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    Transcriptomic Identification of a Unique Set of Nodule-Specific Cysteine-Rich Peptides Expressed in the Nitrogen-Fixing Root Nodule of Astragalus sinicus
    (The American Phytopathological Society in cooperation with the International Society for Molecular Plant-Microbe Interactions, 2022-10-08) Wei F; Liu Y; Zhou D; Zhao W; Chen Z; Chen D; Li Y; Zhang X-X
    Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis.
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    The detection of plasmid transfer genes in Rhizobium species : a thesis presented in partial fulfilment of the requirement for the degree Master of Science in Microbiology at Massey University, New Zealand
    (Massey University, 1994) Fisher, Paul Jamie
    In order that Rhizobium tra genes responsible for Sym plasmid transfer might be found, DNA probes were constructed from Agrobacterium tra genes. Three probes were constructed from DNA containing;- 1) traR, a gene which regulates other tra genes on the Agrobacterium tumefaciens plasmid pTiC58, 2) an OriT site, at which nickases cleave the plasmid before conjugal transfer can take place, and 3) part of a gene required for construction of a mating bridge. All three probes were constructed by the ligation of tra areas from the A.tumefaciens strain C58 plasmid pTiC58 into broad host range plasmid vectors and subsequent electroporation into E.coli cells. The genomic DNA digests of several Rhizobium and Agrobacterium strains were blotted and probed with the three probes under various washing and hybridisation stringencies. A.tumef aciens strain LMG64 was the only Agrobacterium strain aside from strain C58 to have DNA homologous to any of the probes. Neither R.leguminosarum bv trifolii strain ICMP2163, nor R. leguminosarum bv trifolii strain ICMP2163::Tn5, nor R.leguminosarum bv trifolii strain PN165 had any DNA homologous to any of the probes. However, R.leguminosarum bv trifolii strain ATCC14480 showed homology to the tra I probe (containing traR), and R.loti strain ATCC33669, phylogenetically the most distant relative to A.tumefaciens strain C58 shared homology with the tra III probe (containing DNA responsible for mating bridge assembly). Therefore the distribution of tra genes from the Ti plasmid of A.tumefaciens strain C58 among the agrobacteria and rhizobia used in this study did not correlate to their phylogenetic relatedness to A.tumefaciens strain C58 or to one another.
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    Construction and utilisation of a bidirectional reporter vector in the analysis of two nod-boxes in of Rhizobium loti : a thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in Molecular Genetics at Massey University
    (Massey University, 1993) Parry, Simon Keith
    The nod-box is a 47bp cis-acting regulatory region which has been conserved amongst every species of Rhizobium studied to date. In species such as R. meliloti and R. leguminosarum, the nod-box has been shown to promote constitutive activity towards the regulatory nodD gene, and flavonoid-inducible expression towards the divergently-transcribed nodABCIJ operon. This bidirectional regulation of the so-called common nod genes was not observed in R. loti. A previous analysis of this species had shown that its nod-box promoted inducible activity towards the truncated 'nodD' gene, as well as the nodACIJ operon. It was the unusual arrangement of these R. loti nod genes that had initially aroused interest in this bacteria. To further investigate the role of the nod-box in the regulation of the R. loti common nod genes, a bidirectional reporter vector (pSPV4) was constructed. This novel vector allowed the promoter activity of a cloned nod-box-containing fragment to be concurrently measured in either direction using the same culture of cells. To achieve this construct, the gusA gene from pRAJ260 was blunt-end ligated into pUC21. An in-frame ribosome binding site (rbs) was cloned upstream of the gusA coding sequence to facilitate transcriptional fusions. The rbs and gusA gene were later excised as a functional unit and blunt-end ligated into pMP220 alongside the B-galactosidase reporter gene but in the opposite orientation. Hence, both reporter genes could be divergently transcribed from a common regulatory region cloned into the multiple cloning site that separated the genes. The fragments of DNA that were eventually cloned into the bidirectional vector were generated through the polymerase chain reaction. Each DNA insert contained the nod-box bracketed by differing lengths of flanking region. Once these PCR-generated fragments had been sequenced in pUC118 and subcloned into pSPV4, the resulting constructs were transformed into R. loti cells by electroporation. As the electroporation of these cells had not previously been reported, the conditions for this procedure were established and optimised. The results obtained from the bidirectional reporter assays disagreed with those observed in the earlier assays by Teo (1990). Neither the nodACIJ nod-box of NZP2037 nor the nodB nod-box of NZP2213, showed bidirectional inducible expression. In fact, both nod-boxes showed constitutive expression in the 'nodD' direction and inducible expression in the opposite direction. This indicates that the control of the nod genes in R. loti is fundamentally the same as that seen in other fast-growing Rhizobium species. Three regulatory elements affecting the levels of nod gene expression have tentatively been identified outside the nod-box sequence, though the results indicating their presence may simply be·due to spacing differences between the nod-box and the reporter gene.
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    The use of LAC fusions to analyse the regulation of NOD gene region of Rhizobium loti : a thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 1990) Gillman, Michelle Ann
    Two approaches where used in the analysis of common and host specific nod gene expression in Rhizobium loti strains NZP2213 and NZP2037. The first approach using the Tn3-HoHol transposon to generate lacZ transcriptional/translational fusions, produced 290 insertions within the 8.3kb EcoRI nod fragment of R.loti strain NZP2213. The position and orientation of all but one of these insertions was determined using restriction enzyme mapping and hybridisation. The sites of the insertion and orientation were generally found to be random. The lacZ fusions were transferred into R.loti strain NZP2213 where their B-­galactosidase activity was measured in the presence and absence of Lotus tenuis seed exudate. All insertions had a low level of B-galactosidase activity that was the same as the controls. This activity was independent of position or orientation. This lack of expression could be a result of the fusions being in regions that are not transcribed ie not downstream of either a nod inducible or other promoter, or that the appropriate conditions for constitutive or inducible activity were not achieved. The second approach to construct lacZ transcriptional fusions was less random and involved the cloning of three separate nod gene fragments: i) a 4.1kb Sall fragment that overlaps the nod region of the 8.3kb EcoRI fragment of R.loti strain NZP2213, ii) a 0.65kb EcoRl fragment isolated from the 4.1kb Sall fragment of R.loti strain NZP2213, and iii) a 1.4kb Sall fragment isolated from the 7.1kb EcoRI nod region of R.loti strain NZP2037. These three fragments (4.1kb, 0.65kb and 1.4kb) were isolated and cloned into pMP190, pMP220 and pMP190 respectively, in both orientations. Each lacZ fusion was transferred into the R.loti strains from which the fragment had originated, ie either NZP2213 or NZP2037. The B-galactosidase activity of these transconjugants was measured in the presence and absence of Lotus tenuis seed exudate. The 4.1kb Sall construct from R.loti NZP2213 was found to have consititutive activity in both orientations indicating that at least two consititutive promoters are located on this fragment. The activity of one orientation, corresponding to pPN38, was twice that of the reverse orientation corresponding to pPN37. V The smaller 0.65kb EcoRI fragment, that lies within the larger 4.1kb Sall fragment, contains a "nod box" and part of a nodD-like gene (Scott et al., In prep.). No significant 13-galactosidase activity was observed in either orientation with or without seed extract. These experiments showed that the "nod box" alone was insufficient for plant inducible expression. The 1.4kb Sall fragment from R.loti NZP2037, that was known to contain a nodA promoter (Emerson-Colins et al., pers. comm.) showed inducible expression for pPN39, corresponding to a fusion between nodA and lacZ. No significant activity was detected in the reverse orientation, pPN40, either with or without plant exudate.
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    Application of DNA hybridization to the taxonomy of rhizobium trifolii and related species : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1979) Dick, Allan George
    Rhizobia were grown in Yeast Mannitol broth in the presence and absence of K2HPO4. Increasing the concentration of yeast extract in the medium resulted in an increase in growth up to a certain concentration of yeast extract. Growth of rhizobia was inhibited by further increases in yeast extract concentration, especially in the presence of K2HPO4. The inhibition is due to glycine present in the yeast extract and is increased by the presence of monovalent cations. The molecular weight of DNA extracted from. rhizobia was determined by centrifugation in alkaline sucrose gradients. It was found that 32 P-labelled DNA was more fragile under shear than unlabelled DNA. Unlabelled DNA required 75 seconds and 32P-labelled DNA required 56 seconds sonication to reduce the fragment size to 230,000 Daltons. Labelled DNA prepared by a phenol-chloroform method was contaminated with polysaccharide and only low homologous hybridisation could be obtained. A hydroxyapatite-urea method of purifying DNA was developed which produced polysaccharide-free DNA. When labelled DNA was prepared by this method homologous hybridisation averaged 72%. Polysaccharide contamination of unlabelled DNA preparations did not effect the % relative reassociation. Complete reassociation of heterologous DNA required a longer incubation time than did' homologous DNA. The homologous reaction was complete after 32 hours (Cot 200) whereas heterologous DNA required an incubation of 40 hours (Cot 250) to achieve maximum reass ociation. Deoxyribonucleic acid homologies were determined among 27 strains of Rhizobium trifolii, 4 strains of R. Jeguminosarum and 4 of R. phaseoli. Results from related strains indicate that DNA homologies correlate with serological relationships and ability to form nodules on legume roots can be lost without detectable change in homology with an independent reference strain. All rhizobia which nodulated effectively on. T. repens, T. subterranoum, T. ambiguum, and Vicia hirsuta formed one population with an average relatedness of 70% (range 49-94%) and ∆Tm(e) of 0.2-7.5°C with respect to reference strains capable of nodulating the first two clover species. Two strains from African .Trifolium species and one from a Japanese species were less closely related. Th average relatedness of strains from Phaseolus vulgaris with clover rhizobia was 46% (range 37-50%) and ∆ Tm(e) 6.5- 10.0°C. Taxonomic revisions consistent with these observations are discussed. It is proposed that R. trifolii and R. leguminosarum should be combined and called Rhizobium leguminosarum Frank. Within this species various biotypes should be designated according to their plant specificity R.phaseoli should be retained as separate species and examined in more detail. The results are discussed in relation to proposed genetic basis for plant specificity.