Dissection of the Cytokinin dependent 3’,5’-cAMP response in the α-proteobacterium Sinorhizobium meliloti
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Cytokinins (CK) are a group of plant hormones, which were shown to be a major player during the development of root nodules, harboring N2 fixing rhizobia like Sinorhizobium meliloti, to ensure the survival of leguminous plants in nitrogen-depleted soil. Recently it was shown that S. meliloti supports this symbiotic interaction by regulating occurrence of secondary root hair infections through the Clr-3’,5’-cAMP dependent autoregulation of infection (AOI) pathway.
This thesis presents a connection between CK phytohormone perception and a concomitant cAMP response of S. meliloti via screening of an AC/GC (for adenylate/ guanylate cyclase) mutant library. Subsequent gene complementation studies led to the identification of CyaB as the sole CK-responding AC/GC, responding to the compounds iP (N6-(∆2-isopentenyl)adenine), tZ (trans-Zeatin), kinetin and 6-BAP (6-benzylaminopurine). Promoter analysis, protein truncations and investigation of the population response helped characterizing the CK-CyaB response at different levels. Inspection of CK-, but also CyaB-dependent physiological short- and long-term changes was carried out via analysis of the S. meliloti whole cell proteome after application of exogenous iP. Detected proteomic changes not only displayed a CyaB-dependent CK response of S. meliloti, resulting in increased abundance of the Clr-regulon protein SMb20495, but also uncovered a CyaB-independent CK response, potentially hinting on a defense mechanism against compounds of the isoquinoline alkaloid group, a known toxin implicated in anti-bacterial plant defense.
Phylogenetic analysis revealed a conservation of CyaB among representatives of Rhizobiaceae. Subsequent complementation studies were carried out utilizing cyaB gene orthologs from a free-living, plant-symbiotic and plant-pathogenic species. This revealed an overall conserved, but also varying catalytic response of the here tested orthologs to the CK compound iP. Additionally, co-immunoprecipitation (CoIP) and bacterial two hybrid experiments revealed the MEP pathway protein 1-deoxyxylulose-5-phosphate synthase Dxs as an interaction partner of CyaB. The interaction with Dxs was also conserved for the tested CyaB orthologs. Further, HPLC analysis of MEP pathway metabolites revealed a CK- and CyaB-dependent increase of DMAPP, a crucial downstream product of the MEP pathway. On the other hand, loss of CyaB resulted in the decreased abundance of proteins of the thiamine synthesis pathway, another pathway Dxs activity is involved in. This interaction suggests a connection between the CK-CyaB-cAMP response and the central Dxs-dependent metabolic MEP and thiamine synthesis pathways.
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