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The actinorhizal plant Datisca glomerata: interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences. Stockholm University.
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 [en]
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: urn:nbn:se:su:diva-172315ISBN: 978-91-7797-813-8 (print)ISBN: 978-91-7797-814-5 (electronic)OAI: oai:DiVA.org:su-172315DiVA, id: diva2:1346033
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
List of papers
1. Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis
Open this publication in new window or tab >>Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis
Show others...
2018 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 361, no 6398, article id 1743Article in journal (Refereed) Published
National Category
Biological Sciences
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-159071 (URN)10.1126/science.aat1743 (DOI)000438449200035 ()29794220 (PubMedID)
Available from: 2018-08-27 Created: 2018-08-27 Last updated: 2019-08-27Bibliographically approved
2. Comparative Analysis of the Nodule Transcriptomes of Ceanothus thyrsiflorus (Rhamnaceae, Rosales) and Datisca glomerata (Datiscaceae, Cucurbitales)
Open this publication in new window or tab >>Comparative Analysis of the Nodule Transcriptomes of Ceanothus thyrsiflorus (Rhamnaceae, Rosales) and Datisca glomerata (Datiscaceae, Cucurbitales)
2018 (English)In: Frontiers in Plant Sciences, Vol. 9, no 1629Article in journal (Refereed) Published
Abstract [en]

 Two types of nitrogen-fixing root nodule symbioses are known, rhizobial and actinorhizal

symbioses. The latter involve plants of three orders, Fagales, Rosales, and Cucurbitales.

To understand the diversity of plant symbiotic adaptation, we compared the nodule

transcriptomes of Datisca glomerata  (Datiscaceae, Cucurbitales) and Ceanothus

thyrsiflorus  (Rhamnaceae, Rosales); both species are nodulated by members of the

uncultured Frankia  clade, cluster II. The analysis focused on various features. In

both species, the expression of orthologs of legume Nod factor receptor genes

was elevated in nodules compared to roots. Since arginine has been postulated as

export form of fixed nitrogen from symbiotic Frankia  in nodules of D. glomerata,  the

question was whether the nitrogen metabolism was similar in nodules of C. thyrsiflorus .

Analysis of the expression levels of key genes encoding enzymes involved in arginine

metabolism revealed up-regulation of arginine catabolism, but no up-regulation of

arginine biosynthesis, in nodules compared to roots of D. glomerata,  while arginine

degradation was not upregulated in nodules of C. thyrsiflorus . This new information

corroborated an arginine-based metabolic exchange between host and microsymbiont

for D. glomerata,  but not for C. thyrsiflorus.  Oxygen protection systems for nitrogenase

differ dramatically between both species. Analysis of the antioxidant system suggested

that the system in the nodules of D. glomerata  leads to greater oxidative stress than

the one in the nodules of C. thyrsiflorus,  while no differences were found for the

defense against nitrosative stress. However, induction of nitrite reductase in nodules of

C. thyrsiflorus  indicated that here, nitrite produced from nitric oxide had to be detoxified.

Additional shared features were identified: genes encoding enzymes involved in thiamine

biosynthesis were found to be upregulated in the nodules of both species. Orthologous

nodule-specific subtilisin-like proteases that have been linked to the infection process

in actinorhizal Fagales, were also upregulated in the nodules of D. glomerata  and 

 C. thyrsiflorus. Nodule-specific defensin genes known from actinorhizal Fagales and

Cucurbitales, were also found in  C. thyrsiflorus. In summary, the results underline the

variability of nodule metabolism in different groups of symbiotic plants while pointing at

conserved features involved in the infection process.

Place, publisher, year, edition, pages
United States of America: , 2018
Keywords
nitrogen-fixing root nodules, actinorhiza, nitrogen metabolism, divergent evolution, subtilase, defensin, Nod factor receptor
National Category
Bioinformatics and Systems Biology
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-172313 (URN)10.3389/fpls.2018.01629 (DOI)
Available from: 2019-08-26 Created: 2019-08-26 Last updated: 2019-08-27Bibliographically approved
3. A bioassay to analyze the expression of orthologues of legume genes involved in nodulation of the actinorhizal plant Datisca glomerata: auxins and cytokinin induce NIN expression
Open this publication in new window or tab >>A bioassay to analyze the expression of orthologues of legume genes involved in nodulation of the actinorhizal plant Datisca glomerata: auxins and cytokinin induce NIN expression
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Two types of nitrogen-fixing root nodules are known, legume nodules and actinorhizal nodules. During the induction of nitrogen-fixing root nodules, phytohormones are involved in regulating gene expression to shape essential processes. One example is the expression of the early nodulin NODULE INCEPTION (NIN), which in the model legume Lotus japonicus is induced by cytokinin. In order to develop a bioassay for a quick and superficial analysis how phytohormones affect the expression of nodulation-related genes in different root nodule-forming plants, an axenic liquid culture system was established. The bioassay was used to examine responses in roots of the actinorhizal plant Datisca glomerata to phytohormones using quantitative RT-PCR. The synthetic cytokinin 6-Benzylaminopurine (BAP), the natural auxin phenyl acetic acid (PAA), and the synthetic auxin 1-Naphthaleneacetic Acid (NAA) were used. Marker genes for auxins and cytokinin responses were identified. PAA induced the expression of DgSAUR1, whereas NAA induced DgGH3.1.

Induction of Lotus japonicus NIN was analysed for proof of concept, and the bioassay showed induction by cytokinin and auxin (NAA). D. glomerata NIN1 was induced by PAA, NAA, and more weakly by BAP. The gene encoding a transcription factor that activates NIN expression, CYCLOPS, was induced by PAA and NAA. To see whether induction of NIN1 expression led to the activation of target genes of NIN, orthologs of the transcription factors NF-YA1, NSP1 and NSP2, and of ERN1 were identified in D. glomerata and the transcription of the corresponding genes was analysed for regulation by BAP, PAA or NAA. Induction by PAA was found for all genes examined. Additionally, BAP and PAA were used individually or in combination with antagonists of ethylene or gibberellin biosynthesis, respectively. Both antagonists abolished the induction of NIN by BAP or PAA; furthermore, no induction of NIN was found when BAP and PAA were applied together.

Altogether, these results show that the bioassay system can be used to identify target genes of NIN in legumes and actinorhizal plants for further analysis of the role of phytohormones in nodule induction.

National Category
Plant Biotechnology
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-172324 (URN)
Available from: 2019-08-27 Created: 2019-08-27 Last updated: 2019-08-27Bibliographically approved
4. DgDef1, a nodule-specific defensin-like peptide from the actinorhizal plant Datisca glomerata, induces membrane disruption and transcriptome changes in Sinorhizobium meliloti 1021
Open this publication in new window or tab >>DgDef1, a nodule-specific defensin-like peptide from the actinorhizal plant Datisca glomerata, induces membrane disruption and transcriptome changes in Sinorhizobium meliloti 1021
(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:nbn:se:su:diva-172325 (URN)
Available from: 2019-08-27 Created: 2019-08-27 Last updated: 2019-08-27Bibliographically approved
5. Microsymbionts are supplied with citrate in actinorhizal root nodules of Casuarina glauca and Datisca glomerata
Open this publication in new window or tab >>Microsymbionts are supplied with citrate in actinorhizal root nodules of Casuarina glauca and Datisca glomerata
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Two types of root nodule symbioses between nitrogen-fixing soil bacteria and higher plants are known, rhizobia/legume symbioses and the symbiosis between soil actinobacteria of the genus Frankia and more than 200 species from three different orders, collectively called actinorhizal plants. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen. Based on studies with bacterial mutants, it is known that rhizobia are supplied with dicarboxylates, specifically malate, but no legume malate exporters involved in the symbiosis have been identified thus far. For Frankia, a malate exporter from the NPF transporter family that locates to the perisymbiont membrane was identified in Alnus glutinosa (AgDCAT1; Jeong et al., 2004).   

In this study, the substrate specificity of two homologs of AgDCAT1 from nodules of two different actinorhizal hosts was analysed: CgDCAT1 from Casuarina glauca (Casuarinaceae, Fagales), a close relative of A. glutinosa, and DgDCAT1 from Datisca glomerata (Datiscaceae, Cucurbitales). A Multidrug And Toxic compound Extrusion (MATE) type protein from D. glomerata nodules (DgMATE1) was included in the analysis. The transcription of the corresponding genes of all three proteins was induced strongly in nodules compared to roots. Experiments using Xenopus laevis oocytes pre-loaded with 13C-labeled metabolites showed that all three proteins represent citrate exporters. Studies on the levels of citrate cycle intermediates showed that both citrate and malate were present at dramatically higher levels that 2‑oxoglutarate, succinate and fumate in roots and nodules of C. glauca as well as D. glomerata. The Casuarina-nodulating strain Frankia casuarinae CcI3 grew equally well on malate or citrate. In short, the results of this study led to the conclusion that intracellular Frankia bacteria are fed with citrate, not malate, in nodules of C. glauca and D. glomerata.

AgDCAT1 and CgDCAT1 represent orthologs, but DgDCAT1 maps to another subclade of the NPF family, showing that the NPF carboxylate exporters in actinorhizal nodules are polyphyletic. DgMATE1 represents a member of the detoxification (DTX) subgroup that seems to have been recruited for supplying Frankia with citrate. The data suggest that in different symbiotic lineages, carbon source exporters for the symbiosis were recruited from different transporter families independently.

National Category
Plant Biotechnology
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-172326 (URN)
Available from: 2019-08-27 Created: 2019-08-27 Last updated: 2019-08-27Bibliographically approved

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