Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

BACKGROUND: Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca(2+) concentration, which is a fundamental prerequisite for any Ca(2+)-based signalling system, is accomplished by complex mechanisms including Ca(2+) binding proteins acting as Ca(2+) buffers. In this work we investigated the occurrence of Ca(2+) binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus. RESULTS: A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca(2+)-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca(2+)-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca(2+) binding ability of the M. loti protein was demonstrated by (45)Ca(2+)-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca(2+) adducts. CONCLUSIONS: The present data indicate that ferredoxin II is a major Ca(2+) binding protein in M. loti that may participate in Ca(2+) homeostasis and suggest an evolutionarily ancient origin for protein-based Ca(2+) regulatory systems. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users

Evidence for calcium-mediated perception of plant symbiotic signals in aequorin-expressing Mesorhizobium loti

<p>Abstract</p> <p>Background</p> <p>During the interaction between rhizobia and leguminous plants the two partners engage in a molecular conversation that leads to reciprocal recognition and ensures the beginning of a successful symbiotic integration. In host plants, intracellular Ca²⁺changes are an integral part of the signalling mechanism. In rhizobia it is not yet known whether Ca²⁺can act as a transducer of symbiotic signals.</p> <p>Results</p> <p>A plasmid encoding the bioluminescent Ca²⁺probe aequorin was introduced into <it>Mesorhizobium loti </it>USDA 3147^Tstrain to investigate whether a Ca²⁺response is activated in rhizobia upon perception of plant root exudates. We find that <it>M. loti </it>cells respond to environmental and symbiotic cues through transient elevations in intracellular free Ca²⁺concentration. Only root exudates from the homologous host <it>Lotus japonicus </it>induce Ca²⁺signalling and downstream activation of nodulation genes. The extracellular Ca²⁺chelator EGTA inhibits both transient intracellular Ca²⁺increase and inducible <it>nod </it>gene expression, while not affecting the expression of other genes, either constitutively expressed or inducible.</p> <p>Conclusion</p> <p>These findings indicate a newly described early event in the molecular dialogue between plants and rhizobia and highlight the use of aequorin-expressing bacterial strains as a promising novel approach for research in legume symbiosis.</p

Transcriptional analysis of calcium-dependent and calcium-independent signalling pathways induced by oligogalacturonides

alpha-1,4-linked oligogalacturonides (OGs) are pectic fragments of plant cell walls that are able to induce defence and developmental responses. To understand plant responses to OGs at the transcriptional level, changes in gene expression were examined using oligonucleotide-based microarrays that cover almost the entire Arabidopsis transcriptome. In suspension-cultured Arabidopsis thaliana (L.) Columbia hypocotyl cells, approximately 4% of the total transcriptome exhibited significant change in abundance in response to treatment with OGs for 2 h. Steady-state changes in the abundance of transcripts encoding stress- and disease-related proteins, signalling components, and transcription factors were particularly noteworthy. As in other plant cell types, OGs elicit a rapid, but transient, elevation in cytosolic free Ca(2+). The Ca(2+) transient can be abolished by the protein kinase inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and by the Ca(2+) channel inhibitor La(3+), thereby facilitating a distinction between Ca(2+)-dependent and -independent transcriptional responses. Among the 244 transcripts that were up-regulated by OGs, the response of 93 (38%) was selectively sensitive to abolition of the Ca(2+) transient. These OG-up-regulated, Ca(2+)-dependent transcripts included two noteworthy classes, the first comprising genes involved in cell wall modification following pathogen attack, and the second consisting of genes involved in the biosynthesis of jasmonate and C6 volatile compounds. These results support the notion of an important role for cytosolic Ca(2+) signalling in jasmonate biosynthesis following OG perception. Promoter analysis of OG-induced, inhibitor-sensitive and -insensitive genes identified several putative cis-elements that might be involved specifically in Ca(2+)-dependent transcriptional regulation

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride

<p>Abstract</p> <p>Background</p> <p>Calcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. <it>Trichoderma </it>spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.</p> <p>Results</p> <p>Here, we investigated the effects on plant cells of metabolite complexes secreted by <it>Trichoderma atroviride </it>wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca²⁺and activation of defense responses. <it>Trichoderma </it>culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen <it>Botrytis cinerea</it>, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (<it>Glycine max </it>L.) cell suspension cultures, <it>Trichoderma </it>and <it>Botrytis </it>metabolite mixtures were distinctively perceived and activated transient intracellular Ca²⁺elevations with different kinetics, specific patterns of intracellular accumulation of reactive oxygen species and induction of cell death. Both Ca²⁺signature and cellular effects were modified by the culture medium from the knock-out mutant of <it>Trichoderma</it>, defective for the production of the secreted 42 kDa endochitinase.</p> <p>Conclusion</p> <p>New insights are provided into the mechanism of interaction between <it>Trichoderma </it>and plants, indicating that secreted fungal molecules are sensed by plant cells through intracellular Ca²⁺changes. Plant cells are able to discriminate signals originating in the single or two-fungal partner interaction and modulate defense responses.</p

Monitoring calcium handling by the plant endoplasmic reticulum with a low‐Ca 2+ ‐affinity targeted aequorin reporter

Precise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca2+-affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non-cleavable N-terminal ER signal peptide (fl2). The correct targeting of fl2-AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well-established in animal cells – consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling – was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+]ER) were recorded in response to different environmental stresses, displaying stimulus-specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set-up of an ER-targeted aequorin chimera extends and complements the currently available toolkit of organelle-targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system

The Hydrophobin HYTLO1 Secreted by the Biocontrol Fungus Trichoderma longibrachiatum Triggers a NAADP-Mediated Calcium Signalling Pathway in Lotus japonicus

Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum, which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca2+ increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3, WRK33, and CP450. The Ca2+-dependence of these gene expression was demonstrated by using the extracellular Ca2+ chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca2+ elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca2+-mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca2+ release in a signalling pathway triggered by a biotic stimulus