1) is a negative regulator of plant disease resistance, and loss-of-function in the EDR1 gene confers enhanced disease resistance to powdery mildew pathogen Golovinomyces cichoracearum. In an edr1 suppressor screen, we recently found that a mutation in HPR1, a component of the THO/TREX complex, suppresses edr1-mediated disease resistance, however the hpr1 mutation enhances the ethylene-induced senescence in edr1. The hpr1 single mutant displays enhanced susceptibility, indicating that HPR1 is involved in plant defense responses. THO/TREX is a conserved protein complex that functions in pre-mRNA processing and mRNA export. Several components of THO/TREX complex in Arabidopsis have been identified. By searching Arabidopsis database, we found that Arabidopsis (Columbia-0) has two copies of UAP56, another component of the THO/TREX complex, and the UAP56 proteins are highly conserved. Similar to human UAP56 protein, Arabidopsis UAP56 also localizes to the nucleus, showing a pattern similar to the splicing speckles. Further characterization of the components of THO/TREX in Arabidopsis will provide new insights into the role of THO/TREX in defense responses in plants.

HPR1, a component of the THO/TREX complex, plays an important role in disease resistance and senescence in Arabidopsis.

ENHANCED DISEASE RESISTANCE 1 (EDR1) is a negative regulator of powdery mildew resistance, cell death and ethylene-induced senescence. To identify components involved in EDR1 signaling, we performed a forward genetic screen for edr1 suppressors. In this screen, we identified the hpr1-4 mutation, which partially suppresses edr1-mediated resistance to the powdery mildew pathogen Golovinomyces cichoracearum and mildew-induced cell death. However, the hpr1-4 mutation enhanced the ethylene-induced senescence phenotype of edr1. The hpr1-4 single mutant displayed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Arabidopsis HPR1 encodes a homolog of human HPR1, a component of the conserved THO/transcription export (THO/TREX) complex that is required for mRNA export in yeast and humans. HPR1 is expressed in various organs and throughout all developmental stages. HPR1 localizes to the nucleus, and, significantly, mRNA export is compromised in the hpr1-4 mutant. Taken together, these data demonstrate that HPR1 plays an important role in disease resistance in plants, and that the THO/TREX complex is functionally conserved among plants, yeast and humans. Our data indicate a general link between mRNA export, defense responses and ethylene signaling in plants.