Organization and characterization of the virCD genes from Agrobacterium rhizogenes

Summary We have precisely localized virulent (vir) genes of the hairy root-inducing plasmid pRiA4b on the basis of sequence similarity with the tumor-inducing plasmid pTiA6NC, and shown that the overall organizations of vir genes in both plasmids are fairly analogous, although sizes and spacer lengths in some genes differ from each other. Among the vir genes thus mapped, the virC and virD loci were characterized in detail. Transposon insertions in virD led to loss of tumorigenicity on Kalanchoe stems and carrot discs, and one within virC exhibited an attenuated pathogenicity. The avirulent phenotype of the virD2 strain among these mutants was due to the lack of ability to recombine T-DNA border repeats in Agrobacterium cells. The nucleotide sequence of most parts of the virCD loci were similar in both plasmids. The virCD genes of these two plasmids, therefore, seem comparable both functionally and structurally. Phylogeny of pRi and pTi has also been discussed from the sequence data.     

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng

 Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA₃, the embryos were transferred to 1/2-MS medium without GA₃. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield. Received: 27 March 1999 / Revision received: 18 May 1999 / Accepted 8 July 1999     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

Genetic Transformation of Cucumis sativus by Agrobacterium rhizogenes

Hairy roots were obtained in vitro 10 days after inoculation of cucumber ( Cucumis sativus L. ) cotyledon explants with the strains of Agrobacterium rhiwgenes R1000 and R1601. The frequency of the cotyledon explants transformed by R1000 and R1601 was up to 87.5% and 88.9%, respectively. All hairy roots induced by the strains of R1000 and R1601 grew rapidly on solid hormone-free MS medium. The roots incited by A. rhizogenes R1000 could be divided into three phenotypes. The roots of phenotype Ⅰ were similar to the normal ones, but had more numerous lateral roots. Roots of phenotype m were much stouter and shorter, they elongated very slowly and were more highly branched than roots of phenotype Ⅰ . Roots of phenotype Ⅱ were of intermediate in appearance. However, the roots incited by A. rhizogenes R1601 appeared similar to phenotype Ⅰ roots incited by A. rhizogenes R1000. Transformation was confirmed by opine detection.     

A Ti-plasmid determined function is responsible for chemotaxis of Agrobacterium tumefaciens towards the plant wound product acetosyringone

Agrobacterium tumefaciens C58C¹[pTiB6S3] harbouring the octopine Ti-plasmid pTiB6S3, showed positive chemotaxis towards the phenolic plant wound exudate acetosyringone (AS). Maximal attraction was observed at 10⁻⁷ M. In contrast, A. tumefaciens C58C¹ lacking a Ti-plasmid, exhibited no chemotactic response to AS. However, chemotaxis did occur towards the plant phenolic vanillyl alcohol, but at higher concentrations (10⁻² M) and in both Ti-plasmid-containing and cured A. tumefaciens.These results indicate that at least one Ti-plasmid function is involved in the specific chemotactic response to AS, although chemotaxis per se is not Ti-plasmid-encoded. This correlates well with the specific induction of vir-operons mediated by this plant wound product [1].     

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane

Summary The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).Deceased June 5, 1988     

Effect of phenolic compounds on cellulose degradation by some white rot basidiomycetes

Abstract Cellulose degradation by several white rot fungi was investigated. In most fungi cellulase production was stimulated by lignin-related phenolics. Detailed investigation of Tremetes versicolor showed that this stimulation was not directly effected by phenols but was due to an indirect induction. The phenol was oxidized by laccase to quinone. The quinone was then reduced by the enzyme cellobiose: quinone-oxidoreductase while cellobiono-lactone was formed from cellobiose. The cellobiono-lactone was responsible for the increased cellulase production in submerged cultures with cellulose as the sole carbon source.     

Transformation of Gynostermma pentaphyllum by Agrobacterium rhizogenes and Saponin Production in Hairy Root Cultures

Authors report here the establishment of an efficient transformation system for Gynosternrna pentaphyllum (Thunb.) Makino using Agrobacteriurn rhizogenes R1600. Hairy roots appeared on leaf explants 10 days after inoculation with the bacteria . Frequency of the explants transformed by R1600 was up to 94%. Transformation was confirmed by Southern analysis. Biomass of hairy root cultures suspended in hormone-free MS medium increased 9 times after 20 days of incubation. There was no callus formation on the hairy roots during suspension culture. Saponin content in the hairy root cultures was about 2 times as much as in the natural roots, saponins of the hairy root cultures were also released into growth medium as well.     

High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens

 Agrobacterium tumefaciens efficiently transforms most plants. A few dicotyledonous plants and most monocotyledonous plants are, however, recalcitrant to A. tumefaciens infection. We investigated whether the constitutive synthesis of a high level of the T-strand DNA intermediate can improve the transformation efficiency of plants. We previously described a mutation in the vir gene regulator virG, virGN54D, that allows constitutive expression of the vir genes. We also described the isolation of a mutant plasmid that is present at a significantly high level in A. tumefaciens. The two mutations were combined to produce an A. tumefaciens strain that synthesizes a high level of T-strand DNA in an inducer-independent manner. DNA transfer efficiency of the mutant was measured by monitoring β-glucuronidase (GUS) expression in a transient transfer assay. A significant increase in the efficiency of DNA transfer to both rice and soybean was observed with the double mutant. The presence of virGN54D had a major positive effect on transformation efficiency. Received: 4 August 2000 / Revision received: 9 October 2000 / Accepted: 12 October 2000     

Getting to the root: The role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots

Agrobacterium tumefaciens and A. rhizogenes are the causative agents of the crown gall and hairy root diseases, respectively. The pathogenicity of both species is caused by an inter-kingdom transfer of DNA from the bacteria to wounded plant cells. This 'transfer-DNA' (T-DNA) contains oncogenes whose expression transforms the plant recipient cell into a rapidly dividing tumour cell. In the case of A. tumefaciens , three of these oncogenes have been shown to encode enzymes catalyzing the biosynthesis of the plant growth hormones auxin and cytokinin. Therefore, the unorganized cell division in the crown gall tumour can be largely explained by an unregulated overproduction of these plant growth regulators. In contrast, the hairy root disease is characterized by a massive growth of adventitious roots at the site of infection. Because of the similarities of the infection processes, and because A. rhizogenes and A. tumefaciens are very closely related, it has been suggested that the most important A. rhizogenes oncogenes, the so called rol genes, are also encoding proteins involved in the regulation of plant hormone metabolism. However, recent data indicate that this is not the case. Thus the rol genes have functions that most likely are different from producing mere alterations of plant hormone concentrations. This review summarizes recent results concerning the expression and function of the rol genes, and presents a model for the role of these genes, especially rolB and rolC , in the A. rhizogenes infection process.     

发根农杆菌转化大豆的研究

本文利用发根农杆菌感染大豆不同外植体,在成熟胚靠近子叶节部位诱导产生毛状根。经冠瘿碱检测表明,毛状根及由此产生的愈伤组织均有甘露碱存在,说明Ri质粒的T-DNA已整合到大豆的转化根及愈伤组织中。转化根再生实验表明,在含NAA和IAA8mg/L的MS培养基上得到不定根的分化,MS和B_3培养基及6％蔗糖对转化丛生芽的诱导有利。转化的丛生芽在MS基本培养基上进一步长成小植株。     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions

Summary DNA transfer fromAgrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyledonous plantZea mays, was analysed using the recently developed technique of agroinfection. Agroinfection ofZ. mays with maize streak virus using strains ofA. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterialvirC genes. In contrast, agroinfection of the control hostBrassica rapa with cauliflower mosaic virus was less dependent on thevirC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intactvirA, B, D and G functions were absolutely necessary, mutants invirE were attenuated. Agroinfection of maize was effective in the absence of an exogenously suppliedvir gene inducer, and indeed woundedZ. mays tissues were found to produce substance(s) which induced the expression ofA. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function ofAgrobacterium vir genes.     

Disarming and sequencing of Agrobacterium rhizogenes strain K599 (NCPPB2659) plasmid pRi2659

Agrobacterium rhizogenes strain K599 (pRi2659), a causative agent of hairy root disease, effectively induces hairy root formation in a variety of plant species, including numerous soybean (Glycine max) cultivars. Because Agrobacterium-mediated transformation of soybean remains challenging and labor intensive, K599 appeared a suitable progenitor for new agrobacteria strains for plant transformation. In this paper, we report the disarming and sequencing of pRi2659 and the usefulness of the resulting disarmed strain in plant transformation studies of Arabidopsis thaliana, maize (Zea mays), tomato (Lycopersicon esculentum), and soybean (G. max). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biotransformation of phenolic compounds by the cultured cells of Catharanthus roseus

The cultured cells of Catharanthus roseus were able to convert 2-, 3-, and 4-hydroxybenzyl alcohols into their corresponding hydroxybenzyl-β- -glucopyranosides or β- -glucopyranosylbenzyl alcohols, and then convert 2- and 3-hydroxybenzyl-β- -glucopyranosides into primeverosides and vicianosides. Further, the C. roseus cells were capable of hydroxylation of 2-hydroxybenzoic acid to afford 2,5-dihydroxybenzoic acid and then glucosylation of the newly introduced phenolic hydroxyl group.     

用农杆菌Ri诱导蒙古黄芪发根培养的研究

用蒙古黄芪(Astragalus membranaceus Bunge var mongolicus (Bunge) Hsiao) 无菌籽苗的不同部位作为外植体, 用发根农杆菌(Agrobacterium rhizogenes) R1601进行感染, 该品系对不同外植体诱导毛状根的能力是不同的。由下胚轴成功的诱导出毛状根。在附加100μmol/L乙酰丁香酮(acetosyringone)、0 3 mg/L IBA的条件下, 诱导率达42 2%。用硅胶薄层层析法检测到毛状根中含有冠瘿碱, 用硅胶薄层扫描法(TLCS) 测定了蒙古黄芪野生根、栽培根及毛状根中的黄芪甲甙( astragaloside IV) 含量, (用黄芪甲甙标准品作对照), 它们分别为0 2654% (DW), 0 2071% (DW) 和0 2535% (DW)。     

Comparative analysis of bioactive phenolic compounds composition from 26 medicinal plants

Bioactive phenolic compounds are powerful antioxidants in traditionally used medicinal and industrial crop plants and have attracted increased interest in the last years in their application and role in non-destructive methodology for pre-screening analysis of some stress factors. In this study the qualitative target was linked with future possible applications of received data for improving non-destructive methodology as well as for improving existing knowledge regarding antioxidant content in some plant species. Comparative analysis of total phenolics, flavonoid contents, phenolic acid composition, and antioxidant activity in known east central Europe medicinal and industrial crop plants of 26 species of families Asteraceae, Rosaceae and Lamiaceae was done. Among the investigated leaf extracts the highest total phenolic, total flavonoid contents and antioxidant activity have been seen for Stachys byzantine L. (Lamiaceae), Calendula officinalis L. (Asteraceae) and for Potentilla recta L. (Rosaceae). The highest syringic acid content has been found in the leaf extracts of plant family Asteraceae – in the range from 0.782 to 5.078 mg g^?1 DW. The representative’s family Rosaceae has a higher content of p-anisic acid in the range 0.334–3.442 mg g^?1DW compared to the leaf extracts of families Lamiaceae and Asteraceae. The comparative study showed significant differences of content of phenolic acids in the leaf extracts of different representative’s families Rosaceae, Asteraceae and Lamiaceae. We suggest that the presence of some phenolic acids can be used as a possible marker for family botanical specifications of representative families Asteraceae and Rosaceae. It was supposed that some pharmacological effects can be connected with the analyzed data.     

Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g ⋅ l^–1 agar, 7.5 mg ⋅ l^–1 6-benzylaminopurine, 1.0 mg ⋅ l^–1 -naphthaleneacetic acid, 300 mg ⋅ l^–1 cefotaxime and 80 mg ⋅ l^–1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil. Received: 15 March 1997 / Revision received: 30 December 1997 / Accepted: 19 January 1998