WO1997020470A1 - Regulation du developpement et de la physiologie de plantes par l'intermediaire du transport macromoleculaire par les plasmodesmes de proteines et d'oligonucleotides - Google Patents

Regulation du developpement et de la physiologie de plantes par l'intermediaire du transport macromoleculaire par les plasmodesmes de proteines et d'oligonucleotides Download PDF

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WO1997020470A1
WO1997020470A1 PCT/US1996/019260 US9619260W WO9720470A1 WO 1997020470 A1 WO1997020470 A1 WO 1997020470A1 US 9619260 W US9619260 W US 9619260W WO 9720470 A1 WO9720470 A1 WO 9720470A1
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cell
gene
protein
plant
tissue
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William J. Lucas
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to the field of plant biology. More particularly, the present invention is directed to compositions and methods for use in regulation of plant growth.
  • Partitioning of assimilates in plants is an important and highly regulated process [Wardlaw I. F. (1990) The control of carbon partitioning in plants. New Phytologis t 116, 341-381] . It involves regulation of photosynthesis, intracellular and intercellular transport of metabolites, phloem loading and unloading, storage and other interrelated biochemical processes. Partition of assimilates is closely related to the regulation of growth and development, in as much as growth of different plant parts and organs often requires the import of assimilates from elsewhere in the plant. The relationship between root and shoot biomass is an excellent example of regulation of partition of assimilates.
  • Root-to-shoot ratios vary from plant species-to-species, and are influenced by the environment [Geiger D. R. & Servaites J. S. (1991) Carbon allocation and response to stress. In Response of plants to mul tiple stresses (eds. H. A. Mooney, W. E. Winner & E. J. Pell ) pp. 103-127. Academic Press, New York; Mooney H. A. & Winner W. E. (1991) Partitioning
  • transgenic plants exhibited a decrease in translocation of assimilates, from source leaves, during the day [Lucas et al. 1993, supra] .
  • sucrose Similar considerations are involved in understanding the distribution of other plant products, such as sucrose.
  • Sucrose synthesis occurs within the cytosol of tobacco mesophyll cells, but the pathway followed by sucrose during its movement from the site of synthesis to the cells of the phloem remains equivocal.
  • the prevailing view is that solute movement between mesophyll cells occurs via a symplasmic route through plasmodesmata [Giaquinta, R.T. (1983) Phloem loading of sucrose. Ann. Rev. Plant Physiol . 34, 347-387; Tucker, J.E., Mauzerall, D., Tucker, E.B. (1989) Symplastic transport of carbxyfluorescin in staminal hairs of Setcreasea purpurea is diffusive and includes loss to the vacuole.
  • a plant regulatory composition (as hereinafter defined) is administered in a manner such that plasmodesmal transport of the composition in a
  • TMV-MP is shown to interfere with an endogenous signal transduction pathway that involves macromolecular trafficking through plasmodesmata to regulate biomass partitioning between the source and various sink tissues.
  • GLOBOSA A homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis.
  • EMBO J. 11, 4693-4704] have now been shown to interact with plasmodesmata to mediate in their cell-to-cell transport.
  • the first direct experimental proof that a plant mRNA-encoded protein can mediate in the plasmodesmal transport of itself and its own mRNA such that the mRNA can undergo extensive cell-to-cell movement is provided.
  • a further example illustrates that plant growth response to light intensity is altered by a viral movement protein.
  • selective cell-to-cell movements of proteins through plasmodesmata are shown to potentiate cellular interactions between cells in adjacent cell layers, such as: between layers of meristematic tissue; and, between vascular tissue cells and cells in adjacent mesophyll and epidermal layers.
  • Figs. 1A, 1B and 1C illustrate diurnal changes in 14 C-photosynthates in leaves of TMV-MP transgenic ( ⁇ ) and vector control (O) tobacco plants, experiments being performed on fully expanded source leaves (#5 and 6) (1A and 1B) or on younger, expanding source leaves (#2 and 3) (1C);
  • Fig. 3 is a schematic diagram illustrating sites where the TMV-MP might interact with the plant's endogenous control network to cause the observed alterations in sugar metabolism and reallocation of photosynthate to yield a reduction of root-to-shoot ratio in transgenic plants expressing the TMV-MP gene.
  • Fig. 4 is a schematic diagram, in conventional single-letter amino acid code, showing the positions of mutations for different alanine scanning mutants of wild type KN1.
  • compositions and methods which enable one skilled in the art to control this macromolecular trafficking and, thereby, control the rate and direction (e.g., to particular organs of the plant) of resource allocation.
  • the regulatory composition comprises at least one active agent which affects the growth and/or metabolism of the plant.
  • the composition may comprise one or more polypeptides and/or oligonucleotides encoding such polypeptides as an active agent.
  • the plant regulatory composition comprises at least one polypeptide and at least one oligonucleotide operatively encoding the polypeptide.
  • the active agent may comprise an endogenous or exogenous polypeptide or glycoprotein, including both heretofore known products and newly-engineered ones (e.g., mutant forms, fusion or chimeric proteins, etc.).
  • exemplary classes of products include, but are not limited to, the following:
  • growth factors e.g., KNOTTED- 1 as discussed in detail herein
  • herbicides e.g., KNOTTED- 1 as discussed in detail herein
  • insecticides e.g., actyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-N-phenyl-N-N-phenyl-N-N-N-phenyl-N-N-phenyl-N-N-N-phenyl-N-N-N-phenyl-N-N-N-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N
  • Systemin is a peptide of 18 amino acids [McGurl B., Pearce G., Orozco
  • a plant regulatory composition in the case of this herbivory blocker, would comprise an oligonucleotide coding for: systemin; defense genes such as Proteinase Inhibitor I and II; and, a movement protein specific for movement of its coding oligonucleotide through plasmodesmata.
  • Modulations in plant growth or metabolism can be any suitable modululations in plant growth or metabolism.
  • transgenic plants which express an endogenous or exogenous gene, from a plant or other source.
  • modified pathogenic genes and/or the development and expression of artificial genes that can mimic or override the functions performed by endogenous plant proteins encoded by the parent gene are also all clearly contemplated as within the scope of the present invention.
  • novel molecules can be engineered to utilize this cell-to-cell and phloem long-distance transport route.
  • Such a strategy will allow for the effective delivery of xenobiotic agents for control of a wide range of pests, as well as modified or genetically engineered plant proteins that will potentiate control over gene expression and
  • Example 1 the influence of the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) on carbon
  • the TMV-MP thus interferes with an endogenous signal transduction pathway that involves
  • Example 2 transgenic tobacco plants (Ni cotiana tabacum L. cv. Xanthi) expressing wild-type or mutant forms of the 30-kDa movement protein of tobacco mosaic virus
  • TMV-MP TMV-MP
  • 12 C-export from young leaves of TMV-MP plants, where the MP is yet to influence plasmodesmal size exclusion limit indicated a similar pattern, in that daytime 14 C export was slower in TMV-MP plants as compared to equivalentaged leaves on control plants.
  • Pulse-chase experiments were used to monitor radioactivity present in the different carbohydrate fractions, at specified intervals following 14 CO 2 labeling. These studies established that the TMV-MP can cause a significant adjustment in short-term 14 C-photosynthate storage and export. That these effects of the TMV-MP on carbon metabolism and phloem function were not attributable to the effect of this protein on plasmodesmal size exclusion limits, per se, was established using
  • transgenic tobacco plants expressing temperature-sensitive and C-terminal deletion mutant forms of the TMV-MP.
  • Example 4 it is shown that TMV-MP alters plant growth response to light intensity.
  • vector control wild type and deletion mutant forms of TMV-MP transgenic plants were grown under high light conditions, the wild type showed slight reductions in height and weight compared with the control. But, the deletion mutant exhibited an extremely different phenotype from the other two: plant height and total dry weight were greatly reduced.
  • mean mean:
  • Example 5 an analysis of movement of the protein and RNA encoded by the maize knottedl ( knl ) homeobox gene (8) is reported.
  • the protein product, KN1 moves between adjacent cell layers.
  • Microinjection studies in maize and tobacco show similar results.
  • comparison of wild type with various alanine scanning mutants of KN1 showed the mutants to have a reduced capacity to dilate plasmodesmata and potentiate their own cell-to-cell transport.
  • the present invention has immediate practical value in more precise characterization of the molecular steps involved in the process of macromolecular transport. This will then potentiate efficient manipulation of resource allocation without the complications associated with traditional genetic approaches .
  • Transgenic Ni cotiana tabacum L. cv Xanthi expressing the TMV-MP and various deletion mutant forms of the MP were obtained from Dr. Roger Beachy, Scripps Institute, La Jolla, California .
  • Transgenic line 277 expressed the TMV-MP, while line 306 was a control which contained only the plasmid vector [Deom et al. 1987, supra] . Details relating to the different mutant forms of the TMV-MP utilized in this example are provided in Table 1. Transgenic lines 277 and 306 were used in the graft experiments. Most plant lines were R 5 to R 8 and were homozygous for the MP construct. In addition, it was already confirmed that the effects of the TMV-MP on carbon metabolism and biomass partitioning were not due to somaclonal variation or position effects associated with insertion sites [Lucas et al. 1993, supra] . The results obtained on the independent transformed tobacco plants used in the present study, which involved a wide range of TMV-MP mutants (see Table 1), further confirmed this finding.
  • Seeds were germinated on a soil mix, and after four weeks, seedlings were transplanted into 17.5 cm diameter plastic pots containing Yolo sandy loam. Plants were watered with 1/2X Hoagland's solution [Hoagland D. R. & Arnold D. I. (1938) The water-culture method for growing plants without soil. California Agri cul tural Experimental Student Circular 357, 1-39] twice daily. Five to six weeks after
  • a "v" shaped notch was cut in the stem of the stock plant after removing the shoot at the second or third internode above the soil line.
  • the lower remaining leaves on the stock were removed and the scion stem base was cut to a wedge and then inserted into the notch made in the stock.
  • Both scion and stock were held together at the graft region using tygon tubing.
  • the lower mature leaves on the scion were removed to reduce transpiration.
  • the grafted plants were maintained in a mist chamber for two to three weeks until the graft had fused. Day/night temperatures in the mist chamber were 25°C/18°C, respectively. Grafted plants were then transferred to the greenhouse where they were grown for a further three weeks before being harvested for dry matter partitioning analysis.
  • leaf tissues of transgenic plant lines RMN-1, RMn-1, 277 and of vector control line 306 were extracted in lithium-phosphate buffer (50 mM) containing iodo-acetic acid (2.0 mM) and
  • TMV movement protein Role of the C-terminal 73 amino acids in subcellular localization and function. Virology 182, 682-689; Gafny R., Lapidot M., Berna A., Holt C A., Deom C. M. & Beachy R. N. (1992)
  • polyacrlamide gels to nitrocellulose sheets Procedure and some applications. Proc . Natl . Acad. Sci . USA 76, 4350-4354] with some modifications. Proteins were transferred onto Immobilon membrane (Millipore), which was then blocked in bovine serum albumin for one hour. The membrane was then incubated in phosphate-saline buffer containing antibody raised against the TMV-MP. The cross-reaction between the 30-kDa TMV-MP and the antibody against the TMV-MP was
  • Tobacco plants (lines 306 and 277) having six fully expanded leaves (mid-summer-grown plants) were inoculated on the 4th leaf (counting from the top) with 0.5 mg ml -1 TMV in phosphate buffer (pH 7.2) using Carborundum (400 mesh).
  • Plants were transferred to shade conditions (light intensity approx. 150 ⁇ mol m -2 s -1 ) for two days before being returned to normal full sunlight conditions in the greenhouse (see above for growth conditions). Symptom development was recorded and at the time of harvesting the plants had 12-14 systemically infected leaves, showing normal chlorotic-mosaic symptoms associated with TMV infection.
  • transgenic plants were produced by the putative endogenous control system that regulates carbon partitioning.
  • TMV-MP in these plant lines was confirmed by western blot analysis using antisera against the TMV-MP. As illustrated in the data presented in Table 3, when the
  • TMV-MP was localized to the phloem, such plants were similar in phenotype to the vector control line (cf. lines RMn-1, 306 and 277) . Note that although the leaf biomass was similar in lines 306, 277 and RMn-1, stem and root mass was
  • plasmodesmata was also restricted to the mesophyll and mesophyll/bundle-sheath boundaries [Ding et al . 1992, supra] .
  • experiments were performed on a range of MP deletion mutants that spanned the domain in the TMV-MP (amino acids 195-213) responsible for the dilation of secondary plasmodesmata [Berna et al . 1991, supra] . Since these mutant forms of the TMV-MP were expressed in tobacco genotypes nn and NTS.
  • transgenic tobacco line C-terminal 33 and 55 amino acids deleted, respectively, but they still retained the ability to dilate plasmodesmata to the level detected in TMV-MP transgenic plants [Berna et al . 1991, supra]. As shown in Table 4, transgenic tobacco line
  • MN-1 showed a slight reduction in its ability to override the endogenous control over carbon partitioning, when compared with that of the full-length TMV-MP.
  • transgenic tobacco line MN-2 a minor alteration in the ability of the mutant TMV-MP to reduce carbon allocation to the lower stem and roots was also observed.
  • a deletion within the SEL domain of the TMV-MP (line Mn-3) had no significant effect on growth or root-to-shoot ratios, when compared with values obtained on line 277. Furthermore, a similar result was obtained when the entire SEL domain was removed (line Mn-4; see Table 4). Note that in mutant lines Mn-3 and Mn-4 the plasmodesmal SEL was identical to line 306, being approx. 900 Da.
  • Transgenic line Mn-6 represented an ideal internal control for the latter
  • transgenic tobacco plants expressing the various mutant forms of the TMV-MP, plant height and number of leaves were selected from the various mutant forms of the TMV-MP, plant height and number of leaves.
  • TMV-MP has when constitutively expressed in transgenic tobacco plants
  • the effect of the TMV-MP produced during normal plant infection by TMV was explored.
  • TMV infected 306 plants had reduced root biomass and their subsequent root-to-shoot ratio was also lowered from the uninfected control value of 0.11 to 0.07 (i.e., a value identical to that measured on the uninfected 277 plant line).
  • TMV infection of plant line 277 failed to further reduce root mass or root-to-shoot ratio.
  • TMV-MP is required, specifically, in mesophyll tissue in order to exert its effects over biomass partitioning.
  • TMV-MP could traffic from vascular into mesophyll tissue, a change should have been observed in root-to-shoot ratio in plant line RMn-1 and RMN-1.
  • the absence of such an effect is consistent with the observation that macromolecular trafficking through plasmodesmata is always correlated with an increase in SEL to 9.4 kDa or above.
  • the inability of the TMV-MP to interact with phloem plasmodesmata to cause such an increase in SEL [Ding et al . 1992, supra] implies that the protein would not be able to traffic within this tissue, nor would it be capable of exiting across the bundle-sheath plasmodesmata to enter the mesophyll.
  • mesophyll would similarly be restricted to plasmodesmal transport within this tissue, but also would include
  • transgenic tobacco plants expressing the TMV-MP was observed in plants expressing a mutant form in which the N-terminal 3-5 amino acids were deleted. These plants exhibited an overall reduction in plant morphology in comparison to control line 306 (see Table 5). This effect is quite
  • CMV-MP cucumber mosaic virus
  • the present results establish that the TMV-MP exerts its effect on carbon allocation and biomass partitioning from a site located within the mesophyll tissue. Further, the mode of action of the TMV-MP, in terms of altering biomass
  • source leaves of TMV-MP transgenic tobacco plants have elevated levels of sugars and starch, although their photosynthetic rates are equivalent to control lines,
  • the TMV-MP may exert its effects directly, or
  • the TMV-MP would act as a competitive analogue to an endogenous protein that functions as an essential component of the input signal (s) that traffics, via plasmodesmata, from the mesophyll to the companion cell-sieve element complex.
  • These two protein homologue (the TMV-MP and the endogenous input signaling protein) would compete for a common plasmodesmal binding site, with the TMV-MP reducing the transport of the endogenous input signal.
  • transgenic plants expressing mutant forms of the TMV-MP exhibited an overall reduction in plant growth. This was the direct result of expressing a mutant form of the TMV-MP in which amino acids 3,4 & 5 had been deleted (mutant Mn-5 in Tables 4 & 5). In these transgenic tobacco plants, the presence of a dysfunctional Mn-5 TMV-MP gave rise to a down-regulation of overall plant growth. Whereas leaf number was little affected, total plant weight and plant height were reduced to 41% and 55%, respectively, of control plants
  • respiration was measured by covering the leaf chamber with a black cloth and measurements were started immediately after an increase in CO 2 concentration was observed. Negative values of photosynthesis were interpreted as dark
  • Carbohydrate content within leaves was determined as previously described [Lucas et al. 1993, supra] .
  • soluble sugars were extracted in 80% ethanol from leaf discs (1.5 cm 2 ). After evaporating the supernatant to dryness, sugars were redissolved in H 2 O and then filtered through a 0.45 ⁇ m membrane (Whatman, Clifton, New Jersey, USA).
  • Sugar separation was carried out on an analytical HPLC system (LDC Anal., Reviera Beach, Florida, USA), fitted with a Sugar-Pak I column (6.5 mm ⁇ 300 mm; Waters Associates, Milford, Mass., USA) using an LDC refractive-index detector (Refractor
  • Starch content was determined on the ethanol-water extracted leaf discs following starch conversion by amyloglucosidase (Cat. No. A-7255; Sigma Chemical Co., St. Louis, Missouri, USA). Starch content, as glucose equivalents, was measured using the Sigma (HK) quantitative glucose determination kit.
  • Rate of reduction in radioactivity of either total leaf 14 C or 14 C-labeled carbohydrates was determined from time course measurements that commenced immediately after a leaf was released from the 14 CO 2 -labeling chamber.
  • Radioactivity within an intact leaf was assayed using the portable ⁇ counter system following 14 CO 2 feeding. Then, three leaf discs were punched from the detection site and dissolved for 3-5 days in
  • TMV-MP transgenic plants (line 277) accumulated much higher amounts of carbohydrate during the day as compared to control tobacco plants (line 306); however, over the dark period, the level of all
  • Radioactivity within intact leaves was assayed non- destructively with a "Rotem" portable ß counter.
  • Experiments were performed on fully expanded source leaves (#5 and 6) (A, B) and on younger, expanding source leaves (#2 and 3) (C). Plant lines 277 and 306 were used for experiments in A and C, while plant lines 2004 and 3001 were employed in the
  • plant line 2004 [Deom et al 1991, supra] , were chosen for these studies. As illustrated in Fig. 1B, plant line 2004 also exhibited a slower rate of reduction in the level of radioactivity that remained in the source leaves compared with equivalent leaves on the respective vector control plants (line 3001).
  • Radioactivity in this component increased in both lines, probably due to turnover of cell wall components.
  • TMV-MP mutant MPP154A
  • ts temperature-sensitive (ts) TMV-MP, mutant MPP154A
  • alanine a temperature-sensitive strain of tobacco mosaic virus defective in cell-to-cell movement generates an altered viral-coded protein.
  • the SEL was elevated to levels identical to those measured in wild-type TMV-MP transgenic plants (line 277).
  • the SEL in plant line 2-72 was similar to that detected in control plants (line 306) [Wolf et al. 1991, supra] .
  • this plant line was selected for the present study as homozygous plants were shown to express the ts-form of the TMV-MP to yield levels that were equivalent to those present in wild-type TMV-MP plants (lines 277, 274, etc.).
  • previous studies on plant line 2-72 established that the ts TMV-MP did not undergo accelerated degradation under elevated
  • transgenic plants expressing either the ts mutant or wild-type TMV-MP, but was slower compared to control plants (line 306) .
  • Plants were grown in the greenhouse under natural sunlight with a midday average photon flux density of 1500 ⁇ mol.m -2 . s -1 , under two temperature regimes of 25°/18°C
  • a mutant form of the TMV-MP in which the C-terminal 10 amino acids were deleted, retained full wild-type function, supporting viral infection and causing an increase in
  • Sucrose is the major translocated sugar in many plant species, including tobacco [Giaquinta 1983, supra]. As demonstrated by analysis of the data presented in Table 8, the actual turnover of 14 C in the sucrose pool is retarded by the presence of the TMV-MP. Similar trends were also
  • TMV-MP may interact at any of the regulatory sites involved in controlling the compartmentation and/or the loading of sucrose into the sieve element-companion cell complex.
  • TMV-MP plasmodesmata would have remained dilated by the TMV-MP (see also Table 8).
  • the influence of the ts TMV-MP on sucrose levels strongly supports the hypothesis that the TMV- MP is pleiotropic in its effects within transgenic tobacco plants; i.e., this viral protein contains domains that allow it to interact with secondary plasmodesmata to potentiate the cell-to-cell transport of viral RNA [Lucas and Gilbertson 1994, supra ; Waigmann et al .
  • T ⁇ indicates control sites where the effect of the TMV-MP is overridden when sink strength is altered by raising the growth
  • PD plasmodesmata
  • SEL size exclusion limit
  • the temperature-induced convergence of net carbon export from lines 2-72, 277 and 306 may reflect the influence of an hierarchical control site, involving a feedback (or feedforward) mechanism whose input signal can override some of the sites where the TMV-MP interacts with the endogenous regulatory pathway(s) that controls
  • Knotted in young leaves allows cells that would normally be determinate in nature to undergo continued cell division, resulting in aberrant morphology [Hake S., Freeling M. (1986) Analysis of genetic mosaics shows that the extra epidermal cell divisions in Knot ted mutant maize plants are induced by adjacent mesophyll cells. Nature 320, 621-623; Smith et al . 1992, supra) .
  • KN1 has the ability to mediate in its own cell-to-cell transport
  • whether KN1 has the ability to interact with its own mRNA to allow the mRNA to undergo transport from the site of synthesis to the cells where KN1 controls tissue development was next tested.
  • Table 13 coinjection of KN1 and fluorescently labelled Knotted mRNA resulted in the efficient transport of mRNA from the target cell into the cells of the surrounding tissues.
  • injection of fluorescently labelled Knotted mRNA alone resulted in the confinement of the
  • TMV-MP transgenic plants exhibit varying degrees of reduction in both plant height and root biomass.
  • Plant line Mn-5 (expressing a mutant TMV-MP in which 3 amino acids [#3 -#5] from the N terminal were deleted [Lapidot et al. 1993 supra . ]) exhibited the most striking phenotype when grown under high light conditions. Plant height as well as total dry weight were approx. 40 and 50 percent lower than those of plant lines 277 (wild type) and 306 (control), respectively. Interestingly, mean internodal length was not significantly different between the three plant lines. It is important to note that the root-to-shoot ratio of plant line Mn-5 was similar to that of plant line 277, despite its specific phenotype. Finally, plants
  • TMV-MP transgenic and vector control plants undergo a significant reduction in root-to-shoot ratio (Table 14).
  • Table 14 The important point to note is that under these light conditions all plant lines tested had equivalent root-to-shoot ratios; i.e., in the presence of limiting light, the endogenous control system(s) involved in biomass partitioning appears to override the influence of the TMV-MP. However, the presence of the TMV-MP still appeared to influence other developmental processes. For example, while control plants responded to low light conditions by
  • Floricaula which affects meristem identity in Antirrhinum majus (snapdragon), in only the outer (epidermal) layer (LI) of the meristem, activates down-stream genes involved in flower development
  • KN1 immunolocalization of KN1, revealed the presence of KN1 in L1 cells in which its mRNA was not detected.
  • the shoot apical meristem was flanked by leaf primordia and older expanding leaves in which knl mRNA and KN1 were not detected. Regions in the shoot apical meristem that lacked KN1 predicted the position of leaf primordial development. KN1 was present in a few cells across the base of each developing leaf.
  • KN1 is a nuclear-localized transcription factor
  • Goat anti-rabbit-alkaline phosphatase (Boehringer Mannheim) was used as the secondary antibody (1:600 dilution) and visualized according to Jackson et al. [D. Jackson, B. Veit, S. Hake, Development 120, 405 (1994)] . Sections were lightly counterstained in basic fuchsin (0.005 % w/v).
  • fluorescently-labeled Escherichia coli -expressed KN1 (FITC-KN1) was microinjected into the cytoplasm of plant cells. Wild-type and mutant KN1 were expressed, extracted and labeled with fluorescein
  • FITC isothiocyanate
  • proteins were extracted and FITC-labeled from an E. coli preparation which did not contain the knl cDNA.
  • Alanine scanning mutants were created in groups of charged amino acids, which are likely to be present in surface domains (PC gene software, Intelligenetics).
  • the knl cDNA (BamHI-NcoI partial digest) from pKOC10 was inserted into the pET23-d(+) vector (Novagen) to create pDJX-1. Single-stranded virions were produced in the CJ236 (dut ung) strain of E.
  • K ⁇ 1 must be capable of interacting with
  • plasmodesmata to potentiate its own movement from cell to cell.
  • Tobacco offers another system in which to study K ⁇ 1, as ectopic meristems are also obtained when K ⁇ 1 is overexpressed in tobacco [ ⁇ . Sinha, R. Williams, S. Hake, Genes & Dev. 7 , 787 (1993)].
  • FITC-K ⁇ 1 microinjected into mesophyll cells of tobacco leaves also moved to neighboring cells. See Table 16.
  • SEL plasmodesmal size exclusion limit
  • KN1 and its mutant derivative, M6 were expressed in E. coli and extracted
  • KN1-induced increase in plasmodesmal SEL also permitted the cell-to-cell movement of a labeled, coinjected, 20 kDa soybean cytosolic protein, soybean trypsin inhibitor (Table 16). Occasionally, KN1 permitted the movement of a 39 kDa FITC-dextran, and so the upper plasmodesmal SEL associated with KN1 transport is greater than 20 and close to 39 kDa.
  • Protein domains essential for KN1 cell-to-cell movement were investigated using a series of alanine scanning mutants (created as described above). (Fig. 4) . Of the 9 mutants studied, only one (M6) showed a significant reduction in ability to move from cell to cell (Table 16) .
  • the M6 mutation resides in a potential nuclear localization sequence present in the N-terminal region of the homeodomain [E. Vollbrecht, R. Kerstetter, B. Lowe, B. Veit, S. Hake, in Evolutionary
  • FITC-KN1 could be detected in the neighboring cells was also short (a few seconds), subsequent movement into the second layer of cells required from 3 to 5 minutes. Furthermore, rarely was fluorescence detected beyond this second layer of mesophyll cells. Analysis of plant viral movement proteins
  • KN1 mutants showed that alanine scanning mutants either exhibited normal movement, or were incapable (0% movement) of cell-to-cell transport.
  • the varied response of KN1 mutants may reflect the presence of multiple domains involved in mediating efficient plasmodesmal transport or interaction with the plasmodesmata.
  • KN1 interacts with plasmodesmata to increase SEL and mediate in its own cell-to-cell
  • Sense knl RNA was TOTO-labeled and coinjected into mesophyll cells with
  • Knl sense or antisense RNA was transcribed using T3 or T7 RNA polymerase from linearized pKOC10 plasmid which contained the full length cDNA.
  • the DNA template was digested with RQ1 DNase (Promega) and the RNA was phenol extracted and ethanol precipitated.
  • Knl RNA (1.6 kb) was resuspended in 20 ⁇ l DEPC-H 2 O and concentration and purity was determined by spectroscopy.
  • Sense and antisense RNA 500 ⁇ g/ml were labeled with the nucleotide-specific fluorescent probe, TOTO-1 (Molecular Probes), as previously described. All knl RNA-TOTO preparations were adjusted to 225 ⁇ g/ml for use in microinjection experiments.
  • CMV RNA-TOTO was adjusted to 250 - 500 ⁇ g/ml.
  • RNAl 3.3 kb
  • RNA2 3.0 kb
  • RNA3 2.2 kb
  • the procedures of Ding et al. were used to prepare and FITC-label the CMV 3a movement protein.
  • KN1 would presumably have trafficked into surrounding cells, it failed to transport the CMV RNA-TOTO.
  • KN1 was selective in terms of the RNA that it would traffic as shown by coinjection of TOTO-labeled cucumber mosaic virus (CMV) single-stranded sense RNA and KN1 (Table 17).
  • CMV TOTO-labeled cucumber mosaic virus
  • the CMV movement protein in contrast, potentiated cell-to-cell transport both of its own RNA and of knl RNA (Table 17), consistent with the known non-specificity of viral movement proteins.
  • KN1 has the capacity to move from cell to cell provides a plausible explanation for the non-cell autonomy of the dominant Knl mutation, as well as the lack of autonomy found with other developmental mutations [P. W.

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Abstract

L'invention concerne des procédés et des mécanismes de régulation du transport macromoléculaire entre des cellules communiquant les unes avec les autres par les plasmodesmes. La protéine de mouvement du virus de la mosaïque du tabac (TMV-MP) est présentée sous des formes de type sauvage et mutantes et utilisée afin de modifier la taille des plantes, le métabolisme du carbone et la séparation de la biomasse. L'utilisation d'une protéine pour induire son propre transport de cellule à cellule par les plasmodesmes, est illustrée par des formes mutantes et de type sauvage de protéine KNOTTED (noueuse) provenant du gène de boîte de croissance du maïs Knotted (noueux).
PCT/US1996/019260 1995-12-04 1996-12-04 Regulation du developpement et de la physiologie de plantes par l'intermediaire du transport macromoleculaire par les plasmodesmes de proteines et d'oligonucleotides WO1997020470A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058487A2 (fr) * 1999-03-31 2000-10-05 Wisconsin Alumni Research Foundation Vecteurs à base de virus d'insecte et leurs utilisations
WO2000060088A2 (fr) * 1999-04-07 2000-10-12 E.I. Du Pont De Nemours And Company Genes de proteines de mouvement virales vegetales
WO2006137064A2 (fr) * 2005-06-21 2006-12-28 Ramot At Tel Aviv University Ltd. Structures et procedes destines a generer des plantes manifestant une conductance plasmodesmatale modifiee
US7186885B1 (en) 1999-04-07 2007-03-06 E.I. Du Pont De Nemours And Company Plant viral movement protein genes
WO2008001126A2 (fr) * 2006-06-30 2008-01-03 Plant Bioscience Limited Compositions et procédés se rapportant au ciblage cellulaire
KR101568342B1 (ko) 2013-02-07 2015-11-12 경상대학교산학협력단 원형질연락사를 통해 이동 가능한 복합체 및 그를 이용하여 식물 세포 간 목적 물질을 이동시키는 방법

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FEBS LETTERS, August 1990, Vol. 269, No. 1, TAKAMATSU et al., "Production of Enkephalin in Tobacco Protoplasts Using Tobacco Mosaic Virus RNA Vector", pages 73-76. *
LETTERS TO NATURE, 12 November 1987, Vol. 300, HILDER et al., "A Novel Mechanism of Insect Resistance Engineered Into Tobacco", pages 160-163. *
SCIENCE, 20 March 1992, Vol. 255, McGURL et al., "Structure, Expression and Antisense Inhibition of the Systemin Precursor Gene", pages 1570-1573. *
THE PLANT CELL, August 1992, Vol. 4, No. 8, DING et al., "Secondary Plasmodesmata are Specific Sites of Localization of the Tobacco Mosaic Virus Movement Protein in Transgenic Tobacco Plants", pages 915-928. *
THE PLANT CELL, December 1993, Vol. 5, No. 12, FUJIWARA et al., "Cell-to-Cell Trafficking of Macromolecules Through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein", pages 1783-1794. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058487A2 (fr) * 1999-03-31 2000-10-05 Wisconsin Alumni Research Foundation Vecteurs à base de virus d'insecte et leurs utilisations
WO2000058487A3 (fr) * 1999-03-31 2001-02-08 Wisconsin Alumni Res Found Vecteurs à base de virus d'insecte et leurs utilisations
US6700038B1 (en) 1999-03-31 2004-03-02 Wisconsin Alumni Research Foundation Plant expression vectors based on the flock house virus genome
WO2000060088A2 (fr) * 1999-04-07 2000-10-12 E.I. Du Pont De Nemours And Company Genes de proteines de mouvement virales vegetales
WO2000060088A3 (fr) * 1999-04-07 2001-04-26 Du Pont Genes de proteines de mouvement virales vegetales
US7186885B1 (en) 1999-04-07 2007-03-06 E.I. Du Pont De Nemours And Company Plant viral movement protein genes
US7572951B2 (en) 1999-04-07 2009-08-11 E. I. Du Pont De Nemours And Company Plant viral movement protein genes
WO2006137064A2 (fr) * 2005-06-21 2006-12-28 Ramot At Tel Aviv University Ltd. Structures et procedes destines a generer des plantes manifestant une conductance plasmodesmatale modifiee
WO2006137064A3 (fr) * 2005-06-21 2007-02-15 Univ Ramot Structures et procedes destines a generer des plantes manifestant une conductance plasmodesmatale modifiee
WO2008001126A2 (fr) * 2006-06-30 2008-01-03 Plant Bioscience Limited Compositions et procédés se rapportant au ciblage cellulaire
WO2008001126A3 (fr) * 2006-06-30 2008-03-27 Plant Bioscience Ltd Compositions et procédés se rapportant au ciblage cellulaire
KR101568342B1 (ko) 2013-02-07 2015-11-12 경상대학교산학협력단 원형질연락사를 통해 이동 가능한 복합체 및 그를 이용하여 식물 세포 간 목적 물질을 이동시키는 방법

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