Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens
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Arachis hypogaea
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Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine. The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs. [14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized [14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14C]mannopine, was in the form of agropine.
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To develop an efficient protocol for Agrobacterium -mediated transformation of common bean ( Phaseolus vulgaris L.) and tepary bean ( P. acutifolius A. Gray), we have tested the susceptibility of six genotypes to eight Agrobacterium tumefaciens and two A. rhizogenes strains. The virulence of the Agrobacterium strains was shown to be genotype dependent. In general, the tumors observed on common bean cultivars were larger than those observed on tepary bean cultivars. The A. tumefaciens AT8196 and Ach5 strains and the A. rhizogenes 8196 strain induced the best responses in all genotypes tested. Polymerase chain reaction (PCR) analysis confirmed the presence of T-DNA in tumors derived from inoculation with three A. tumefaciens strains in common beans. Apical meristems of P. vulgaris cv. Jalo were bombarded with tungsten microprojectiles and then inoculated with an A. tumefaciens wild-type strain (Ach5). One month later, the explants showed a high frequency of tumor formation (50% to 70%). Similarly, when bombarded meristems were inoculated with an A. tumefaciens disarmed strain (LBA4404/p35SGUSINT), 44% of them showed substantial sectors of GUS activity, suggesting the expression of introduced gene. The bombardment/ Agrobacterium system appears to be a promising method to stably transform bean through the regeneration of plants directly from transformed apical meristems.
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Spontaneous transfer of the Ti plasmid from Agrobacterium tumefaciens to strain K84 of A. radiobacter was observed and studied for the first time in an experiment on biological control of crown gall. This transfer was detected in a tumor from a K84-treated plant grown in soil inoculated with a nopaline strain of A. tumefaciens biovar 1 sensitive to agrocin 84. The transconjugant strain was virulent and produced agrocin 84. Southern blot hybridization analysis with several probes (T-DNA and right adjacent regions and vir genes) showed important changes at the Ti plasmid, suggesting that recombination between Ti plasmid and pAtK84b in K84 could have happened, resulting in a new Ti plasmid. Transfer of both plasmids of strain K84, pAtK84b and pAgK84, responsible for nopaline catabolism and agrocin 84 production, respectively, to A. tumefaciens also was detected in isolates from the same tumor. Southern blot hybridization of plasmids from one of these avirulent isolates with a nopaline plasmid-specific probe of strain K84 indicated there was a replacement of Ti plasmid by pAtK84b in A. tumefaciens, explaining its avirulence. These results show that plasmid exchanges can occur spontaneously between A. tumefaciens and A. radiobacter. This kind of transfer generates genetic diversity in Agrobacterium and may influence the biocontrol efficiency of A. radiobacter.
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We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean ( Glycine max ), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6. Images
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Agrobacterium tumefaciens is generally used to achieve genetic transformation of plants. The temperatures that have been used for infection with Agrobacterium in published transformation protocols differ widely and, to our knowledge, the effect of temperature on the efficiency of T‐DNA transfer to plants has not been investigated systematically. Agrobacterium tumefaciens strains harbouring a binary vector with the β‐glucuronidase ( uidA ) gene and either a nopaline‐, an octopine‐ or an agropine/ succinamopine‐type helper plasmid were tested in two transformation systems at temperatures between 15 and 29°C. One system involved cocultivation of Phaseolus acutifolius callus whereas in the other system Nicotiana tabacum leaves were vacuum‐infiltrated. In both situations, irrespective of the type of helper plasmid, the levels of transient uidA expression decreased notably when the temperature was raised above 22°C. Expression was low at 27°C and undetectable at 29°C. We anticipate that the efficiency of many published transformation protocols can be improved by reconsidering the factor of temperature.
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Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,β-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria.
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