Callose-mediated regulation of Plasmodesmata during the establishment of Medicago Truncatula-Sinorhizobium Meliloti Symbiotic interaction

Legumes, such as Medicago truncatula, can fix atmospheric nitrogen by forming symbiotic associations with soil-borne bacteria collectively called rhizobia. As a result of this relationship, specific roots organs called nodules, are developed that houses rhizobia and where the nitrogen fixation process occurs. Nodule formation is tightly regulated by complex signalling mechanisms and environmental cues, such as nitrate availability. Molecular signals move between the site of infection and the cortex/pericycle to coordinate nodule organogenesis and also systemically along the vascular system to coordinate root and shoot responses. Despite recent progress in the identification of some of these signals very little is known about the pathways for intercellular transport. In this project, the role of the cell-wall polysaccharide callose in the establishment of symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti was addressed. Callose metabolism regulates transport through plasmodesmata: intercellular channels that form a symplastic path for transport. Using immuno-histochemistry we found that callose is downregulated as early as 16 hours post-bacterial inoculation. Concomitantly, the expression of a plasmodesmata located callose degrading enzyme (Medtr3g083580), identified using phylogeny, was induced. Roots constitutively expressing either Medtr3g083580 or its Arabidopsis orthologue PdBG1, showed reduced callose levels and a higher rate of infection and nodulation, even when grown in nitrate concentrations that inhibit nodulation. The effects were stronger when using a promoter active early after rhizobial infection and were mimicked, in high nitrate conditions, by the ectopic expression of a novel plasmodesmata receptor-like kinase (Medtr1g073320). The results suggest an important role for callose in the control of nodulation, both under nitrate deprived or sufficient conditions, likely associated with the regulation of transport via plasmodesmata. The relevance of the findings is discussed in light of potential applications in crop improvement and in reducing the use of nitrogen fertilizers

Callose deposition and symplastic connectivity are regulated prior to lateral root emergence.

Root growth is critical for the effective exploitation of the rhizosphere and productive plant growth. Our recent work(1) showed that root architecture was dependent upon the degree of symplastic connectivity between neighboring cells during the specification of lateral root primordia and was affected by genes regulating callose deposition at plasmodesmata (PD). Here we provide additional evidence that both symplastic connectivity and callose are also important during the later phase of lateral root development: emergence. Callose immunolocalization assays indicated that transient symplastic isolation of the primordium occur immediately prior to emergence through the overlaying tissues to produce the mature lateral root.(1) Here we could corroborate these results by analyzing the mobility of a symplastic tracer and the expression of PD genes in lateral roots and in response to auxins. Moreover, we show that altering callose deposition affects the number of emerged lateral roots suggesting that PD regulation is important for emergence

A comparative meta-proteomic pipeline for the identification of plasmodesmata proteins and regulatory conditions in diverse plant species

Background A major route for cell-to-cell signalling in plants is mediated by cell wall-embedded pores termed plasmodesmata forming the symplasm. Plasmodesmata regulate the plant development and responses to the environment; however, our understanding of what factors or regulatory cues affect their structure and permeability is still limited. In this paper, a meta-analysis was carried out for the identification of conditions affecting plasmodesmata transport and for the in silico prediction of plasmodesmata proteins in species for which the plasmodesmata proteome has not been experimentally determined. Results Using the information obtained from experimental proteomes, an analysis pipeline (named plasmodesmata in silico proteome 1 or PIP1) was developed to rapidly generate candidate plasmodesmata proteomes for 22 plant species. Using the in silico proteomes to interrogate published transcriptomes, gene interaction networks were identified pointing to conditions likely affecting plasmodesmata transport capacity. High salinity, drought and osmotic stress regulate the expression of clusters enriched in genes encoding plasmodesmata proteins, including those involved in the metabolism of the cell wall polysaccharide callose. Experimental determinations showed restriction in the intercellular transport of the symplasmic reporter GFP and enhanced callose deposition in Arabidopsis roots exposed to 75-mM NaCl and 3% PEG (polyethylene glycol). Using PIP1 and transcriptome meta-analyses, candidate plasmodesmata proteins for the legume Medicago truncatula were generated, leading to the identification of Medtr1g073320, a novel receptor-like protein that localises at plasmodesmata. Expression of Medtr1g073320 affects callose deposition and the root response to infection with the soil-borne bacteria rhizobia in the presence of nitrate. Conclusions Our study shows that combining proteomic meta-analysis and transcriptomic data can be a valuable tool for the identification of new proteins and regulatory mechanisms affecting plasmodesmata function. We have created the freely accessible pipeline PIP1 as a resource for the screening of experimental proteomes and for the in silico prediction of PD proteins in diverse plant species

Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development

The formation of nitrogen-fixing nodules in legumes involves the initiation of synchronized programs in the root epidermis and cortex to allow rhizobial infection and nodule development. In this study, we provide evidence that symplastic communication, regulated by callose turnover at plasmodesmata (PD), is important for coordinating nodule development and infection in Medicago truncatula. Here, we show that rhizobia promote a reduction in callose levels in inner tissues where nodules initiate. This downregulation coincides with the localized expression of M. truncatula β-1,3-glucanase 2 (MtBG2), encoding a novel PD-associated callose-degrading enzyme. Spatiotemporal analyses revealed that MtBG2 expression expands from dividing nodule initials to rhizobia-colonized cortical and epidermal tissues. As shown by the transport of fluorescent molecules in vivo, symplastic-connected domains are created in rhizobia-colonized tissues and enhanced in roots constitutively expressing MtBG2. MtBG2-overexpressing roots additionally displayed reduced levels of PD-associated callose. Together, these findings suggest an active role for MtBG2 in callose degradation and in the formation of symplastic domains during sequential nodule developmental stages. Interfering with symplastic connectivity led to drastic nodulation phenotypes. Roots ectopically expressing β-1,3-glucanases (including MtBG2) exhibited increased nodule number, and those expressing MtBG2 RNAi constructs or a hyperactive callose synthase (under symbiotic promoters) showed defective nodulation phenotypes. Obstructing symplastic connectivity appears to block a signaling pathway required for the expression of NODULE INCEPTION (NIN) and its target NUCLEAR FACTOR-YA1 (NF-YA1) in the cortex. We conclude that symplastic intercellular communication is proactively enhanced by rhizobia, and this is necessary for appropriate coordination of bacterial infection and nodule development

Callose-regulated symplastic communication coordinates symbiotic root nodule development

Raw and analyzed data presented in the manuscript of Gaudioso-Pedraza et al. 2018 Current Biology

Raw data for "A comparative meta-proteomic pipeline for the identification of plasmodesmata proteins and regulatory conditions in diverse plant species"

This repository contains the raw data for the publication listed in the title. Abstract: A major route for cell-to-cell signaling in plants is mediated by cell wall-embedded pores termed plasmodesmata forming the symplasm. Plasmodesmata regulate plant development and responses to the environment however, our understanding of what factors or regulatory cues affect their structure and permeability is still limited. In this paper, a meta-analysis was carried out for the identification of conditions affecting plasmodesmata transport and for the in silico prediction of plasmodesmata proteins in species for which the plasmodesmata proteome has not been experimentally determined