Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes

Summary To analyse the respective role of TL- and TR-DNA in root induction by agropine-type Agrobacterium rhizogenes Ri plasmids, deletions covering the TL- or the TR-regions were constructed in vitro and introduced into pRiA4 by marker exchange. Each T-region of pRiHRI was also cloned separately on an independent replicon and used in a binary system with the virulence functions of either an Ri or a Ti plasmid provided in trans. Transformed roots were induced on tobacco and tomato explants by TL-DNA as well as by TR-DNA, suggesting that agropine type Ri plasmids from strains A4 and HRI can induce root proliferation by two independent transformation mechanisms. The root induction by the TR-DNA is probably due to auxin biosynthesis by gene products of aux loci homologous to the tms genes of Ti plasmid T-DNA. The molecular mechanism of root proliferation induced by the TL-DNA is probably equivalent to that of mannopine type Ri plasmid T-DNA.     

Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes.

The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions. The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955. The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5' and 3' flanking sequences. The mas1' and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2' proteins are only 59% identical. Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4. An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region.     

A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains.

The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown. The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids. Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine. Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine. Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli. Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags. Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes. Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes. ags is preceded by a promoter that functions only in the plant. Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium. Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence. Antibodies raised against the agcA product cross-reacted with the anabolic enzyme. These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium.     

Free auxin level and inheritance of introduced markers in tobacco transformed by binary vector eased on A4 Ri plasmid

Tobacco cv. Mamont was transformed by leaf disc method with A.tumefaciens C58C1 (pRiA4b) (pGA472). Transformed leaf segments were cultivated on medium with kanamycin as a selection agent, but without phytohormones. Transformed tissues grew as roots which later regenerated plants. Kanamycin in the medium ensured that only doubly transformed tissues, which contain in their genome both pRi T-DNA as well as pGA472 chimeric kanamycin resistance gene were recovered. Regenerated plants showed clear-cut morphological deviations, but only a slight increase of the auxin content. Kanamycin resistance in the progeny segregated in the Mendelian ratio 3: 1 or 2: 1.     

Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).     

发根农杆菌Ri质粒在药用植物生物工程中的应用

发根农杆菌(Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ)与根癌农杆菌(Ａ.ｔｕｍｅｆａｃｉｅｎｓ)属于根瘤菌科,均为革兰氏阴性菌[1]。它们在侵染植物后引起的症状不同,含有Ｔｉ质粒的根癌农杆菌在侵染植物后形成冠瘿瘤(ｃｒｏｗｎｇａｌｌ),而含有Ｒｉ质粒的发根农杆菌表现与其不同,它在感染植物后在植物的伤口部位诱发产生毛状根(ｈａｉｒｙｒｏｏｔ)。由发根农杆菌侵染植物诱导产生的毛状根具有生长快、分枝多、根毛多等特点。发根农杆菌Ｒｉ质粒与根癌农杆菌Ｔｉ质粒结构相似,都具有高效率转移的Ｔ-ＤＮＡ区和致病的Ｖｉｒ区…     

Development of transformed phenotype induced by a human ras oncogene is inhibited by interferon

Mouse IFN inhibited the development of transformed foci in NIH 3T3 cultures transfected with the viral Ha-MuSV(ras) and Mo-MuSV(mos) oncogenes, or with the human bladder carcinoma ras EJ/T24 DNA. IFN treatment five or seven days after transfection was still effective in inhibiting the oncogenic transformation, but did not inhibit significantly the biochemical transformation induced by pSV2-neo or Ecogpt DNA, so that inhibition of ras-induced transformation was not a result of a general effect on the transfection process. Treatment with IFN did not alter the expression of ras EJ/T24 DNA after the transformed phenotype had been established.     

Binary plant vector based on Ri plasmid and part of T-DNA of the Ti plasmid

A binary vector system inA. tumefaciens for the introduction of foreign genes into the plant genome was developed. The first component is the Ri plasmid and the second component is a small vector plasmid, replicating inAgrobacterium, which carries the 25 bp terminal sequence of the Ti plasmid, theNos gene as the selectable marker and neighbouring sequences of the Ti plasmid. Functions necessary for integration are providedin trans by the virulence region of the Ri plasmid. Transformed cells are selected on the basis of hairy root tumor proliferation and agropine synthesis. They also showNos activity coded by the gene on the small part of Ti T-DNA.     

Induction of nitrate reductase by chloramphenicol in detached cucumber cotyledons

Summary Chloramphenicol (CAP) induced nitrate reductase activity (NRA) in detached, etiolated cucumber (Cucumis sativus L.) cotyledons. The effect was reduced by cycloheximide. Light was not necessary for induction of the enzyme but potentiated the effect of CAP as an inducer of NRA, despite the fact that the antibiotic inhibited chlorophyll accumulation.CAP at suboptimal concentrations (2.5–5 mM) acted synergistically with succinic acid-2,2-dimethylhydrazide (B-Nine) and benzylaminopurine (BAP) in respect to induction of NRA, the effect being especially marked in darkness. The highest NRA was found in cotyledons treated for 24 h with a mixture of CAP+BAP+B-Nine. The effects of KNO₃ and CAP on NRA were neither synergistic nor additive.KNO₃ and BAP induced NADH-NR (EC 1.6.6.1). The same was true for CAP and B-Nine, but the latter two compounds induced, in addition, some activity of another NR that could utilize NADPH as an electron donor.     

Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid

Summary Kanamycin-resistant plants of belladonna (Atropa belladonna) were obtained after Agrobacterium mediated transformation. When a rolC gene, which is one of the loci located on Ri plasmid of Agrobacterium rhizogenes, was co-introduced with a kanamycin resistant (NPT II) gene under control of a cauliflower mosaic virus 35S promoter, the rolC gene was expressed strongly in leaves, flowers, stems and roots. The transformed plants exhibited dramatic promotion of flowering, reduced apical dominance, pale and lanceolated leaves and smaller flowers. On the other hand, when native rolC gene was co-introduced with NPT II, the transgenic plants obtained did not exhibit the altered phenotypes observed in 35S-rolC transformants, and the expression level of the rolC gene was much lower than in 35S-rolC transformants. These results suggest that the morphological changes in transgenic Atropa belladonna were related to the degree of expression of the rolC gene.Abbreviations native rolC rolC gene under control of its own promoter - 35S-rolC rolC gene under control of a cauliflower mosaic viras 35S promoter     

Effect of the hypocotyl hook on greening in etiolated cucumber cotyledons

Four-day-old etiolated cucumber cotyledons (Cucumis sativus, L.) were excised and allowed to green in white fluorescent light at 28 C. Cotyledons excised with a full hypocotyl hook exhibited a lag phase of 1 hour before entering the rapid greening phase, whereas cotyledons excised without any hypocotyl hook exhibited a lag phase of 6 hours. Cotyledons excised with varying lengths of hypocotyl hook accumulated chlorophyll roughly in proportion to the hook length. When cotyledons were excised with a full hook and were partially or totally shielded from light with aluminum foil, the samples with the hook covered accumulated more chlorophyll than the wholly exposed samples. The samples with the cotyledons covered showed no net accumulation of chlorophyll irrespective of hook's exposure to light. These data suggest the contribution of some factor or factors by the hypocotyl hook which reduce the lag phase during greening.     

Transgenic roots produced by introducing Ri-rol genes into cucumber cotyledons by particle bombardment

Transformed roots ofCucumis sativus were obtained from cotyledon tissues that had been bombarded with gold particles coated with plasmid pE7.4 using a pneumatic particle gun. This plasmid containsrolA, rolB, rolC genes and ORF 13 of the 7.4 kbEco RI fragment of T-DNA of pRi 1724 isolated fromAgrobacterium rhizogenes MAF 03-01724. The nature of the tissue and the composition of the culture media used greatly influenced the recovery of transformed roots. The transgenic nature of the derived roots was confirmed by the vigorous. highly-branched growth seen on a phytohormone-free medium. The stable integration ofrol genes into the cucumber genome was confirmed by Southern blot analysis.     

Metabolic alterations in cotyledons of Cucurbita pepo infected by cucumber mosaic virus

Changes in the capacities of enzymes in various metabolic pathwayshave been measured during infection of cotyledons of Cucurbitapepo L. with cucumber mosaic virus (CMV). Starch accumulationand low sucrose content, which are characteristic of the earlystages of infection, are reversed in the later stages of infection.The decline in starch correlated with a reduced capacity forstarch synthesis (ADP-glucose pyrophosphorylase) and a risein the capacity for starch degradation (total starch hydrolase,starch phosphorylase). ¹⁴CO₂ feeding experiments, conductedat saturating CO₂ concentration, show that the newly-assimilatedcarbon was lost at a lower rate from infected cotyledons andless was incorporated into structural carbohydrates, phosphorylatedintermediates plus organic acids, more into soluble sugars,amino acids and proteins. At a later stage of infection therewere dramatic increases in respiratory capacity and a substantialalteration of carbohydrate metabolism. The infection had a largestimulatory effect on the capacity for oxidative pentose-phosphatepathway (glucose-6-phosphate dehydrogenase, 6-phospho-gluconatedehydrogenase), glycolysis (ATP- and pyrophosphate-dependentphosphofructokinases), tri-carboxylic acid cycle (isocitratedehydrogenase, fumarate hydratase), anaplerotic reactions (NAD-dependentmalic enzyme, phosphoenolpyruvate car-boxylase) and oxidativeelectron transport (cytochrome c oxidase). While there wereno overall changes in photosynthetic rate (measured in saturatingCO₂), infection either reduced (Rubisco and glycerate kinase)or did not affect (chloroplastic fructose bis-phosphatase andhydroxypyruvate kinase) the capacities of the photosyntheticcarbon reduction pathway or the photosynthetic carbon oxidationpathway. Key words: Plant-virus interaction, sucrose, starch, enzymes, ¹⁴CO₂ incorporation, O₂ flux     

Effects of tentoxin on enzymic activities in cucumber and cabbage cotyledons

Tentoxin, produced by Alternaria alternata (Fr.) Keissl. causes severe variegated chlorosis in germinating seedlings of certain dicotyledonous species. However, it does not impair radicle and hypocotyl elongation or cotyledon expansion. Effects of the toxin on the activity of selected enzymes from both chloroplasts and cytoplasm were determined. Cucumber (Cucumis sativus L.), which is highly sensitive to tentoxin and cabbage (Brassica oleracea L.), which is resistant, were used as test plants. The activities of chloroplastic, but not cytoplasmic, enzymes were decreased by treatment of cucumber cotyledons with tentoxin. Neither group of enzymes was affected by the toxin in cabbage cotyledons. The decreased enzymic activities are probably related to reported inhibition of photophosphorylation by tentoxin.     

Effect of light on nucleotide modifications in the transfer RNA of cucumber cotyledons

The effect of light on nucleotide modifications in the tRNA of cucumber (Cucumis sativus L. var. Guntur) cotyledons was studied by chromatographic, electrophoretic and immunological methods. The tRNA from light-grown tissue showed the absence of 2-methylguanosine and a decrease in the relative proportions of ribothymidine and cytokinin-active ribonucleosides when compared to those produced from dark-grown tissue. On the other hand, a significant amount of one species of 2′-O-methyldinucleotide was observed in the tRNA of light-grown tissue which was not detected in the dark-grown tissue. Also, tRNA from light-grown tissue had higher levels of another species of 2′-O-methyldinucleotide. The results showed no difference in the amounts of other modified nucleosides in tRNA between tissues grown under the two conditions. 2′-O-Methyl-l-methyladenosine, a nucleotide modified both in the base and the ribose, apparently specific to plant tRNAs, has been found to be present in the RNA of both light- and dark-grown tissues. These results on the variation in modified nucleotides suggest that light has some role in nucleotide modification and, consequently, in cellular functions.