Results Plasmid pSfr64b is required for symbiosis

but pSfr64a is dispensable Strain GR64 contains two plasmids: pSfr64a (183 kb) and pSfr64b (~400 kb) (Figure Selleck MAPK Inhibitor Library 1A, Table 1). A band corresponding to a megaplasmid (~1300 kb), has been visualized [13], but is not always clearly apparent in the gels. Plasmid pSfr64b was identified as the symbiotic plasmid [13], because it hybridizes with the nifH gene. Nodulation assays confirmed that the genetic information in pSfr64b is necessary and sufficient to establish symbiosis. Table 2 shows that all derivatives carrying pSfr64b, were able to form nodules (GR64, CFN2001-1, GMI9023/pSfr64b), and that the construct lacking pSfr64b (GR64-4) was unable to nodulate beans. Consistent with previous findings

[14, 15], the number of nodules was decreased in an Agrobacterium genomic background. On the other hand, lack of pSfr64a had no effect HDAC activity assay on the symbiotic process (GR64-2), and its presence in Agrobacterium did not confer nodulation capacity to the receptor, indicating that pSfr64a encodes none of the essential symbiotic genes. Figure 1 Eckhardt type gel showing the plasmid profile of S. fredii strain Progesterone GR64 and derivatives, in comparison to R. etli CFN42. Panel A. Ethidium bromide stained Eckhardt gel. Lane 1: CFN42, lane 2: wild type GR64, lane 3: GR64-2, lane 4: GR64-3, lane

5: GR64-4, lane 6: GR64-5, lane 7: GR64-6, lane 8: GMI9023/pSfr64a, lane 9: GMI9023/pSfr64b, lane 10: CFN2001, lane 11: CFN2001-1, lane 12: CFN2001- 2, lane 13: CFN2001-3. Panel B. Ethidium bromide stained Eckhardt gel (lanes 1 and 2), and Southern blot of the plasmid profiles probed with pSfr64a (lanes 3 and 4). Lanes 1 and 3: GR64-1 (GR64/pSfr64a::Tn5-GDYN, pSfr64b::Tn5mob), lanes 2 and 4: GR64-2 (pSfr64a-, pSfr64b::Tn5mob). Table 1 Strains and plasmids used in this study Strain Relevant characteristic Source Rhizobium     CFN42 wild type R. etli (pRet42a to pRet42f) [58] CFN2001 CFN42 lacking pRet42a and LY3039478 purchase pRet42d [37] CFNX195 CFN42 derivative cured of pRet42a, pRet42d::Tn5mob [32] GR64 wild type bean-nodulating S.