Cytoplasmic calcium influx mediated by plant MLKLs confers TNL-triggered immunity.

The plant homolog of vertebrate necroptosis inducer mixed-lineage kinase domain-like (MLKL) contributes to downstream steps in Toll-interleukin-1 receptor domain NLR (TNL)-receptor-triggered immunity. Here, we show that Arabidopsis MLKL1 (AtMLKL1) clusters into puncta at the plasma membrane upon TNL activation and that this sub-cellular reorganization is dependent on the TNL signal transducer, EDS1. We find that AtMLKLs confer TNL-triggered immunity in parallel with RPW8-type HeLo-domain-containing NLRs (RNLs) and that the AtMLKL N-terminal HeLo domain is indispensable for both immunity and clustering. We show that the AtMLKL HeLo domain mediates cytoplasmic Ca2+ ([Ca2+]cyt) influx in plant and human cells, and AtMLKLs are responsible for sustained [Ca2+]cyt influx during TNL-triggered, but not CNL-triggered, immunity. Our study reveals parallel immune signaling functions of plant MLKLs and RNLs as mediators of [Ca2+]cyt influx and a potentially common role of the HeLo domain fold in the Ca2+-signal relay of diverse organisms.

FLOWERING REPRESSOR AAA+ ATPase 1 is a novel regulator of perennial flowering in Arabis alpina.

Arabis alpina is a polycarpic perennial, in which PERPETUAL FLOWERING1 (PEP1) regulates flowering and perennial traits in a vernalization-dependent manner. Mutagenesis screens of the pep1 mutant established the role of other flowering time regulators in PEP1-parallel pathways. Here we characterized three allelic enhancers of pep1 (eop002, 085 and 091) which flower early. We mapped the causal mutations and complemented mutants with the identified gene. Using quantitative reverse transcriptase PCR and reporter lines, we determined the protein spatiotemporal expression patterns and localization within the cell. We also characterized its role in Arabidopsis thaliana using CRISPR and in A. alpina by introgressing mutant alleles into a wild-type background. These mutants carried lesions in an AAA+ ATPase of unknown function, FLOWERING REPRESSOR AAA+ ATPase 1 (AaFRAT1). AaFRAT1 was detected in the vasculature of young leaf primordia and the rib zone of flowering shoot apical meristems. At the subcellular level, AaFRAT1 was localized at the interphase between the endoplasmic reticulum and peroxisomes. Introgression lines carrying Aafrat1 alleles required less vernalization to flower and reduced number of vegetative axillary branches. By contrast, A. thaliana CRISPR lines showed weak flowering phenotypes. AaFRAT1 contributes to flowering time regulation and the perennial growth habit of A. alpina.

PUB1 Interacts with the Receptor Kinase DMI2 and Negatively Regulates Rhizobial and Arbuscular Mycorrhizal Symbioses through Its Ubiquitination Activity in Medicago truncatula.

PUB1, an E3 ubiquitin ligase, which interacts with and is phosphorylated by the LYK3 symbiotic receptor kinase, negatively regulates rhizobial infection and nodulation during the nitrogen-fixing root nodule symbiosis in Medicago truncatula In this study, we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2, the key symbiotic receptor kinase of the common symbiosis signaling pathway, required for both the rhizobial and the arbuscular mycorrhizal (AM) endosymbioses. We also show here that PUB1 expression is activated during successive stages of root colonization by Rhizophagus irregularis that is compatible with its interaction with DOES NOT MAKE INFECTIONS 2. Through characterization of a mutant, pub1-1, affected by the E3 ubiquitin ligase activity of PUB1, we have shown that the ubiquitination activity of PUB1 is required to negatively modulate successive stages of infection and development of rhizobial and AM symbioses. In conclusion, PUB1 represents, to our knowledge, a novel common component of symbiotic signaling integrating signal perception through interaction with and phosphorylation by two key symbiotic receptor kinases, and downstream signaling via its ubiquitination activity to fine-tune both rhizobial and AM root endosymbioses.

An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing.

Rhizobium-induced root nodules are specialized organs for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we used a sensitive and comprehensive approach based upon oriented high-depth RNA sequencing coupled to laser microdissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942 million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole-genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of our approach. We illustrate the use of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while the plant mRNAs most enriched in nodules compared with roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated website, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest.

MtbHLH1, a bHLH transcription factor involved in Medicago truncatula nodule vascular patterning and nodule to plant metabolic exchanges.

This study aimed at defining the role of a basic helix-loop-helix (bHLH) transcription factor gene from Medicago truncatula, MtbHLH1, whose expression is upregulated during the development of root nodules produced upon infection by rhizobia bacteria. We used MtbHLH1 promoter::GUS fusions and quantitative reverse-transcription polymerase chain reaction analyses to finely characterize the MtbHLH1 expression pattern. We altered MtbHLH1 function by expressing a dominantly repressed construct (CRES-T approach) and looked for possible MtbHLH1 target genes by transcriptomics. We found that MtbHLH1 is expressed in nodule primordia cells derived from pericycle divisions, in nodule vascular bundles (VBs) and in uninfected cells of the nitrogen (N) fixation zone. MtbHLH1 is also expressed in root tips, lateral root primordia cells and root VBs, and induced upon auxin treatment. Altering MtbHLH1 function led to an unusual phenotype, with a modified patterning of nodule VB development and a reduced growth of aerial parts of the plant, even though the nodules were able to fix atmospheric N. Several putative MtbHLH1 regulated genes were identified, including an asparagine synthase and a LOB (lateral organ boundary) transcription factor. Our results suggest that the MtbHLH1 gene is involved in the control of nodule vasculature patterning and nutrient exchanges between nodules and roots.

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

The Medicago truncatula E3 ubiquitin ligase PUB1 interacts with the LYK3 symbiotic receptor and negatively regulates infection and nodulation.

LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogen-fixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box-dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant-microbe interactions and development through interacting with receptor-like kinases is discussed.

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth.

The Saccharomyces cerevisiae protein Knr4 is composed of a globular central core flanked by two natively disordered regions. Although the central part of the protein holds most of its biological function, the N-terminal domain (amino acids 1-80) is essential in the absence of a functional CWI pathway. We show that this specific protein domain is required for the proper cellular localization of Knr4 at sites of polarized growth during vegetative growth and sexual differentiation (bud tip and 'shmoo' tip). Moreover, Knr4 N-terminal domain is also necessary for cell cycle arrest and shmoo formation in response to pheromone to occur at the correct speed. Thus, the presence of Knr4 at the incipient mating projection site seems important for the establishment of the following polarized growth. Cell wall integrity (CWI) and calcineurin pathways are known to share a common essential function, for which they can substitute for one another. Searching for Knr4 partners responsible for survival in a CWI-defective background, we found that the catalytic subunit of calcineurin Cna1 physically interacts with Knr4 in the yeast two-hybrid assay, in a manner dependent on the presence of the Knr4 N-terminal domain. In addition, we present evidence that Knr4 protein participates in the morphogenesis checkpoint, a safety mechanism that holds the cell cycle in response to bud formation defects or insults in cytoskeleton organization, and in which both the CWI pathway and calcineurin are involved.

A remorin protein interacts with symbiotic receptors and regulates bacterial infection.

Remorin proteins have been hypothesized to play important roles during cellular signal transduction processes. Induction of some members of this multigene family has been reported during biotic interactions. However, no roles during host-bacteria interactions have been assigned to remorin proteins until now. We used root nodule symbiosis between Medicago truncatula and Sinorhizobium meliloti to study the roles of a remorin that is specifically induced during nodulation. Here we show that this oligomeric remorin protein attaches to the host plasma membrane surrounding the bacteria and controls infection and release of rhizobia into the host cytoplasm. It interacts with the core set of symbiotic receptors that are essential for perception of bacterial signaling molecules, and thus might represent a plant-specific scaffolding protein.

Experimental evolution of a plant pathogen into a legume symbiont.

Rhizobia are phylogenetically disparate alpha- and beta-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis.

RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector.

Bacterial wilt, a disease impacting cultivated crops worldwide, is caused by the pathogenic bacterium Ralstonia solanacearum. PopP2 (for Pseudomonas outer protein P2) is an R. solanacearum type III effector that belongs to the YopJ/AvrRxv protein family and interacts with the Arabidopsis thaliana RESISTANT TO RALSTONIA SOLANACEARUM 1-R (RRS1-R) resistance protein. RRS1-R contains the Toll/Interleukin1 receptor-nucleotide binding site-Leu-rich repeat domains found in several cytoplasmic R proteins and a C-terminal WRKY DNA binding domain. In this study, we identified the Arabidopsis Cys protease RESPONSIVE TO DEHYDRATION19 (RD19) as being a PopP2-interacting protein whose expression is induced during infection by R. solanacearum. An Arabidopsis rd19 mutant in an RRS1-R genetic background is compromised in resistance to the bacterium, indicating that RD19 is required for RRS1-R-mediated resistance. RD19 normally localizes in mobile vacuole-associated compartments and, upon coexpression with PopP2, is specifically relocalized to the plant nucleus, where the two proteins physically interact. No direct physical interaction between RRS1-R and RD19 in the presence of PopP2 was detected in the nucleus as determined by Förster resonance energy transfer. We propose that RD19 associates with PopP2 to form a nuclear complex that is required for activation of the RRS1-R-mediated resistance response.

Arabidopsis katanin binds microtubules using a multimeric microtubule-binding domain.

Katanin is a heterodimeric protein that mediates ATP-dependent destabilization of microtubules in animal cells. In plants, the catalytic subunit of Arabidopsis thaliana katanin (AtKSS, Arabidopsis thaliana Katanin Small Subunit) has been identified and its microtubule-severing activity has been demonstrated in vitro. In vivo, plant katanin plays a role in the organization of cortical microtubules, but the way it achieves this function is unknown. To go further in our understanding of the mechanisms by which katanin severs microtubules, we analyzed the functional domains of Arabidopsis katanin. We characterized the microtubule-binding domain of katanin both in vitro and in vivo. It corresponds to a poorly conserved sequence between plant and animal katanins that is located in the N-terminus of the protein. This domain interacts with cortical microtubules in vivo and has a low affinity for microtubules in vitro. We also observed that katanin microtubule-binding domain oligomerizes into trimers. These results show that, besides being involved in the interaction of katanin with microtubules, the microtubule-binding domain may also participate in the oligomerization of katanin. At the structural level, we observed that AtKSS forms ring-shaped oligomers.

A rice calcium- and calmodulin-dependent protein kinase restores nodulation to a legume mutant.

The Medicago truncatula DMI3 gene encodes a calcium- and calmodulin-dependent protein kinase (CCaMK) that is necessary for the establishment of both rhizobial and mycorrhizal symbioses. The two symbiotic signaling pathways diverge downstream of DMI3; therefore, it has been proposed that legumes have evolved a particular form of CCaMK, acting like a switch able both to discriminate between rhizobial and mycorrhizal calcium signatures and to trigger the appropriate downstream signaling pathway. To test this hypothesis, we examined whether a CCaMK gene from a nonlegume species was able to restore the rhizobial symbiotic properties of a M. truncatula dmi3 mutant. Our results show that a CCaMK gene from rice can restore nodule formation, indicating that CCaMKs from nonlegumes can interpret the calcium signature elicited by rhizobial Nod factors and activate the appropriate downstream target. The nodules did not contain bacteria, which suggests that DMI3 is also involved in the control of the infection process.

Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection.

The penetration of arbuscular mycorrhizal (AM) fungi through the outermost root tissues of the host plant is a critical step in root colonization, ultimately leading to the establishment of this ecologically important endosymbiotic association. To evaluate the role played by the host plant during AM infection, we have studied in vivo cellular dynamics within Medicago truncatula root epidermal cells using green fluorescent protein labeling of both the plant cytoskeleton and the endoplasmic reticulum. Targeting roots with Gigaspora hyphae has revealed that, before infection, the epidermal cell assembles a transient intracellular structure with a novel cytoskeletal organization. Real-time monitoring suggests that this structure, designated the prepenetration apparatus (PPA), plays a central role in the elaboration of the apoplastic interface compartment through which the fungus grows when it penetrates the cell lumen. The importance of the PPA is underlined by the fact that M. truncatula dmi (for doesn't make infections) mutants fail to assemble this structure. Furthermore, PPA formation in the epidermis can be correlated with DMI-dependent transcriptional activation of the Medicago early nodulin gene ENOD11. These findings demonstrate how the host plant prepares and organizes AM infection of the root, and both the plant-fungal signaling mechanisms involved and the mechanistic parallels with Rhizobium infection in legume root hairs are discussed.