The T-DNA oncogene A4-orf8 from Agrobacterium rhizogenes A4 induces abnormal growth in tobacco

The related orf8 and iaaM T-DNA genes from Agrobacterium are each composed of two distinct parts. The 5' parts (called Norf8 or NiaaM) encode a 200-amino-acid (aa) sequence with homology to various T-DNA oncoproteins such as RolB, RolC, and 6b. The 3' parts (Corf8 or CiaaM) encode a 550-aa sequence with homology to IaaM proteins from Pseudomonas and Pantoea spp. Whereas iaaM genes encode flavin adenine dinucleotide (FAD)-dependent tryptophan 2-monooxygenases that catalyze the synthesis of indole-3-acetamide (IAM), A4-orf8 from Agrobacterium rhizogenes A4 does not. Plants expressing a 2x35S-A4-Norf8 construct accumulate soluble sugars and starch. We now have regenerated plants that express the full-size 2x35S-A4-orf8 and the truncated 2x35S-A4-Corf8 gene. 2x35S-A4-Corf8 plants accumulate starch and show reduced growth like 2x35S-A4-Norf8 plants but, in addition, display a novel set of characteristic growth modifications. These consist of leaf hypertrophy and hyperplasia (blisters); thick, dark-green leaves; thick stems; and swollen midveins. Mutations in the putative FAD-binding site of A4-Orf8 did not affect the blister syndrome. Plants expressing 2x35S-A4-Corf8 had a normal phenotype but contained less starch and soluble sugars than did wild-type plants. When 2x35S-A4-Corf8 plants were crossed to starch-accumulating 2x35S-A4-Norf8 plants with reduced growth, A4-Corf8 partially restored growth and reduced starch accumulation. A4-Corf8xA4-Norf8 crosses did not lead to the blister syndrome, suggesting that this requires physical linkage of the A4-NOrf8 and A4-COrf8 sequences.     

The rolB-like part of the Agrobacterium rhizogenes orf8 gene inhibits sucrose export in tobacco

Many Agrobacterium T-DNA genes belong to the highly diverse rolB family. The mode of action of most of these genes is still unknown. rolB-like sequences also are present at the 5' ends of the T-DNA-located iaaM genes and the iaaM homolog orf8, whereas iaaM genes from Pseudomonas and Erwinia spp. lack such sequences. iaaM genes encode tryptophan monooxygenases; these enzymes convert tryptophan into indole-3-acetamide, a precursor of indole-3-acetic acid. Tobacco plants expressing the rolB-like part of the A4 orf8 gene (2x35S-A4-Norf8 plants) accumulate glucose, fructose, sucrose, and starch and resemble sucrose transporter (NtSUT1) antisense plants. Different lines of evidence indicate that 2x35S-A4-Norf8 plants export less sucrose from source leaves. Glucose, fructose, sucrose, and starch accumulate in source leaves during sink-source transition, whereas sink tissues like petioles and midveins contain lower levels than normal. Petiole exudation experiments demonstrate a significant decrease in export of label after 14C-sucrose infiltration and after 14CO2 labeling. Grafting of stunted homozygous 2x35S-A4-Norf8 plants onto wild-type rootstocks restores growth, indicating that unloading is not affected. Growth of 2x35S-A4-Norf8 seedlings is inhibited on naphthalene acetic acid-containing media, suggesting a link between sucrose transport and auxin sensitivity.     

IAA synthesis and root induction with iaa genes under heat shock promoter control

We have devised a heat shock-inducible indole-3-acetic acid (IAA) synthesis system for plant cells, which is based on the iaa genes of the Agrobacterium tumefaciens T-DNA and the heat shock promoter hsp70 of Drosophila melanogaster.Two DNA constructs were tested: one contains the iaaM gene linked to the hsp70 promoter (hsp 70-iaaM) and encodes the production of indoleacetamide (IAM), the other contains hsp 70-iaaM and the wild-type iaaH gene which codes for the conversion of IAM into IAA (hsp 70-iaaM/iaaH). Heat shock-controlled IAM and IAA synthesis was tested on two levels: biochemically by measuring IAM and IAA levels in Kalanchoe stem segments infected with the two constructs, and morphologically by IAA-dependent root formation on Kalanchoe plants, on carrot discs and on tobacco leaf fragments. At both levels the responses were found to be controlled by the heat shock promoter. IAM levels of segments infected with hsp 70-iaaM increased 6-fold upon heat shock induction to 240 pmol IAM per stem segment. The accumulation of IAA in segments infected with hsp 70-iaaM/iaaH and heat-shocked was found to be more variable, possibly due to IAA transport and metabolism. Heat shock treatment of Kalanchoe plants and tobacco leaf fragments infected with hsp 70-iaaM/iaaH led to a strong increase in root formation. On carrot discs, heat shock-specific root induction was also demonstrated, but the responses differed between individual carrots.     

The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity

The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4. The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein. The N-terminal domain shows homology to the A. rhizogenes rolB protein and may modulate the auxin responsiveness of host cells. The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m). We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants. Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls. We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity.     

35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

Introduction of a novel pathway for IAA biosynthesis to rhizobia alters vetch root nodule development

We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.     

Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene

The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L. x Populus tremuloides Michx.). We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA. In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression. Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA. Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants. This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth.     

Evidence that Indole-3-Acetic Acid is Not Synthesized Via the Indole-3-Acetamide Pathway in Pea Roots

The biosynthetic route of the key plant hormone, indole-3-acetic acid (IAA) has confounded generations of biologists. Evidence in higher plants has implicated two auxin intermediates with roles established in bacteria: indole-3-acetamide (IAM) and indole-3-pyruvic acid. Herein, the IAM pathway is investigated in pea (Pisum sativum), a model legume. The compound was not detected in pea tissue, although evidence was obtained for its presence in Arabidopsis, tobacco, and maize. Deuterium-labeled tryptophan was not converted to IAM in pea roots, despite being converted to IAA. After feeds of deuterium-labeled IAM, label was recovered in the IAA conjugate IAA-aspartate (IAAsp), although there was little or no labeling of IAA itself. Plants treated with IAM did not exhibit high-IAA phenotypes, and did not accumulate IAA. This evidence, taken together, indicates that although exogenous IAM may be converted to IAA (and further to IAAsp), the IAM pathway does not operate naturally in pea roots.     

大白菜orf224基因植物表达载体的构建及遗传转化

为了创制新的大白菜雄性不育材料,双酶切重组质粒pMD18-T-CMS7311-orf224和表达载体pWR306后,将CMS7311-orf224基因与线性表达载体pWR306进行定向连接,构建了细胞质雄性不育线粒体基因CMS7311-orf224的植物表达载体pWR-CMS7311-orf224,通过酶切和PCR验证pWR-CMS7311-orf224载体构建正确.采用快速冻融法,将表达载体导入农杆菌EHA105,转化大白菜材料06J28,对转化植株的GUS、PCR和RT-PCR检测表明,获得了2个大白菜转基因植株.     

The Pseudomonas savastanoi tryptophan-2-mono-oxygenase is biologically active in Nicotiana tabacum

It has been proposed that the eukaryotic T-DNA-encoded indole-3-acetic acid (IAA) biosynthesis genes of Agrobacterium tumefaciens and their prokaryotic counterpart in Pseudomonas savastanoi originated from common ancestor genes. This paper provides additional evidence for the functional similarity between the gene products. We have demonstrated that a chimeric gene consisting of the coding sequence of the P. savastanoi tryptophan-2-mono-oxygenase (iaaM gene) and a plant promoter encodes an active enzyme in Nicotiana tabacum. Transformants obtained with this chimeric gene grew as a callus on hormone-free media. No stably transformed plantlets could be isolated. The callus tissues contained extremely high levels of indole-3-acetamide and slightly elevated levels of IAA. Either indole-3-acetamide by itself has a low auxin activity or, alternatively, it is converted aspecifically and at low rates into IAA. The P. savastanoi tryptophan-2-mono-oxygenase activity in plants is also able to detoxify the amino-acid analogue 5-methyltryptophan. This property can be used for positive selection of transformed calli.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IAM indole-3-acetamide - NAA naphthalene-1-acetic acid - NPT-II neomycin phosphotransferase II - T-DNA transferred DNA     

Targeted engineering of Azospirillum brasilense SM with indole acetamide pathway for indoleacetic acid over-expression

Rhizospheric bacterial strains are known to produce indole-3-acetic acid (IAA) through different pathways, and such IAA may be beneficial to plants at low concentrations. IAA biosynthesis by a natural isolate of Azospirillum brasilense SM was studied and observed to be tryptophan-inducible and -dependent in nature. While our work demonstrated the operation of the indole pyruvic acid pathway, the biochemical and molecular evidence for the genes of the indole acetamide (IAM) pathway were lacking in A. brasilense SM. This led us to use the IAM pathway genes as targets for metabolic engineering, with the aim of providing an additional pathway of IAA biosynthesis and improving IAA levels in A. brasilense SM. The introduction of the heterologous IAM pathway, consisting of the iaaM and iaaH genes, not only increased the IAA levels by threefold but also allowed constitutive expression of the same genes along with efficient utilization of IAM as a substrate. Such an engineered strain showed a superior effect on the lateral branching of sorghum roots as well as the dry weight of the plants when compared with the wild-type strain. Such an improved bioinoculant could be demonstrated to enhance root proliferation and biomass productivity of treated plants compared with the parental strain.     

Genetic evidence that the tryptophan 2-mono-oxygenase gene of Pseudomonas savastonoi is functionally equivalent to one of the T-DNA genes involved in plant tumour formation by Agrobacterium tumefaciens

Summary The combined activities of the Agrobacterium tumefaciens T-DNA genes 1 and 2 are sufficient to induce tumorous growth on several plants, by introducing a new auxin biosynthetic pathway in infected cells. We have isolated Nicotiana tabacum plants containing only gene 1 or gene 2. These plants, respectively called rG1 and rG2, grow and develop in a normal fashion, indicating that neither the gene 1 nor the gene 2 activity by itself interferes with the endogenous auxin metabolism in plants. Previous evidence indicated that the auxin biosynthetic pathway of Pseudomonas savastanoi and that proposed to be encoded by the T-DNA of Agrobacterium tumefaciens are similar. When rG2 plants were infected with non-oncogenic A. tumefaciens or Escherichia coli strains that harbour the P. savastanoi iaaM gene (responsible for indole-3-acetamide synthesis) root and callus formation at the infection site was readily observed. This shows that the product of iaaM, indole-3-acetamide, is an in vivo substrate for the gene 2 encoded enzyme and supports the proposal that the gene 1-encoded enzyme is involved in the synthesis of indole-3-acetamide in transformed plants. This result offers new insights in evolution of bacteria and plants involved in pathogenic and symbiotic interactions.Abbreviations IAM indole-3-acetamide - IAA indole-3-acetic acid     

The NtAMI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells can be grown in medium containing indole-3-acetamide (IAM). Based on this finding, the NtAMI1 gene, whose product is functionally equivalent to the AtAMI1 gene of Arabidopsis thaliana and the aux2 gene of Agrobacterium rhizogenes, was isolated from BY-2 cells. Overexpression of the NtAMI1 gene allowed BY-2 cells to proliferate at lower concentrations of IAM, whereas suppression of the NtAMI1 gene by RNA interference (RNAi) caused severe growth inhibition in the medium containing IAM. These results suggest that IAM is incorporated into plant cells and converted to the auxin, indole-3-acetic acid, by NtAMI1.     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

Phenotypes of Tobacco Plants Expressing Genes for the Synthesis of Growth Regulators

The expression of genes for synthesis of auxin (iaaM and iaaH) and cytokinins (ipt) was studied in tobacco plants transformed by two Agrobacterium tumefaciens strains C 58 and LBA 4404. The strain LBA 4404 carried binary vector plasmid pCB 1334 (ipt gene) and plasmid pCB 1349 (iaaM, iaaH and ila genes). Both plasmids carried reportered gene for npt II. Obtained plants expressed incorporated genes. New proteins with molecular masses of about 74, 40, 26, 25, 21 and 17 kDa for wild plasmid pTi C58; 60, 36, 31.5, 27, 26 and 17 kDa for binary vector plasmid pCB 1334 and 74, 49, 36, 31.5, 26 and 25 kDa for binary vector plasmid pCB 1349 were found in the patterns of soluble proteins. Significant changes in the content of chlorophylls, especially chlorophyll a, were detected in the plants carrying ipt gene and in plants transformed by the wild strain C58 of A. tumefaciens. Tobacco plants expressing ipt gene and genes from T-DNA of pTi C58 plasmid were dwarf, and in comparison to the controls, they had thicker stems, and the surface of the leaf blades was reduced to 20 - 50 %. Adventitious roots, growing from the stem, were typical for transformants overproducing auxins. Regenerants and transformants expressing genes from T-DNA of plasmid pTi C58 differed in the shape of the flowers and their fertility. This revised version was published online in July 2006 with corrections to the Cover Date.     

苏云金杆菌辅助蛋白基因p19和orf1-orf2对杀虫晶体蛋白Cyt1 Aa表达的影响(英文)

为检测苏云金杆菌辅助蛋白P19和ORF1 ORF2对杀虫晶体蛋白Cyt1Aa表达的影响 ,构建了 5个重组表达质粒。 5个质粒都含有cyt1Aa基因 ,但pT1只含有cyt1Aa基因 ,pT2同时含有p19基因 ,pT3同时含有orf1 orf2串联基因 ,pT4同时含有p19基因和p2 0基因 ,pT5同时含有orf1 orf2串联基因和p2 0基因。将这 5个表达质粒和质粒pWF4 5电转化到苏云金杆菌晶体缺陷型 4Q7中 ,分别获得转化菌株Bt T1、Bt T2、Bt T3、Bt T4、Bt T5和Bt WF4 5。SDS PAGE结果显示 ,菌株Bt T1、Bt T2和Bt T3只产生少量的 2 7kDCyt1Aa蛋白 ,而且部分降解为大约 2 4kD的蛋白。而Bt T4和Bt T5能产生大量的Cyt1Aa蛋白 ,但Bt T4和Bt T5的Cyt1Aa蛋白产量都明显少于Bt WF4 5。电镜观察和生物测定结果表明Bt T4和Bt T5与Bt WF4 5的晶体大小和杀蚊毒力没有显著性差异。研究表明P19和ORF1 ORF2对Cyt1Aa蛋白的合成显示可能有抑制作用。