Investigation of diazotrophic bacteria association with plants
Date
2022-07-08
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Abstract
Lämmastik on hädavajalik enamiku orgaaniliste ühendite biosünteesiks. Kolm peamist bakterirühma – tsüanobakterid (Nostocaceae), mügarbakterid (Rhizobiaceae ja Burkholderiaceae sugukondade proteobakterid) ja Frankia „kiirikseened“ (Frankiaceae aktinobakterite sugukond) – seovad õhulämmastikku nii iseseisvalt kui ka taimejuurtega sümbioosis olles. Käesolevas doktoritöös tutvustan morfoloogilistel ja fülogeneetilistel tõenditel põhinevat lämmastikku siduvate taimede andmebaasi NodDB. Geenijärjestusi kasutades uurisin varem kirjeldatud juuremügaraid moodustavate proteobakterite perekondade nod-faktorit ja hindasin sümbioosi arengut fülogenoomika põhjal. Prognoosisin, et mügarbakterite süsinikku siduv sümbioos arenes Rhizobiaceae sugukonnas ligi 51 miljonit aastat tagasi ja kandus horisontaalselt edasi mitmele α- ja β-proteobakteri taksonile. Kogusin ebatüüpilisi juurenooduleid moodustavate liikide Tribulus terrestris (seiglehelised, Zygophyllaceae) juureproove Lähis-Idast ja Roystonea regia (palmilised, Arecaceae) juureproove Kariibi mere saartelt. Pindsteriliseeritud juurenoodulitest eraldati ja sekveneeriti viie bakteritüvi genoomid ning metagenoomi andmed loodi 14 liikide T. terrestris ja R. regia juurenooduli proovile. Fülogenoomiline analüüs näitab, et taimeliikide T. terrestris ja R. regia proovidest eraldatud peamised bakteritaksonid on vastavalt Klebsiella ja Kosakonia (γ-proteobakterid). Taimeliigi T. terrestris juurenoodulite proovidest leidsin amplifitseerimise ja sekveneerimise tulemusel Sinorhizobium meliloti struktuurseid nod/nif geene. Erinevalt varasematest töödest ma ei tuvastatud metagenoomi andmete analüüsi põhjal T. terrestris juurenoodulites tsüanobakteriaalset infektsiooni. Järeldan, et taimeliikidel T. terrestris ja R. regia arenes loomulike “tühjade noodulite” teke rakusisese mügarbakterite infektsioonita. Taimeliikidel T. terrestris ja R. regia esineva bioloogilise lämmastiku sidumist võib seostada vastavalt S. meliloti ja K. sacchari bakteritega, mis hõivavad noodulilaadseid struktuure epi- või endofüütidena – kasvades kudedes sees või kudede pinnal. Selle töö tulemused lükkavad ümber hüpoteesi, mille kohaselt nodulatsiooni teke või selle eelsoodumus arenes teadaolevate lämmastikku siduvate katteseemnetaimede klaadi ühisel esivanemal ainult ühel korral. Pigem tekkis juursümbiontide toel õhulämmastiku sidumine õistaimedel korduvalt.
Nitrogen (N) is essential to life because it is required for the biosynthesis of all N-containing organic compounds, such as proteins. Three main groups of bacteria – Nostocaceae (Cyanobacteria), rhizobia (Proteobacteria), and Frankia (Actinobacteria) fix atmospheric N both in a free-living form and in root symbiosis with plants. Here, we introduce the NodDB database of N-fixing plants based on morphological and phylogenetic evidence and discuss plant groups with conflicting reports and interpretations. We screened the Nod factor production capacity of nodule-inducing Proteobacteria genera using their genome sequences and assessed the evolutionary history of symbiosis based on phylogenomics. Based on molecular clock analysis, we estimate that rhizobial N-fixing symbiosis appeared for the first time in about 51 Mya (Eocene epoch) in Rhizobiaceae, and it was laterally transferred to multiple symbiotic taxa in the α- and β-Proteobacteria. We collected root samples of Tribulus terrestris L. (Zygophyllaceae) and R. regia Cook (Arecaceae) forming an atypical root nodule from the Middle East and the Caribbean, respectively. Genomes of five bacterial strains isolated from surface-sterilized root nodules were sequenced and metagenome data were generated for 14 nodule samples from T. terrestris and R. regia. Phylogenomic analysis indicates that the dominant bacterial species isolated from T. terrestris and R. regia are clustered with Klebsiella and Kosakonia (γ-Proteobacteria), respectively. structural nod/nif genes identified to Sinorhizobium meliloti could be amplified from T. terrestris root nodule samples. In contrast to previous reports, cyanobacterial infection in root nodules of T. terrestris was not detected based on metagenome sequence data analysis. We conclude that T. terrestris and R. regia evolved to produce natural 'empty nodules' without intracellular rhizobial infection. The demonstrated biological N fixation of T. terrestris and R. regia can be attributed to S. meliloti and K. sacchari respectively, which colonize the nodule-like structures as epi- or endophytes. Knowledge generated here adds to the body of evidence rejecting the hypothesis that nodulation was gained or predisposed on a single occasion in the common ancestor of the currently known N-fixing clade of angiosperms.
Nitrogen (N) is essential to life because it is required for the biosynthesis of all N-containing organic compounds, such as proteins. Three main groups of bacteria – Nostocaceae (Cyanobacteria), rhizobia (Proteobacteria), and Frankia (Actinobacteria) fix atmospheric N both in a free-living form and in root symbiosis with plants. Here, we introduce the NodDB database of N-fixing plants based on morphological and phylogenetic evidence and discuss plant groups with conflicting reports and interpretations. We screened the Nod factor production capacity of nodule-inducing Proteobacteria genera using their genome sequences and assessed the evolutionary history of symbiosis based on phylogenomics. Based on molecular clock analysis, we estimate that rhizobial N-fixing symbiosis appeared for the first time in about 51 Mya (Eocene epoch) in Rhizobiaceae, and it was laterally transferred to multiple symbiotic taxa in the α- and β-Proteobacteria. We collected root samples of Tribulus terrestris L. (Zygophyllaceae) and R. regia Cook (Arecaceae) forming an atypical root nodule from the Middle East and the Caribbean, respectively. Genomes of five bacterial strains isolated from surface-sterilized root nodules were sequenced and metagenome data were generated for 14 nodule samples from T. terrestris and R. regia. Phylogenomic analysis indicates that the dominant bacterial species isolated from T. terrestris and R. regia are clustered with Klebsiella and Kosakonia (γ-Proteobacteria), respectively. structural nod/nif genes identified to Sinorhizobium meliloti could be amplified from T. terrestris root nodule samples. In contrast to previous reports, cyanobacterial infection in root nodules of T. terrestris was not detected based on metagenome sequence data analysis. We conclude that T. terrestris and R. regia evolved to produce natural 'empty nodules' without intracellular rhizobial infection. The demonstrated biological N fixation of T. terrestris and R. regia can be attributed to S. meliloti and K. sacchari respectively, which colonize the nodule-like structures as epi- or endophytes. Knowledge generated here adds to the body of evidence rejecting the hypothesis that nodulation was gained or predisposed on a single occasion in the common ancestor of the currently known N-fixing clade of angiosperms.
Description
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Keywords
plants (botany), root system, nitrogen fixation, symbiosis, rhizobia, phylogeny, environmental genomics