The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is ...unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified inAMfungal exudates and shown to activate nuclear calcium (Ca2+) spiking in the rice root epidermis.
An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca2+ signaling following root treatment with either crude fungal exudates or purified Myc-COs.
Compared with wild-type rice, Ca2+ spiking responses toAMfungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs.
These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.
Spatiotemporal changes in cellular calcium (Ca
) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca
...oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca
cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca
signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca
changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca
sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca
signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic
roots and
were used to successfully monitor Ca
responses to microbial biotic and abiotic elicitors. In
, we demonstrate that GECOs detect symbiosis-related Ca
spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both
and
, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca
signaling
. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca
signaling dynamics in
in response to different stimuli in multi-subcellular compartments of plant cells.
Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear ...Ca2+concentration (Ca2+spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca2+spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume–rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca2+spiking and symbiotic gene expression inMedicago truncatularoots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca2+spiking and symbiotic gene expression in both wild-type plants anddmi2mutants, but interestingly not indmi1mutants. Finally, MVA induced Ca2+spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca2+spiking in this heterologous system.
Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines ...representing an estimated 190 000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R₁ lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14 000-16 000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500 000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.
Root endosymbioses are beneficial associations formed between terrestrial plants and either bacterial or fungal micro-organisms. A common feature of these intracellular symbioses is the requirement ...for mutual recognition between the two partners before host-regulated microbial entry. As part of this molecular dialogue, symbiosis-specific microbial factors set in motion a highly conserved plant signal transduction pathway, of which a central component is the activation of sustained nuclear Ca2+ oscillations in target cells of the host epidermis. Here, we focus on recent findings concerning this crucial Ca2+-dependent signalling step for endosymbiotic associations involving either arbuscular mycorrhizal fungi or nitrogen-fixing Frankia actinomycetes, and in particular how this knowledge is contributing to the identification of the respective microbial factors.
Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogénesis, allowing them to fix atmospheric ...nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulationrelated Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues.
• The aim of this study was to investigate Ca²⁺ responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes ...and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. • Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca²⁺ responses in host root tissues. • Ca²⁺ spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca²⁺ spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. • These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca²⁺ response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals.
Although it is now well‐established that decorated lipo‐chitooligosaccharide Nod factors are the key rhizobial signals which initiate infection/nodulation in host legume species, the identity of the ...equivalent microbial signaling molecules in the Frankia/actinorhizal association remains elusive. With the objective of identifying Frankia symbiotic factors we present a novel approach based on both molecular and cellular pre‐infection reporters expressed in the model actinorhizal species Casuarina glauca. By introducing the nuclear‐localized cameleon Nup‐YC2.1 into Casuarina glauca we show that cell‐free culture supernatants of the compatible Frankia CcI3 strain are able to elicit sustained high frequency Ca²⁺ spiking in host root hairs. Furthermore, an excellent correlation exists between the triggering of nuclear Ca²⁺ spiking and the transcriptional activation of the ProCgNIN:GFP reporter as a function of the Frankia strain tested. These two pre‐infection symbiotic responses have been used in combination to show that the signal molecules present in the Frankia CcI3 supernatant are hydrophilic, of low molecular weight and resistant to chitinase degradation. In conclusion, the biologically active symbiotic signals secreted by Frankia appear to be chemically distinct from the currently known chitin‐based rhizobial/arbuscular mycorrhizal signaling molecules. Convenient bioassays in Casuarina glauca are now available for their full characterization.
A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume ...mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.
Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca²⁺ spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine ...whether NFs also elicit Ca²⁺ responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca²⁺ spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca²⁺ oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca²⁺ spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca²⁺ concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca²⁺ oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.
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