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DgDef1, a nodule-specific defensin-like peptide from the actinorhizal plant Datisca glomerata, induces membrane disruption and transcriptome changes in Sinorhizobium meliloti 1021
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Several types of cysteine-rich peptides are involved in the interaction between plants and microorganisms, e.g., anti-bacterial and anti-fungal defensins, characterized by eight conserved cysteine residues, or nodule-specific cysteine-rich peptides that manipulate microsymbiont development in legume nodules, containing five to six conserved cysteine residues. Nodule-specific defensins are found in all actinorhizal plants of Fagales, Rosales as well as Cucurbitales, and a defensin from Alnus glutinosa nodules was shown to affect membrane integrity of a compatible Frankia strain and cause leakage of amino acids (Carro et al., 2015).

Nodules of Datisca glomerata (Datiscaceae. Cucurbitales) contain a gene family encoding nodule-specific defensins with an acidic C-terminal domain. The member expressed at the highest levels, DgDef1, was expressed in young infected cells and, transiently, above the nodule lobe meristem. Neither the complete protein excluding the N-terminal signal peptide produced in Pichia pastoris, nor the synthetically produced cysteine-rich domain had not effect on the growth of Gram-positive Streptomyces coelicolor. However, the cysteine-rich domain alone had a cytotoxic effect on Gram-negative E. coli K-12 and on Sinorhizobium meliloti 1021 with an ID50 of 20.8 µM. RNAseq analysis of Sm1021 cultures treated with this domain, compared to an untreated control, showed a strong effect on the expression of genes encoding transporters, e.g., a dicarboxylate uptake system, consistent with the fact that treated Sm1021 cells had leaky membrane as denoted by propidium iodide staining. However, the expression of the cell cycle regulator ctrA was not affected, suggesting that DgDef1 did not affect the ploidity of Sm1021. Phylogenetic analysis of the cysteine-rich domains showed that legume NCRs and actinorhizal nodule-specific defensins go back to a common ancestor.

National Category
Plant Biotechnology
Research subject
Plant Physiology
Identifiers
URN: urn:nbn:se:su:diva-172325OAI: oai:DiVA.org:su-172325DiVA, id: diva2:1346128
Available from: 2019-08-27 Created: 2019-08-27 Last updated: 2019-08-27Bibliographically approved
In thesis
1. The actinorhizal plant Datisca glomerata: interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms
Open this publication in new window or tab >>The actinorhizal plant Datisca glomerata: interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nitrogen is the element that most often limits plant growth and development. Common agricultural practices rely on the application of large quantities of industrially-produced nitrogen fertilizer, which poses a worldwide environmental threat. Sustainable agriculture encourages the use of biologically fixed nitrogen. However, access to this is still limited to a restricted group of dicotyledonous plants that share among them the ability to form a root nodule symbiosis. After an intricate molecular dialogue, these plants accommodate in the cells of a newly root-derived organ - the nodule - a class of bacteria that produce the nitrogenase enzyme by which they are able to reduce di-nitrogen from air to bioavailable ammonia. This mutualism allows the plant access to nitrogen in exchange for carbon. This thesis focuses particularly on the actinorhizal symbioses established between the North American plant Datisca glomerata (Datiscaceae, Cucurbitales) and Frankia actinobacteria from cluster II (Frankiaceae, Frankiales).

The main aim of this thesis was to improve our understanding about the genetic basis underlying the evolution of root nodule symbioses. Genome-wide comparative analysis indicated that the loss or fragmentation of genes coding for Nodule Inception (NIN) and/or Rhizobium-directed Polar Growth was a major event for the loss of nodulation in close relatives of plants that are able to form a root nodule symbiosis. To acquire more information about the requirements in plant adaptations to meet a symbiosis with Frankia cluster II strains, the nodule transcriptome of D. glomerata was compared with that of Ceanothus thyrsiflorus (Rhamnaceae, Rosales). This study suggested that cluster II Frankia strains use lipochitooligosaccharide Nod factors to signal to their host plants. In addition, it suggested that the nitrogen metabolism likely differs between these symbioses: while transcript profiles from nodules of D. glomerata supports pathways for arginine catabolism, which was previously suggested, those from nodules of C. thyrsiflorus support pathways for asparagine biosynthesis. Since nodules of both plants house Frankia strains from cluster II, the differences in nitrogen metabolism are most likely a feature of the host plant and not of the bacterial symbiont.

As part of an approach to establish D. glomerata as a model organism for actinorhizal Cucurbitales, the effects of phytohormones towards expression of genes putatively involved in signaling for nodule development were investigated. In D. glomerata, similarly to legume plants, the phytohormones cytokinin and auxin were proposed to play a central role in nodule development as they exert a positive effect on the expression of NIN as well as on that of genes whose promoters are presumably transactivated by NIN.

Furthermore, transporter proteins expressed in nodules of D. glomerata and of Casuarina glauca (Casuarinaceae, Fagales), which probably act in supplying C-metabolites to intracellular Frankia, were characterized for their substrate specificity. Results indicated that citrate, and not malate, might be the C-metabolite supplied to both Candidatus Frankia datiscae Dg1 and Frankia casuarinae CcI3 strains in symbiosis.

To explore the option of D. glomerata-mediated control towards its microsymbiont, a nodule-specific defensin-like peptide was characterized (DgDef1). Whereas DgDef1 acts as an antimicrobial peptide against Gram-negative strains in a range compatible with a role in symbiosis, no differentiation was shown in assays with the Gram-positive Streptomyces coelicolor. Nonetheless, DgDef1 induced changes in membrane integrity of the legume symbiont Sinorhizobium meliloti 1021 as well as in its transcription profile, e.g., on transcription of genes associated with dicarboxylate uptake. Thus, a role for DgDef1 in acting against ineffective microsymbionts is suggested. Phylogenetic analysis suggested that actinorhizal nodule-specific defensins and legume nodule-specific cysteine-rich peptides share a common origin, which in an evolutionary scenario of symbiont shift leads to the hypothesis that these peptides have been lost in most legumes lineages.

Collectively, the data presented in this thesis support the idea that root nodule symbioses share more mechanisms than previously assumed, e.g., in the defense against ineffective microsymbionts (“bacterial cheaters”), supporting the new paradigm that the common ancestor of legumes and actinorhizal plants had evolved a symbiosis that was later lost in most lineages.

Place, publisher, year, edition, pages
Stockholm University: Department of Ecology, Environment and Plant Sciences, Stockholm University, 2019. p. 62
Keywords
Root nodule symbiosis, nitrogen fixation, actinorhizal, Datisca glomer-ata, Frankia, nodule development, defensin, antimicrobial, carboxylate transporter, phylogenomics, transcriptomics
National Category
Other Biological Topics Botany
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-172315 (URN)978-91-7797-813-8 (ISBN)978-91-7797-814-5 (ISBN)
Public defence
2019-10-04, Vivi Täckholmssalen (Q-salen), Svante Arrhenius väg 20, Stockholm, 09:30 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2019-09-11 Created: 2019-08-27 Last updated: 2019-09-03Bibliographically approved

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