WO1998012321A1

WO1998012321A1 - Recombinant plant cells expressing heterochromatin 1 (hp1) of drosophila melanogaster

Info

Publication number: WO1998012321A1
Application number: PCT/GB1997/002059
Authority: WO
Inventors: Peter Meyer
Original assignee: The University Of Leeds
Priority date: 1996-09-20
Filing date: 1997-08-01
Publication date: 1998-03-26
Also published as: EP0939811A1; CA2265898A1; CN1230992A; AU3703697A; GB9619633D0

Abstract

A plant or plant cell containing the chromo domain of heterochromatin protein 1, ideally derived from the genus Drosophila, wherein said domain confers selective advantage on said plant or cell.

Description

RECOMBINANT PLANT CELLS EXPRESSING HETEROCHROMATIN 1 (HPI) OF DROSOPHILA MELANOGASTER

The invention relates to recombinantly engineered plant cells which provide for plants with improved vigour; plants including such plant cells; and seeds derived from such plants.

With a view to increasing farming efficiency so as to enhance crop productivity and also investment returns on farming there is a relentless drive towards improving plant vigour. Traditionally, this has been undertaken using conventional plant breeding techniques where two highly inbred strains of plants are cross-bred in order to provide a FI generation exhibiting significant hybrid vigour. Unfortunately, such vigour is typically only characteristic of the FI generation and therefore if productivity is to be maintained, farmers must continually obtain new stocks of other FI generation plants from plant suppliers. The reasons behind this sort of hybrid vigour are largely unknown.

As an alternative to conventional plant breeding, genetic engineering has been used to try and improve plant productivity by selectively engineering into various crop plants genes which are thought to improve the overall yield of a given crop. The estimated market for such genetically engineered plants is expected to reach USS6 billion worldwide by 2005. However, transcriptional silencing of transgenes is a major obstacle in the application of recombinant technology to modern agriculture. The reason for this silencing is largely unknown but may be due to hypermethylation and local condensation of plant chromatin.

The chromosomes of higher eukaryotes are composed of euchromatin and heterochromatin. Heterochromatin is distinguished from euchromatin in that it remains condensed through interphase, replicates late in the cell cycle, is enriched for repetitive sequences and is relatively poor in the number of genes.

If a gene that normally resides in euchromatin is placed next to heterochromatin, as a consequence of chromosomal rearrangement it will undergo cell-specific silencing giving rise to a variegated phenotype.

Thus it would seem that the structure of chromatin has at least a part to play in influencing the expression of transgenes.

In our studies we therefore set out to investigate further the part that DNA hypermethylation and local condensation of chromatin may have to play in transgene expression. However, our work lead to a remarkable discovery in that we found that through trying to identify agents which may have a part to play in transgene silencing we were able to significantly increase plant vigour in a manner that we believe can be passed on from generation to generation.

Our studies concerned the Drosophila polycomb gene product (Pc) and the Drosophila heterochromatin protein 1 (HPI). These two proteins share an almost identical domain termed the "chromo domain", for chromosome organisation modifier. This domain is highly conserved in most eukaryotes.

Whilst HPI is a heterochromatin protein, Pc is part of a complex that maintains the repressed state of homeotic genes.

We thus began our experiments by expressing both these genes in plant cells using a recombinant construct that linked the chromo domains to the green fluorescent protein (GFP) isolated from the bioluminescent jellyfish Aequorea victoria. GFP was linked to the chromo domains of HPI and Pc via a Kinase recognition sequence.

The amino acid homology of HPI and Pc is shown in Figure 1 and it can be seen that there is a considerable of homology as would be expected from such a highly conserved site.

However, we found, surprisingly, that despite the homology between these two proteins, plant cells transfected with HPI grew considerably better than either plant cells transfected with Pc or control cells where no transfection occurred. In fact, we found that cells transfected with HPI showed at least a 10% increase in productivity. This is equatable with the sort of increase one would normally associate with conventional hybrid vigour following typical plant breeding techniques.

Moreover further experimentation with transgenic plants expressing the HPI chromo domain has indicated that the transgene may be able to protect plants from environmental stress.

In the presence of 120 or 150 mM NaCl there is a 1.5 - 2 fold increase in plant biomass yield when compared to plants not expressing the HPI chromo domain. This suggests that the presence of the HPI chromo domain is able to make plant cells refractory to the effects of elevated salt concentration.

This discovery was taken further by analysing the effects of cadmium on seedling growth. The rational for this stems from the observation that many environments where plants would flourish do not because of the presence of elevated levels of heavy metals such as copper, lead and cadmium. If the HPI transgene can protect plants against the harmful effects of heavy metals then these environments may become accessible to plant species that would ordinarily not thrive in these soils.

Experiments to monitor the effects of CdS0₄ were undertaken to establish if expression of the HPI chromo domain in seedlings has protective properties. The inclusion of 50 μM CdS0₄ in culture of medium resulted in a 1.7 - 4.0 fold increase in biomass yield when compared to control seedlings not expressing the HPI transgene.

This indicated that the HPI transgene has protective properties and enables seedlings to tolerate elevated levels of CdS0₄ in growth medium.

Given this information it can be seen that it is an object of the invention to provide a plant cell, plants including such cells and seeds from such plants which exhibit a marked increase in plant productivity, ideally, transferable between generations.

According to a first aspect of the invention there is therefore provided a plant cell including, in expressionable form, the gene, or part thereof, encoding at least the chromo domain of heterochromatin protein 1, or a gene, or part thereof, having greater than 60% homology therewith.

It can be seen from Figure 1 that although HPI and Pc are very similar they differ by approximately 18 amino acids representing a variation of approximately 60% homology (ie 27/45). Since the functional effectiveness of the two proteins, in terms of plant yield, is so distinct it follows that the variations are significant and that at least one of the differences between the two proteins confers a distinct advantage in terms of plant growth. Thus although the two proteins have 27 identical amino acids and five amino acids from the same group, this similarity is not sufficient to confer desirable growth promoting properties on both proteins. It therefore follows that a protein identical to HPI or having greater than 60% and ideally 70% homology therewith, or more ideally, greater than 70% homology therewith, can be used in the invention to advantageously affect plant growth.

In a preferred embodiment of the invention said gene is derived from the genus Drosophila.

According to a yet further aspect of the invention there is provided a construct comprising a gene, or part thereof, encoding at least the chromo domain of heterochromatin protein 1, or a gene, or part thereof, having greater than 60% homology therewith, coupled to a nuclear targeting domain so as to ensure that said HPI gene is imported into the nucleus of the target cell.

According to a further aspect of the invention there is provided a plasmid or vector suitable for effecting transformation of a plant cell with the construct of the invention which vector or plasmid includes the construct of the invention.

According to a further aspect of the invention there is provided a plant including at least one plant cell according to the invention. According to a yet further aspect of the invention there are provided seeds obtained from a plant including at least one plant cell according to the invention.

According to a yet further aspect of the invention there is provided a method for improving plant productivity of a selected plant comprising transforming a plant cell from said selected plant with an agent including a gene, or part thereof, encoding at least the chromo domain of heterochromatin protein 1, or a gene, or part thereof, having greater than 60% homology therewith; and culturing said transformed plant cell, under suitable culture conditions, so as to provide for the growth of a corresponding plant.

In a preferred embodiment of this aspect of the invention said gene, or part thereof, is further coupled to a nuclear targeting domain so as to ensure that the gene, or part thereof, is imported into the nucleus of the target cells.

Although the invention has been described having regard to the chromo domain of HPI it is within the scope of the invention to practise same using the entire HP I gene, or selected parts thereof - providing said parts include the chromo domain.

An embodiment of the invention will now be described by way of example only with reference to the following Figures, tables and methods wherein:

Figure 1 shows the DNA sequence structure and corresponding amino acid sequence structure of the chromo domain of the heterochromatin protein 1 and the chromo domain is represented by sequence 603-738. Figure IB shows the amino acid sequence structure of the chromo domains of the Drosophila polycomb protein and heterochromatin protein 1;

Figure 2 shows the design of the protein fusion construct used in the methods described herein;

Figure 3 illustrates location of the fusion protein in the nucleus of a target cell;

Figure 4A shows improved vigour of expressing line 855/3 compared to wide type SRI;

Figure 4B shows improved vigour of strong expressing line 855/3-4 compared to the weak expressing line 873/2-3;

Figure 5 shows improved growth under salt stress conditions;

Figure 6 shows improved growth of various strains either expressing or not expressing the transgene encoding the chromo domain of heterochromatin protein 1 ;

Figure 7 shows the vector used for the transfer of the chromo domains of polycomb and heterochromatin protein 1 into plant cells;

Table 1 shows the improved biomass yield of seedlings expressing the HPI transgene in the presence of either 150mM NaCl, 1 x Ms medium (A) or 120mM NaCl, 0.5 x Ms medium (B); EXP = expressing plants NONEXP = non-expressing HPI transgenic plants. Table 2 shows improved biomass yield of seedlings expressing the HPI transgeneic (EXP) and non-expressing seedlings (NONEXP) cultured in 0.5 x Ms medium, 50 μM CdS0₄.

Materials and Methods

Protoplast isolation. Sterile shoot cultures of Nicotiana tabacum cv. Petit

Havana SRI (Maliga et al. 1973) grown on LS medium were used for protoplasts isolation. The basal medium used for isolation and incubation of protoplasts was M3, a K3 medium (Nagy and Maliga 1976) supplemented with vitamins, organic acids, sugars and sugar alcohols and vitamin-free casamino acid according to Kao and Michayluk (1975). As described by

Shillito et al (1983) leaf halves without midribs were soaked in M3 medium containing 0.4 M sucrose and cut into thin sections. 2 g of leaf material were incubated in the dark in 30 ml enzyme solution ( 1.3 % Cellulase "Onozuka R- 10" (Serva), Heidelberg, FRG), 0.55% Mazerozyme R-10 (Serva) in M3 medium 0.4 M sucrose) for 20-22 h at room temperature. After lh or moderate swirling the digest was filtered through at lOOμm mesh filter. Protoplasts were floated by centrifugation at 100 g for 10 min and washed twice with osmoticum (0.16 M CaC , 0.5 % w/v 2(n-Morpholino)-ethanesul- phonic acid (MES), 250 mM mannitol, pH 5.6).

Preparation of DNA. The vector used to transfer the chromo domains of polycomb and heterochromatin protein 1 is shown in Figure 7. The vector is referenced 35shgfp. The restriction sites are clearly shown. The vector is based on an EcoRI-Sall fragment from pBR322 containing the bacterial origin of replication and the amp resistance gene for bacterial transformation. The plant selectable marker is a HPT cassette (gift from Dr David Wing and Dr Czaba Concz, Max-Planck-Institute for Breeding Research, Cologne, Germany) which contains a nopaline synthase promoter, a hygromycin resistance gene and a gene 4 polyadenylation region. The GFP construct is inserted between a 35S promoter and terminator.

Protoplast transformation. The fusion method of Hein et al. (1983) was adapted for DNA uptake. About 250,000 protoplasts were washed with osmoticum and resuspended in a final volume of 120 l osmoticum. 10-30μg of DNA dissolved in 20μl sterile water were added. \A0u\ PEG solution (0.5% N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 25% PEG 6000, 0.45 M Mannitol, 0.1M Ca(N0₃)₂ pH 9.5) were added slowly.

After 20 min 5ml of wash-solution (0.5% Hepes, 0.29 M Ca(N0₃)₂, 0.45 M Mannitol, pH 9) were added dropwise. After 10-15 min cells were pelleted and resuspended in 2 ml M3 (0.4 M sucrose, 1 mg/1 NAA, 0.2 mg/1 kinetin).

Cultivation and selection of treated protoplasts. Protoplasts were cultured using the bead type technique of Shillito et al. (1983) which was modified as follows: one week after transformation 3 ml protoplast solution was mixed with 3 ml M3 (0.3 M sucrose, 1 mg/1 NAA, 0.2 mg/ 1 kinetin) containing 1 %

Seaplaque LMT agarose (Marine Colloids, Rockland, USA) in a 5 cm petri dish. After the agarose bead had been transferred to a 10 cm petri dish, 5 ml of M3 (0.3 M sucrose, 1 mg/1 NAA, 0.2 mg/1 kinetin) were added containing

90 mg/ 1 kanamycin acid sulfate to select for kanamycin resistant calli.

Media were changed every 3-4 days lowering the sucrose content by 50 mM per step. After 3-4 weeks, colonies were transferred to solid M3 medium (3% sucrose, 1 mg/1 NAA, 0.2% kinetin, 100 mg/1 kanamycin sulfate). To determine the frequency of transformation calli were grown non selectively, picked at a stage of 2 mm diameter in size and transferred onto solid selection medium containing 100 mg/1 kanamycin.

Regeneration of plants. Shoots were induced by incubation of calli on solid M3 medium (3% sucrose, 0.5 mg/1 benzyl-aminopurine (BAP), 0.1 mg/1 NAA, 100 mg/1 kanamycin acid sulfate (personal communication P.Stabel)). For development of roots, shoots were transferred to solid LS medium

Linsmaier and Skoog (1965) (1 % sucrose, 100 mg/1 kanamycin).

Growth of HP1/GFP Transgeneic Seedlings and Controlled Seedlings in the Presence of NaCl or CdSO,

Seedlings were derived from crosses of HPI transgeneic lines with SP1 wild- type and grown on MS medium containing 150 mM NaCl or 0.5 x MS medium containing 120 mM NaCl. Non-transgeneic controlled plants were grown on corresponding MS medium but lacking NaCl. Seedling growth was monitored periodically at 11 days post sowing, 19 days post sowing and 24 days post sowing as indicated in Figure 5.

To assess the effects of CdS0₄ on seedling growth, transgeneic and controlled seedlings were germinated on 0.5 x MS containing 50 mM CdS0₄.

At the end of each experimental treatment biomass yield was assessed as dry weight.

Results

Referring now to the Figures, and firstly to Figure 2, there is shown the nature of the fusion construct which has been used in the above described methods. Briefly, the protein to be tested, in this case either the chromo domain of heterochromatin protein 1 or polycomb was linked to a nuclear targeting domain, so as to ensure that the fusion construct was targeted to the nucleus of the plant cell, and also a green fluorescent protein to allow visualisation of the transgene product within a transformed cell.

Using confocal microscopy techniques, localisation of transformed cells was visualised using the GFP protein. Specifically it can be seen in Figure 3, that the HP1/GFP fusion protein was localised in the nucleus of the plant cell, as expected.

Transformants were self pollinated and seeds were germinated in the greenhouse and after 5 weeks transformed plants whose cells contained the fusion construct were compared with wide type plants.

It can be seen in Figure 4A that transformed cell line 855/3 grew much better than the wide type SRI .

Shown in Figure 4B is a comparison of what we have termed our strong expressing cell line 855/3 with what we have termed a weak expressing cell line 873/2-3. It can be seen that the strongly expressing cell line shows more growth than the weakly expressing cell line. Moreover, it can also be seen that both cell lines show improved vigour vis-a-vis the wide type.

In Figure 5 we show the results of various experiments to test the ability of wide type and transformed cells to grow under various stress conditions and in particular salt stress conditions. Thus, an ability to survive such stress conditions is of course important if the transformed cell of the invention is to have agricultural application.

Notably, salt stress and desertification is a major limitation for the use of soil in agriculture, indeed, each year 21 million hectares of farm land provide no economic return because of desertification.

The results shown in Figure 5 represent three sampling times the earliest 11 days after sowing, then 19 days after sowing and finally 24 days after sowing. Referring to the earliest sampling time it can be seen that in the absence of salt stress wild type cells, a transformed cell line and also a transgenic control, including just the GFP protein, grew equally well. However, when exposed to 150 mM sodium chloride for a period of 1 1 days, only our transformed cell line only, 855/3-4, showed any sign of exhibiting growth.

At a second sampling time, 19 days after sowing, it again could be seen that the wild type, the transformed cell line and the transgenic control all grew equally well. However, when exposed to 150 mM sodium chloride there was a marked difference in growth, insofar as the transformed cell line showed a significant enhancement in growth compared to either the wild type or the transgenic control.

At the third sampling time, 24 days after sowing, again in the absence of selection pressure, the wild type, the transformed cell line and the transgenic control all grew equally well. However, when exposed to 150 mM sodium chloride there was again a marked difference between the three groups and the transformed cell line was seen to grow significantly better than either of the other two groups. A quantitative analysis of the relative yield of biomass in response to elevated NaCl concentration is presented in Table 1. The transgeneic plant cell-line 855/3-4 was analysed under various growth conditions in the presence of 120 mM or 150 mM NaCl. The data presented in Table 1 is derived from two experiments under two experimental growth conditions. It is apparent that a 1.7 - 2.1 fold increase in biomass yield is achieved by transgeneic plants when compared to controlled seedlings grown under identical salt concentrations.

To assess the effects of cadmium on seedling growth the HPI transgenic plant cell-line 855/3-6 was cultured in 0.5 x MS containing 50 mM CdS0₄.

Plants were harvested after xxxx days of growth and biomass yield was assessed as dry weight. Table 2 represents two experiments showing the protective properties of HPI in the presence of CdS0₄. Those seedlings expressing the HP l/GFP fusion protein show a 1.7 - 4 fold increase in biomass yield when compared to non-expressing controlled seedlings indicating that transgene product expression has protective properties in the presence of cadmium.

This data indicates that the transformed cell line had a developmental advantage which exhibited itself early in germination and which enabled the cell line to tolerate salt stress conditions.

We attributed the improved growth and development of our cell line to the expression of the transgene, or part thereof, of heterochromatin protein 1. Our experiments in Figure 6 were designed to test this. In Figure 6 we show the growth of three different transformed cell lines all including the chromo domain of heterochromatin protein 1. In each cell line those cell expressing HPI showed, in all instances under salt stress conditions, increased growth compared to those transformed cells which did not express the protein. The lowermost data, 873/1-15 represents the control in that it represents cells transformed with only the GFP protein.

The data shown in Figure 6 clearly correlates the expression of the chromo domain of HPI with improved cell vigour.

In summary, our data clearly shows that, unexpectedly, plant cells containing at least the chromo domain of HPI show desirable growth characteristics and so the engineering of said protein into plant cells can be exploited to good effect.

References

Bimboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513.

Hain R, Stabel P, Czernilowsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts. Mol Gen Genet 199: 161-168.

Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126: 105- 1 10.

Linsmaier EM. Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100-27.

Maliga P, Sz-Breznovitis A, Marton L (1973) Streptomycin-resistant plants from callus culture of haploid tobacco. Nature 244:29-30.

Meselson M. Yuan R ( 1968) DNA restriction enzyme from E.coli. Nature 217: 1110.

Nagy JL, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana silvestris. Z Pflanzen-physiol 78:453-455.

Claims

1. A plant cell containing a transcriptionally activated/activatable form of the isolated nucleotide molecule presented in Figure 1A, or part thereof, encoding the chromo domain of heterochromatin protein 1, or a gene, or part thereof, having greater than 60% homology therewith.

2. A plant cell according to Claim 1 wherein said chromo domain of heterochromatin protein 1, is derived from the genus Drosophila.

3. A construct comprising a nucleic acid molecule containing the chromodomain of heterochromatin protein 1 translationally fused to a nuclear localisation domain to target said chromodomain to the nucleus of a plant cell.

4. An expression vector containing the construct according to Claim 3 for effecting transformation of a plant cell.

5. An expression vector according to Claim 4 adapted to contain a selectable marker to confer resistance to a drug/agent.

6. A plant including at least one plant cell according to Claim 1 or 2.

7. A plant including at least one plant cell containing the expression vector according to Claims 3-5.

8. A seed obtained from the plant according to Claim 6 or 7.

8. A seed containing a transcriptionally activated/activatable form of the isolated nucleotide molecule presented in Figure 1 A, or part thereof, encoding the chromo domain of heterochromatin protein 1, or a gene, or part thereof, having greater than 60% homology therewith.

9. A method for improving plant productivity of a selected plant comprising:

i) transforming a plant cell with an expression vector according to Claim 5;

ii) selecting those plant cells transformed with said expression vector, using a drug corresponding to said marker provided in said vector;

iii) culturing said transformed plant cells to maintain and propagate transgenic plants transformed with said expression vector: and, optionally;

iv) selfing or crossing said transgenic plants for seed production.