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.     

Biological activity of the rolB-like 5' end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA

The iaaM gene from different plant-associated bacteria encodes a tryptophan monooxygenase (IaaM) that catalyzes the synthesis of indole-3-acetamide (IAM), a precursor of indole-3-acetic acid (IAA). Unlike the IaaM proteins from other bacteria, Agrobacterium spp. T-DNA-encoded IaaM proteins carry a 200 amino acid N-terminal extension with low homology to various members of the RolB protein family. This family is composed of 18 highly divergent T-DNA-encoded proteins, the basic functions of which are still largely undetermined. Deletion of the 5' rolB-like extension of the iaaM gene from Agrobacterium tumefaciens strain Ach5 did not lead to a reduction in IAM synthesis in plants. When expressed in tobacco, the rolB-like fragment did not affect growth or morphology. An iaaM homolog (A4-orf8) from the TL-DNA of Agrobacterium rhizogenes strain A4 also was investigated. Neither the full-size A4-orf8 gene nor the 5'-truncated form induced detectable IAM synthesis. Plants expressing the rolB-like part of the A4-orf8 gene, however, were dwarfed and mottled to various extents and synthesized abnormally high amounts of glucose, fructose, sucrose, and starch.     

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.     

Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.     

Transformation and regeneration of carrot (Daucus carota L.)

A protocol is presented for the efficient transformation of carrot (Daucus carota L. cv. Nantaise) by Agrobacterium tumefaciens. The binary vector contained the marker gene -glucuronidase (GUS), driven by the 35S promoter of cauliflower mosaic virus, and the nptII gene, which confers kanamycin resistance. Highest T-DNA transfer rates were obtained by co-cultivating bacteria with hypocotyl segments of dark-grown seedlings on solidified B5 medium containing naphthaleneacetic acid and 6-benzylaminopurine. After 2 days, bacterial growth was stopped with antibiotics. Two weeks later, the explants were placed on agar containing the kanamycin derivate geneticin; antibiotic-resistant calli developed during the following 4 weeks. Suspension cultures were obtained from resistant calli and plants regenerated via somatic embryogenesis in liquid culture. The majority of plants were phenotypically normal and, depending on the Agrobacterium strain used, harbored single or multiple copies of the T-DNA. About equal levels of GUS activity were found in different organs of young plants up to 6 weeks after embryogenesis. In leaves of older plants, GUS activity was markedly reduced, whereas the activities in phloem and xylem parenchyma cells of developing tap roots were still high and fairly uniform. Thus, the 35S promoter may be a useful tool to drive the expression of transgenes in developing carrot storage roots.     

Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids.

A physical map was constructed for the 250-kilobase plasmid pRiA4b, which confers the virulence properties of a strain of Agrobacterium rhizogenes for hairy root disease in plants. The complete HindIII and KpnI restriction map was determined from a collection of overlapping HindIII partial digest clones. Homologous regions with two well-characterized plasmids that confer virulence for crown gall disease, plasmids pTiA6 and pTiT37, were mapped on pRiA4b. As much as 160 kilobases of pRiA4b had detectable homology to one or both of these crown-gall-tumor-inducing plasmids. About 33 kilobases of pRiA4b hybridized to the vir region of pTiA6, a segment of DNA required for virulence of Agrobacterium tumefaciens. Portions of pTiA6 and pTiT37 transferred into plant cells in crown gall disease (T-DNA), shared limited homology with scattered regions of pRiA4b. The tumor morphology loci tms-1 and tms-2 from the T-DNA of pTiA6 hybridized to pRiA4b. A T-DNA fragment containing the tml and tmr tumor morphology loci also hybridized to pRiA4b, but the homology has not been defined to a locus and is probably not specific to tmr. A segment of pRiA4b T-DNA which was transferred into plant cells in hairy root disease lacked detectable homology to pTiA6 and had limited homology at one end to the T-DNA of pTiT37.     

Inhibition of agrobacterial oncogenes expression by means of antisense RNA

Plant's infection with soil bacteria Agrobacterium tumefaciens lead to tumour formation, so called crown galls. The reason of tumorigenesis is integration of agrobacterial genes for phytohormone synthesis auxins and cytokinins in plant genome, the most important of them are iaaM and ipt. Obtaining of transgenic plants able to inhibit these genes expression, creates conditions for producing of plants resistant to crown gall disease. With this purpose single and double transformants of tobacco plants with antisense copies of iaaM and ipt genes under the control of single and double promoters of 35S RNA of cauliflower mosaic virus (CaMV 35S and CaMV 35SS) were produced. Infection with virulentA. tumefaciens strains C58 (pTiC58) and A6 (pTiA6) of all types transgenic plants with antisense oncogenes copies showed essential but incomplete inhibition of these genes expression. After agrobacterial transformations of transgenic plants only "weakened" tumours of various morphology, able to regenerate the whole plants, were formed. The analysis data of inhibition of iaaM and ipt genes expression in formed tumour cells were presented. The results indicate perspective RNA-interference strategy for producing of plants resistant to agrobacterial crown gall disease.     

The Initiation of Auxin Autonomy in Tissue from Tobacco Plants Carrying the Auxin Biosynthesizing Genes from the T-DNA of Agrobacterium tumefaciens

Tobacco (Nicotiana tabacum cv Havana 425) plants containing the indole-3-acetic acid biosynthesizing genes (1 and 2) from the T-DNA of Agrobacterium tumefaciens strain T37-ADH₂ (mutated at the cytokinin biosynthesis gene 4) were used to study the physiological basis of the suppression and reinitiation of the auxin autonomous phenotype. The plants, though normal in appearance and cross-fertile with nontransformed, wild type tobacco, are shown to contain multiple copies of genes 1 and 2. Plants carrying these genes respond to inoculation by Agrobacterium strains mutated at genes 1 and 2 in a virulent fashion. Despite the presence and potential in planta activity of these genes, pith explants from such plants require auxin or tryptophan for growth in vitro, as does wild type tobacco. In both cases the indole-3-acetic acid levels increase rapidly in pith explants cultured on tryptophan-containing medium. However, only the tissues containing genes 1 and 2 grow subsequently on auxin-free medium and accumulate indole-3-acetic acid to levels that support growth. The capacity of such tissues to utilize naphthalene acetamide as an auxin suggests that gene 2 is rapidly activated during the reinitiation process.     

Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth

The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.     

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.     

Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1-11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.     

A nuclear gene encoding the beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia.

The beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia is encoded by two nuclear genes, atp2-1 and atp2-2, which are both expressed. The complete nucleotide sequence of atp2-1 has been determined. It contains eight introns ranging from 88 to 1453 bp. The last intron contains a putative insertion element (Inp), of 812 bp bordered by 35-bp inverted repeats which share an 11-bp homology with Agrobacterium tumefaciens T-DNA borders. Sequences homologous to Inp are present in multiple copies in the N. plumbaginifolia and the N. tabacum genome but not in more distant species. The atp2-1 encoded polypeptide is highly homologous to beta subunits from other ATP synthases but it contains an extension at the N terminus which is probably involved in mitochondrial targeting. A sequence homology between exon 4 of atp2-1 and exon 1 of the human ras genes suggests a common ancestral origin for these exons.     

Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes.

The T-DNA regions of the root-inducing (Ri) plasmid pRiA4b of Agrobacterium rhizogenes were characterized. Two regions, designated TL-DNA and TR-DNA, were found to be integrated and stably maintained in the plant genome. The TL-DNA spanned a 15- to 20-kilobase region of pRiA4b and was separated from the TR-DNA region by at least 15 kilobases of nonintegrated plasmid DNA. The TR-DNA region also spanned a 15- to 20-kilobase region of pRiA4b and included a region of homology to the tms morphogenic loci of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Eighteen deletions and 95 transposon insertions were generated in the T-DNA regions and tested for alterations in virulence. Insertions into four loci in the TL-DNA affected the morphology of root formation of Kalancho? diagremontiana leaves and stems, but had no visible effects on other host plants. Insertions into two loci (tms-1 and tms-2) in the TR-DNA eliminated virulence symptoms on all plants tested, with the exception of K. diagremontiana stems, where sparse root formation occurred. Complementation experiments with Ri and Ti plasmid T-DNA mutations indicate that the tms genes of the two plasmids serve similar functions and suggest a functional relationship between one or more genes of the TL-DNA and the cytokinin synthesis locus tmr of the Ti plasmid.     

Cassava Response to Water Deficit in Deep Pots: Root and Shoot Growth, ABA, and Carbohydrate Reserves in Stems, Leaves and Storage Roots

Cassava (Manihot esculenta, Crantz) is an important staple crop for tropical climates worldwide, including drought-prone environments where it is valued for its reliable yield. The extent to which stress tolerance involves regulation of growth and carbon balance aided by remobilization of carbohydrate from various plant parts was investigated. Plants were grown in 1-meter high pots to permit observation of deep rooting while they were subjected to four soil water regimes over a 30-d period. Transpiration declined abruptly in conjunction with leaf ABA accumulation and severe leaf abscission. In water stressed plants, growth of all plant parts decreased substantially; however, a basal rate of leaf growth continued to provide some new leaves, and although growth of fibrous lateral roots was reduced, main root elongation to deeper regions was only modestly decreased by stress. In leaf blades and petioles, sugars were the predominant form of nonstructural carbohydrate and about one third was in starch; these reserves were depleted rapidly during stress. In contrast, stems and storage roots maintained relatively high starch concentrations and contents per organ until final harvest. Stems gradually lost starch and had sufficient reserves to serve as a prolonged source of remobilized carbohydrate during stress. The amount of starch stored in stems represented about 35 % of the reserve carbohydrate in the plant at the onset of water stress (T₀), and 6 % of total plant dry mass. We suggest that this pool of carbohydrate reserves is important in sustaining meristems, growing organs, and respiring organs during a prolonged stress and providing reserves for regrowth upon resumed rainfall.     

Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants.

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N-terminal portions of the potato-derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly-A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50- to 500-fold higher invertase activity compared to non-transformed control plants. This invertase is N-glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.     

Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration.

Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi. The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements.     

Altered Growth and Wood Characteristics in Transgenic Hybrid Aspen Expressing Agrobacterium tumefaciens T-DNA Indoleacetic Acid-Biosynthetic Genes

A key regulator of cambial growth is the plant hormone indoleacetic acid (IAA). Here we report on altered wood characteristics and growth patterns in transgenic hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) expressing Agrobacterium tumefaciens T-DNA IAA-biosynthetic iaaM and iaaH genes. Eighteen lines simultaneously expressing both genes were regenerated. Of these, four lines, verified to be transgenic by northern blot analysis, were selected and raised under controlled growth conditions. All four lines were affected in their growth patterns, including alterations in height and stem diameter growth, internode elongation, leaf enlargement, and degree of apical dominance. Two transgenic lines, showing the most distinct phenotypic deviation from the wild type, were characterized in more detail for free and conjugated IAA levels and for wood characteristics. Both lines showed an altered IAA balance, particularly in mature leaves and roots where IAA levels were elevated. They also exhibited changes in wood anatomy, most notably a reduction in vessel size, an increase in vessel density, and changes in ray development. Thus, the recent development of techniques for gene transfer to forest trees enabled us to investigate the influence of an altered IAA balance on xylem development in an intact experimental system. In addition, the results demonstrate the possibility of manipulating wood properties in a forest tree through controlled changes of IAA concentration and distribution.