Roberto Moscatiello

A Diffusible Signal from Arbuscular Mycorrhizal Fungi Elicits a Transient Cytosolic Calcium Elevation in Host Plant Cells

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca2+ indicator aequorin to detect intracellular Ca2+ changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca2+ were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2+-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca2+ transient were constitutively released in the medium, and the induced Ca2+ signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca2+ response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.

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

BackgroundCalcium 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. Trichoderma 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.ResultsHere, we investigated the effects on plant cells of metabolite complexes secreted by Trichoderma atroviride wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca2+ and activation of defense responses. Trichoderma culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen Botrytis cinerea, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (Glycine max L.) cell suspension cultures, Trichoderma and Botrytis 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 Trichoderma, defective for the production of the secreted 42 kDa endochitinase.ConclusionNew insights are provided into the mechanism of interaction between Trichoderma 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.

Flavonoid‐induced calcium signalling in Rhizobium leguminosarum bv. viciae

• Legume-rhizobium symbiosis requires a complex dialogue based on the exchange of diffusible signals between the partners. Compatible rhizobia express key nodulation (nod) genes in response to plant signals - flavonoids - before infection. Host plants sense counterpart rhizobial signalling molecules - Nod factors - through transient changes in intracellular free-calcium. Here we investigate the potential involvement of Ca(2+) in the symbiotic signalling pathway activated by flavonoids in Rhizobium leguminosarum bv. viciae. • By using aequorin-expressing rhizobial strains, we monitored intracellular Ca(2+) dynamics and the Ca(2+) dependence of nod gene transcriptional activation. • Flavonoid inducers triggered, in R. leguminosarum, transient increases in the concentration of intracellular Ca(2+) that were essential for the induction of nod genes. Signalling molecules not specifically related to rhizobia, such as strigolactones, were not perceived by rhizobia through Ca(2+) variations. A Rhizobium strain cured of the symbiotic plasmid responded to inducers with an unchanged Ca(2+) signature, showing that the transcriptional regulator NodD is not directly involved in this stage of flavonoid perception and plays its role downstream of the Ca(2+) signalling event. • These findings demonstrate a key role played by Ca(2+) in sensing and transducing plant-specific flavonoid signals in rhizobia and open up a new perspective in the flavonoid-NodD paradigm of nod gene regulation.

Plant Cell Suspension Cultures

Plant cell suspension cultures are widely used in plant biology as a convenient tool for the investigation of a wide range of phenomena, bypassing the structural complexity of the plant organism in toto. The homogeneity of an in vitro cell population, the large availability of material, the high rate of cell growth and the good reproducibility of conditions make suspension-cultured cells suitable for the analysis of complex physiological processes at the cellular and molecular levels. Moreover, plant cell cultures provide a valuable platform for the production of high-value secondary metabolites and other substances of commercial interest. Here we describe how to initiate and maintain plant cell cultures starting from explants obtained from in vitro germinated seedlings. Isolation of protoplasts from plant cell suspension cultures and regeneration of plants via organogenesis and somatic embryogenesis are also presented.

Chlorella saccharophila cytochrome f and its involvement in the heat shock response

Cytochrome f is an essential component of the major redox complex of the thylakoid membrane. Cloning and characterization are presented here of a novel partial cDNA (ChspetA) encoding cytochrome f in the psychrophile unicellular green alga Chlorella saccharophila and its involvement in the heat shock (HS) response pathway has been analysed. Semi-quantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in heat-shocked cells and the protein profile of cytochrome f highlighted a release of cytochrome f into the cytosol depending on the time lapse from the HS. Evans Blue assay, analysis of chromatin condensation, and chloroplast alterations showed the induction of cell death in cell suspensions treated with cytosolic extracts from heat-shocked cells. This study identifies cytochrome f in C. saccharophila that seems to be involved in the HS-induced programmed cell death process. The data suggest that cytochrome f fulfils its role through a modulation of its transcription and translation levels, together with its intracellular localization. This work focuses on a possible role of cytochrome f into the programmed cell death-like process in a unicellular chlorophyte and suggests the existence of chloroplast-mediated programmed cell death machinery in an organism belonging to one of the primary lineages of photosynthetic eukaryotes.

TAT-Mediated Aequorin Transduction: An Alternative Approach for Effective Calcium Measurements in Plant Cells

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.

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

BackgroundDuring 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.ResultsA plasmid encoding the bioluminescent Ca2+ probe aequorin was introduced into Mesorhizobium loti USDA 3147T strain to investigate whether a Ca2+ response is activated in rhizobia upon perception of plant root exudates. We find that M. loti cells respond to environmental and symbiotic cues through transient elevations in intracellular free Ca2+ concentration. Only root exudates from the homologous host Lotus japonicus induce Ca2+ signalling and downstream activation of nodulation genes. The extracellular Ca2+ chelator EGTA inhibits both transient intracellular Ca2+ increase and inducible nod gene expression, while not affecting the expression of other genes, either constitutively expressed or inducible.ConclusionThese 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.

Quantitative analysis of the naringenin-inducible proteome in Rhizobium leguminosarum by isobaric tagging and mass spectrometry

The rhizobium–legume interaction is a critical cornerstone of crop productivity and environmental sustainability. Its potential improvement relies on elucidation of the complex molecular dialogue between its two partners. In the present study, the proteomic patterns of gnotobiotic cultures of Rhizobium leguminosarum bv. viciae 3841 grown for 6 h in presence or absence of the nod gene‐inducing plant flavonoid naringenin (10 μM) were analyzed using the iTRAQ approach. A total of 1334 proteins were identified corresponding to 18.67% of the protein‐coding genes annotated in the sequenced genome of bv. viciae 3841. The abundance levels of 47 proteins were increased upon naringenin treatment showing fold change ratios ranging from 1.5 to 25 in two biological replicates. Besides the nod units, naringenin enhanced the expression of a number of other genes, many of which organized in operons, including β(1–2) glucan production and secretion, succinoglycan export, the RopA outer membrane protein with homology to an oligogalacturonide‐specific porin motif, other enzymes for carbohydrate and amino acid metabolism, and proteins involved in the translation machinery. Data were validated at the transcriptional and phenotypic levels by RT‐PCR and an assay of secreted sugars in culture supernatants, respectively. The current approach provides not only a high‐resolution analysis of the prokaryotic proteome but also unravels the rhizobium molecular dialogue with legumes by detecting the enhanced expression of several symbiosis‐associated proteins, whose flavonoid‐dependency had not yet been reported.

The intracellular delivery of TAT‐aequorin reveals calcium‐mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita

Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi.

Calcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis

Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant-microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca(2+) involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca(2+) chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca(2+) influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed.