WO1998003668A1

WO1998003668A1 - Virus resistance in plants

Info

Publication number: WO1998003668A1
Application number: PCT/GB1997/001960
Authority: WO
Inventors: Iossif Grigorievich ATABEKOV; Youri Leonidovich Dorokhov; Sergey Yurievich Morozov
Original assignee: Zeneca Limited
Priority date: 1996-07-22
Filing date: 1997-07-21
Publication date: 1998-01-29
Also published as: AU3627997A; GB9615349D0

Abstract

This invention provides a method for improving or imparting the resistance of an organism to infection by specific viruses. The method involves the production of an RNA transcript mimicking binding sites of the virus specific RNA Dependent RNA Polymerase which is produced by the infecting virus. The said transgenic binding sites compete for the RDRP and binding of this RDRP to the transgenic sequence rather than the infecting virus will result in the inability of the infecting virus to successfully replicate. This will have the effect of severely reducing the virus infection.

Description

VIRUS RESISTANCE IN PLANTS

This invention relates to virus resistance in plants.

Over 75% of all plant viruses (and most of the agronomically important plant viruses) contain a single-stranded (+)-RNA genome. The early stages of infection are common to most of these viruses. First, the virus enters a plant cell, apparently through a wound of some type (while this can occur through mechanical wounding or abrasion, insect-mediated wounding is more common). Second, the virus begins to uncoat. For some viruses, this process occurs concomitant with the third stage - translation of (at least part of) the genomic RNA. The product of this translation is a putative RNA-dependent RNA polymerase (RDRP; sometimes referred to as the viral "replicase"). This enzyme is responsible for synthesising complementary (-)-strand RNA, then using this RNA as a template for synthesis of genomic (+)-strand RNA (which can be packaged into virions) and (for some viruses) sub-genomic (+)-strand RNA's which can encode other structural and non-structural viral proteins. This process is shown diagramatically in Fig 1. Although it appears that host (plant) factors are also involved in viral RNA replication, the RDRP initiates (-)-strand RNA synthesis by binding to a specific sequence at the 3' end of the viral genome, synthesising RNA in the 5'— >3' direction. Synthesis of genomic (+)-strand RNA is initiated at a specific sequence at the 3' end of the (-) strand (this synthesis also proceeds 5'-->3'). If a sub-genomic (ie. internal) RDRP binding site is present, it is also recognised specifically in the (-) strand, and (+)-strand RNA's smaller than genome- length are synthesised.

The present invention seeks to provide, inter alia, a method for the protection of plants by inhibiting the replication of viruses, post infection.

According to the present invention there is provided a DNA construct comprising sequentially a promoter which is operable in plants, a polynucleotide comprising a sequence encoding an RNA capable of binding to an RNA dependent RNA polymerase of a plant virus, said RNA being the promoter region of a gene which when present in the viral genome is sub-genomic, and a terminator sequence, characterised in that the said sequence is heterologous with respect to the plant operable promoter. Further according to the present invention is a DNA costruct in which the polynucleotide is selected from the group of sequences as depicted in SEQ ID Nos. 1 or 2 as provided in the sequence listing herein, or is a polynucleotide which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequences as depicted in SEQ ID Nos. lor 2. Further according to the present invention is a DNA construct as described previously, wherein the said polynucleotides is/are repeated at least once and arranged in tandem. DNA costructs according to the present invention may contain a plant operable promoter which is constitutive, developmentally regulated or inducible. In addition to this the said promoter may be tissue specific and/or inducible.

The present invention further provides a method of imparting to or improving the ability of a plant to inhibit the replication of an infecting virus comprising inserting into the genome of plant material, a construct as described herein, regenerating said plant material, and selecting from the progeny those regenerants which have an improved ability to inhibit the infecting virus replication.

Transformation according to the present invention may be achieved via the Agrobaterium, particle mediated, fibre mediated or direct insertion methods. The invention further provides a plant resulting from any of the aforesaid methods, characterised in that the said plant exhibits an increased inhibition of viral replication in said plant.

Examples of plants according to the present invention are potato, tomato, letuce and tobacco.

While not wishing to be bound by this explanation, the invention may be considered to operate by producing an abundance of the RDRP binding sequence, which upon infection by a specific virus, will actively compete for binding of the RDRP produced by the infecting virus. Binding of the RDRP's to the "transgenic sites"(which are abundant, in-situ and immediately available for binding upon production of the viral RDRP) will reduce the degree of binding to the endogenous infecting virus. This will have the effect of inhibiting the production of infecting viral genomic RNA and thereby interrupting viral replication which depends thereon. Experiments with transgenic tobacco have shown that plants expressing constitutively a chimeric RNA containing the negative (minus polarity) strand of the sub genomic promoter of potato virus X (PVX) coat protein RNA plus the negative strand of the GUS reporter gene RNA , could be induced to express the reporter gene (GUS) when inoculated with PVX. This indicates that viral RNA polymerase can recognise sub-genomic promoters (SgPr.) within chimeric RNA transcripts produced by transgenic plants.

The invention will now be described by way of illustration, in plant systems, with reference to the accompanying drawings of which:

FIGURE 1 - A simplified infection cycle for a prototype (-t-)-strand RNA virus (based on TMV). Although the virus shown here is rod-shaped, not all (+)-strand RNA viruses are rods. A typical infection cycle involves the following steps. Infectious virus ( 1 a) enters the cell (A) and begins to uncoat (B). Translation of this partially uncoated virion (2) ensues (C) and virus uncoating continues (D), resulting in synthesis of RDRP (3) and liberation of genomic RNA (4). The RDRP (in concert with host factors) synthesises (-)-strand RNA (E;5) and subsequently both (+)-strand RNA (F;6) and, in many cases, (+)-strand sub-genomic RNA(s) (7). If sub-genomic RNA's are involved, they are typically used as transcripts for proteins involved in processes such as virus spread as well as the CP (G;8). The CP (8) and newly synthesised (+)-strand genomic RNA (6) can reassemble to form infectious virions (lb). Virus movement throughout the plant may be potentiated by virion-like particles (perhaps involving other proteins such as the movement protein or MP) which could also be assembled at this stage (not shown).

FIGURE 2 - A summary of the principle of virus protection through competition for RDRP binding by means of example involving; A transgenic plant produced containing a chimeric gene (A) that in turn produces a transcript containing (at least 1) "Virus A" RDRP binding sequence (B). This RNA serves no function in the cell unless "Virus A" enters the cell and initiates infection (C). Upon initiation of infection, "Virus A" will produce its RDRP (D). The transgenic RDRP binding sequence transcript will compete for RDRP binding. The result of successful binding to the "transgenic RDRP binding sites", illustrated at (E), will reduce the amount of RDRP available for binding to the endogenous sites thereby interrupting infecting viral replication. FIGURE 3 - Diagram of "X2-TM53", or Construct 1 Construct lb is the plasmid pRTlOl containing "X2-TM53", capable of producing RNA's which are more stable when electroporated into Barley protoplasts

FIGURE 4 - Diagram of "T2-X53", or Construct 2 Construct 2b is the plasmid pRT 101 containing "T2-X53", capable of producing RNA's which are more stable when electroporated into Barley protoplasts

FIGURE 5 - Diagram of Construct 3. This is a negative control and comprises TMV 5' and 3' regions, i.e it contains no sub-genomic promoter binding sequences.

FIGURES 6, 7 & 8 - Diagrams similar to Construct 1 except they contain zero, four (arranged in tandem) and eight (arranged in tandem) SgPr RDRP binding sequences respectively.

FIGURE 9 - Diagram illustrating a multiple resistance model. This construct contains two copies (arranged in tandem) of PVX SgPr.RDRP binding sequence and TMV SgPr.RDRP binding sequence flanked by a promoter and terminator to enable their transcription.

FIGURE 10 - Schematic representation of an inoculated tobacco leaf. The diagram illustrates the leaf regions used for the ELIS A analysis and the distance between the regions.

The constructs illustrated in these examples contain viral genomic promoter binding sequences (eg. Fig. 3. TMV 5' and 3' in the case of construct 1) flanking either side of the sub-genomic promoter (SgPr.) binding sequence Such 5' and 3' sequences are not required for the SgPr binding sequences to be effective as competitors for infecting viral RDRP's In this specific case the 5' and 3' sequences were used in construct preparation as a model for the protection of the transcript from degradation and as a means by which the viral RDRP specificity between genomic and sub-genomic promoters could be tested. Early experiments provided results indicating that viral genomic promoters are not competitive inhibitors for viral RDRP. This inability of viral genomic promoters to inhibit virus replication has been reported elsewhere. For example Dawson 1991, Virology 184, 277-289 and White et al 1992. J. Virol. 66. 3069-3076 This invention, however, relates to the use of Sub-genomic promoters which have demonstrated resistance to virus infection.

In general terms the invention requires the production of transgenic plants in which copies of a RDRP binding site accumulate in the plant. Constitutive accumulation may be sufficient but developmentally regulated and/or tissue-specific accumulation of RDRP binding sites may be preferred. For example, tobacco mosaic virus (TMV) is known to initiate plant infection in epidermal cells, and therefore it may be preferable to block infection specifically in those cells.

For a phloem-limited virus, protection may be produced via accumulation of RDRP binding sites within phloem cells.

Also production of a construct containing inducible promoters would allow transcription to be initiated under specific circumstances.

Another means by which the copy number of the transcript can be produced involves transformation of a plant with multiple copies of the sub-genomic promoter RDRP binding sequence. When the chimeric gene is transcribed, an increased number of binding sites may be produced.

The mode of action of resistance via the RDRP binding competition model can be summarised as follows:

Following inoculation of the transgenic plant with the virus from which the transgenic RDRP was derived, die virus begins to uncoat and RDRP is produced from the genomic RNA, see

Fig.1. parts 1-3. RDRP binding sequences already present in the cytoplasm of these cells (due to transcription of the nuclear transgene) compete with the virus genome for RDRP binding and if sufficient transgenic SgPr. RDRP binding sequences are present, infection should be reduced at this point, see Fig. 2. This would also be true for a multiple resistance model. Transgenic plants producing SgPr.

RDRP binding sites from heterologous viruses would exhibit resistance to either a single virus, or a combination of viruses from which the transgenic SgPr. binding sites were derived.

A collection or selection of the above features will have the effect of providing high levels of resistance via the inhibition of the replication of an infecting virus. The invention will now be further described in the following Examples.

EXAMPLE 1 These experiments used a protoplast transient assay system, as a preliminary model for transgenic plant work. The experiments involved the co-introduction of RNA's containing RDRP binding sequences (synthesised in vitro) and infectious virus RNA. It has previously been shown that, for at least one other virus protection system, inhibition of virus replication can be obtained following co-introduction of the "protecting molecule" with the infectious particle.

Some of the constructs produced for experimental work are as follows:

Construct 1 - For the transient assay experiments, all RNA's are synthesised in vitro using the phage T7 transcription system. The nucleic acids discussed here are the RNA's made using this system and introduced into protoplasts along with infectious RNA's. The RNA from Construct 1 consists of (from 5'— >3'): (i) the 5' end of the TMV genome (containing a sequence complementary to the TMV RDRP binding site), (ii) a tandem repeat of the complement of the PVX sequence (i.e. minus sense) found immediately upstream of the capsid protein (CP) coding region in the virus RNA (each repeat contains a RDRP binding site which is used for synthesis of a sub-genomic RNA that produces PVX CP during infection), and (iii) the 3' end of the TMV genome (containing the TMV RDRP binding sequence). The RNA transcript produced from Construct 1 contains two copies of the minus(-) sense sub-genomic RDRP binding sites. The ability of this transcript to protect against virus infection is then tested by co- introducing TMV RNA or PVX RNA with transcript from Construct 1 into Barley protoplasts and monitoring TMV or PVX infection using an ELISA to the relevant CP. The results are shown in Table 1.

Construct 2 - The RNA from Construct 2 consists of (from 5'->3'): (i) the 5' end of the PVX genome (containing a sequence complementary to the PVX RDRP binding site), (ii) a tandem repeat of the complement of the TMV sequence found immediately upstream of the TMV capsid protein (CP) coding region in the virus RNA (each repeat contains a RDRP binding site which is used for synthesis of a sub-genomic RNA that produces TMV CP during infection), and (iii) the 3' end of the PVX genome (containing the PVX RDRP binding sequence). The RNA transcript produced from Construct 2 contains two copies of the minus(-) sense sub- genomic RDRP binding sites and the ability of this construct to protect against virus infection is tested by co-introducing PVX RNA or TMV RNA with transcript from Construct 2 into tobacco protoplasts and monitoring PVX or TMV infection using an ELISA to the relevant CP. The results are shown in Table 2.

Early work with constructs 1 and 2 was unsuccessful, probably due to the instability of the chimeric RNA's when elecroporated into the protoplasts because the RNA's were lacking a "cap structure". In order to circumvent this problem the constructs 1 and 2 were cloned into the plasmid pRTlOl under the influence of the 35S promoter and a poly a signal (see Fig. 3b and 4b).

Construct 3 - As for construct 1, except without any SgPr. RDRP binding sequences. This construct was produced as a negative control and to identify whether under these circumstances transgenic binding sites using viral genomic promoters will compete for viral RDRP. The construct also aimed to determine whether the binding sites are virus specific.

Construct 4 - As for construct I, except contains 1 copy of the SgPr. RDRP binding sequence from PVX.

Construct 5 - As for construct 1 , except contains 4 copies of the SgPr. RDRP binding sequence from PVX.

Construct 6 - As for construct 1, except contains 8 copies of the SgPr. RDRP binding sequence from PVX

Multiple Resistance Model - This construct comprises from 5' to 3', a 5' region containing a promoter capable of driving the transcription of the coding sequence. Tandem repeats of PVX sub-genomic promoter RDRP binding sites in positive sense (+). Tandem repeats of TMV SgPr. RDRP binding sites in positive sense (+). A 3' region containing a terminator capable of terminating the transcription of the coding sequence. The initial experiments used tobacco mosaic virus (TMV) and potato virus X (PVX), two viruses which are capable of infecting protoplasts. Electroporation was used to introduce the

RNA's into the protoplasts. Barley protoplasts were transformed with chimeπc RNA transcπpts produced in vitro from construct 1 by electroporation and infected with TMV and PVX. See Table 1.

TABLE 1

The influence of the capped transcript from Construct lb (which contains a duplicate copy of the PVX SgPr. and TMV genomic promoters 5' and 3') on PVX and TMV accumulation in electroporated barley protoplasts.

Infecting Virus Relative amounts (μg) of viral Virus concentration (μg) RNA/capped transcript per 2 x 10^s protoplasts^* (electroporated plasmid) (mean ELISA figure for at least three samples)

EXPERIMENT 1

PVX 3 2 (control, viπon DNA only) 2.5

PVX 3 2/12 1.25

TMV 1 0 (control) 100

TMV 1/12 100

EXPERIMENT 2

PVX 1 6 (control) 0 8

PVX 1 6/22 5 0 1

PVX 1 6/20(control, GUS transcπpt) 0.8

PVX 4 0 (control) 1 2

PVX 4.0/15 0 0 8

TMV 1 0 (control) 12.8

TMV 1 0/22.5 12.8

* average of two samples

The results in Table 1 indicated that the PVX sub-genomic promoter binding sequence in the transgene inhibits replication of the PVX but not TMV

In contrast, the TMV genomic promoters (3' and 5') do not inhibit TMV accumulation. Reciprocally, when chimeπc RNA transcπpts from Construct 2b are electroporated into barley protoplasts, infecting TMV is inhibited while PVX is not. Table 2.

TABLE 2 The influence of the capped transcript from Construct 2b (which contains a duplicate copy of the TMV SgPr. and PVX genomic promoters 5' and 3') on the accumulation of PVX and TMV in electroporated barley protoplasts.

Infecting Virus Relative amounts (μg) of viral Virus concentration (μg) RNA/capped transcript per 2 x IO⁵ protoplasts^* (electroporated plasmid)

EXPERIMENT 1

TMV 3.2 (control, virion DNA only) 10.0

TMV 1/10 0.3

PVX 3.2 (control) 2.5

PVX 3.3/10 2.5

EXPERIMENT 2

TMV 1.0 (control) 51.2

TMV 1.0/30.0 6.4

TMV 1.0/20.0 (control, GUS transcript) 51.2

PVX 1.6 (control) 0.8

PVX 1.6/30.0 0.8

PVX 1.6/20.0 (control) 0.8

* average of two samples

The work with Constructs lb and 2b has provided the following conclusions. First, that repUcation of a virus is not inhibited in the presence of RNA transcripts containing solely a viral genomic promoter RDRP binding site from that virus. Second that virus replication is inhibited in the presence of a viral sub-genomic promoter RDRP binding site from that virus, indicating that RDRP's produced by a virus will only bind to sites specific to that virus.

EXAMPLE 2

Constructs containing zero, one, four and eight PVX SgPr. were produced (see Fig. 5,6,7 and 8 respectively), to compare the efficiency of PVX virus inhibition by increasing the RDRP binding sites in the transgenic transcript. The results of constructs containing Zero (negative control), one and four copies (where in the case of two and four copies the sequences are arranged in tandem) are shown in Table 3. Again the RNA's were produced in vitro using the T7 system, and electroporated along with the infecting PVX.

TABLE 3

Inhibition of PVX accumulation in barley protoplasts by chimeric RNA transcripts carrying 1, 2 and 4 SgPr.. RDRP binding sites.

Number of PVX PVX accumulation (DAS ELISA) in 2xl0⁵ protoplasts SgPr.. in transcript*

Exp.l Exp.2 Exp.3 Average

PVX RNA (control) 200** 400 200 266.6 zero 200 400 200 266.6

1 100 200 100 133.3 2 50 200 100 116.3

4 50 100 100 83.3

* 30 μg of each transcript and 1.8 μg of PVX RNA were added per sample of 2x 10⁵ protoplasts

** Each value is a mean of at least three individual samples.

These results clearly show that by increasing the number of SgPr. RDRP binding sites the effectively decreases the infecting virus accumulation.

Western analysis has also indicated progressive inhibition of PVX accumulation with increasing PVX SgPr. RDRP binding sequence copy number.

EXAMPLE 3

The purpose of this experiment was to test the efficiency of electroporating the construct lb as in Fig 3. (containing two arranged PVX SgPr. binding sequences) into barley protoplasts, rather than electroporating the RNA's produced in vitro using the T7 system The results are summarised in Table 4.

- 11

*protoplasts hac low viability

These results indicate that the plasmid containing the PVX SgPr. exhibits a reduced virus concentration when protoplasts were electroporated with plasmid concentrations of l.Oμg. Higher Concentrations of plasmid produced a complete reduction of virus concentration in relation to the control construct containing the GUS reporter gene instead of viral sequence.

EXAMPLE 4.

Tobacco plants (Nicotiana sp.) were transformed with construct lb (pRTlOl "X2-TM53") containing two copies of PVX SgPr. binding sites, arranged in tandem and construct 3 (pRTlOl

'TM53") containing zero copies of SgPr., in effect a negative control.

Transformed tobacco plants selected as containing the above constructs were tested for PVX infection in the following manner; see Fig. 10.

The apical 1/3 sections of 3-4 leaves of the same plant were inoculated with PVX

(concentration of inoculum was 40μg/ml). The virus accumulation was tested by ELISA in the inoculated apical (see Fig. 10 region a) and also the non-inoculated parts of the same leaves

(region b, c and d) and the non-inoculated upper leaves. One line transformed with the construct from Fig. 5 (zero copies of PVX SgPr.) and two lines transformed with construct lb (pRTlOl "X2-TM53") containing two copies of PVX SgPr. binding sites arranged in tandem, were selected for analysis. The virus concentration was determined ten and twenty days after infection, Tables 5 and 6 show PVX accumulation in different parts of inoculated and upper non-inoculated (systemically infected) leaves of control non-transgenic Samsun tobacco ten and twenty days after inoculation, respectively.

These experiments illustrate that transgenic plants containing two copies of PVX SgPr. RDRP binding sites show a level of resistance to infecting PVX. The virus spread within the inoculated leaves and especially the systemic spread into the upper non-inoculated leaves was inhibited ten days and to some extent twenty days after inoculation, which can be seen in tables 5 and 6.

TABLE 5.

Accumulation of PVX (ELISA OD 490nm) in inoculated leaf regions a, b, c and d, and upper non-inoculated leaves of transgenic and control plants (non transgenic Samsun tobacco): 10 days after inoculation with PVX.

Plant Line Inoculated leaf regions Upper non inoculated leaf a b c d non 1.309+/-0.05 0.805+/-0.32 0.263+/-0.12 0.052+/-0.02 1.71747-0.49 transgenic

Pl-0 1.244+/-0.06 0.081+/-0.28 0.757+/-0.25 0.15647-0.12 1.842+/-0.47

P1-2 (I) 1.365+/-0.12 1.105+/-0.18 0.50747-0.25 0.09647-0.03 0.262+/-0.16

P 1-2 (II) 1.266+/-0.10 1.01347-0.27 0.56047-0.26 0.074+/-0.05 0.064+/-0.02

The average data from 21 tested leaves were taken.

TABLE 6.

Accumulation of PVX (ELISA OD 490nm) in inoculated leaf regions a, b, c and d, and upper non-inoculated leaves of transgenic and control plants (non transgenic Samsun tobacco): 20 days after inoculation with PVX. Plant Line Inoculated leaf regions Upper non inoculated leaf a b c d non 1.1294-/-0.09 1.005+/-0.05 0.8914-/-0.12 0.787+/-0.29 1.100+/-0.07 transgenic

Pl-0 1.00347-0.06 0.901+/-0.09 0.8654-/-0.09 0.711+/-0.14 1.074+/-0.09

P1-2 (I) 1.0964-/-0.06 1.021+/-0.45 0.9404-/-0.05 0.594+/-0.21 0.724+/-0.19

Pl-2 (II) 1.042+/-0.06 0.9864-/-0.03 0.9004-/-0.12 0.285+/-0.19 0.59547-0.40

The average data from 21 tested leaves were taken, pi -2 = Plants transformed with construct 3 see fig.5. pi -2(1) = Plant line 1, transformed with construct lb see fig.3. p 1-2(11) = Plant line 2, transformed with construct lb see fιg.3.

The plant lines 1 and 2 were also tested with TMV to further demonstrate that binding of infecting virus RDRP's to SgPr. RDRP binding sites is a process that is virus specific. Clarification of which is illustrated in tables 7 and 8.

TABLE 7.

Accumulation of TMV (ELISA OD 490nm) in inoculated leaf regions a, b, c and d, and upper non-inoculated leaves of transgenic Pl-0 and Pl-2 (lines 1 and 2): 10 days after inoculation with TMV.

Plant Line Inoculated leaf regions Upper non inoculated leaf a b c d

Pl-0 0.54047-0.09 0.471+/-0.05 0.308+/-0.09 0.01547-.005 0.391 47-0.16

Pl-2 (I) 0.6994-/-0.07 0.51047-0.19 0.35647-0.15 0.139+/-.004 0.438+/-0.02

Pl-2 (II) 0.67647-0.12 0.561+/-0.25 0.46247-0.28 0.061+/-.031 0.716+/-0.15

The average data from 15 tested leaves were taken. TABLE 8.

Accumulation of TMV (ELISA OD 490nm) in inoculated leaf regions a, b, c and d, and upper non-inoculated leaves of transgenic Pl-0 and Pl-2 (lines 1 and 2): 22 days after inoculation with TMV.

Plant Line Inoculated leaf regions Upper non inoculated leaf a b c d

Pl-0 0.5644-/-0.12 0.56947-0.13 0. 16+/-0.11 0.643+/-0.09 0.634+/-0.07

Pl-2 (I) 0.6624-/-0.06 0.7724-/-0.05 0.71047-0.13 0.765+/-0.17 0.651+/-0.09

P 1-2 (11) 0.7374-/-0.09 0.776+/-0.08 0.75747-0.08 0.7454-/-0.09 0.619+/-0.10

The average data from 15 tested leaves were taken.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: ZENECA LIMITED

(B) STREET: 15 STANHOPE GATE

(C) CITY: LONDON (D) STATE:

(E) COUNTRY: UNITED KINGDOM

(F) POSTAL CODE (ZIP) : W1Y 6LN

(ii) TITLE OF INVENTION: VIRUS RESISTANCE IN PLANTS (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(v) CURRENT APPLICATION DATA: APPLICATION NUMBER:

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 632 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"

(vii) IMMEDIATE SOURCE: (B) CLONE: TMV COAT PROTEIN GENE SUB-GENOMIC PROMOTER

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

AAGCTTATTG ATAGTGGATA CGTCTGTTTA GCCGGTTTGG TCGTCACGGG CGAGTGGAAC 60

TTGCCTGACA ATTGCAGAGG AGGTGTGAGC GTGTGTCTGG TGGACAAAAG GATGGAAAGA 120 GCCGACGAGG CCACTCTCGG ATCTTACTAC ACAGCAGCTG CAAAGAAAAG ATTTCAGTTC 180

AAGGTCGTTC CCAATTATGC TATAACCACC CAGGACGCGA TGAAAAACGT CTGGCAAGTT 240

TTAGTTAATA TTAGAAATGT GAAGATGTCA GCGGGTTTCT GTCCGCTTTC TCTGGAGTTT 300

GTGTCGGTGT GTATTGTTTA TAGAAATAAT ATAAAATTAG GTTTGAGAGA GAAGATTACA 360

AACGTGAGAG ACGGAGGGCC CATGGAACTT ACAGAAGAAG TCGTTGATGA GTTCATGGAA 420 GATGTCCCTA TGTCGATCAG GCTTGCAAAG TTTCGATCTC GAACCGGAAA AAAGAGTGAT 480

GTCCGCAAAG GGAAAAATAG TAGTAATGAT CGGTCAGTGC CGAACAAGAA CTATAGAAAT 540

GTTAAGGATT TTGGAGGAAT GAGTTTTAAA AAGAATAATT TAATCGATGA TGATTCGGAG 600

GCTACTGTCG CCGAATCGGA TTCGTTTTAA AT 632

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 466 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"

(vii) IMMEDIATE SOURCE: (B) CLONE: PVX COAT PROTEIN SUB-GENOMIC PROMOTER

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TCTAGAGGTA GTTTACCCCA CGTAGGTGAT AACATTCACA GTTTACCACA CGGAGGAGCT 60

TACAGAGACG GCACCAAAGC AATCTTGTAC AACTCACCAA ATCTAGGGTC ACGAGTGAGT 120 CTACACAACG GAAAGAACGC AGCATTTGCT GCCGTTTTAC TACTGACTTT ACTGATCTAT 180

GGAAGCAAAT ACATATCTCA ACGCAATCAT ACTTGTGCTT GTGGTAACAA TCATAGCAGT 240

CATTAGTACT TCCTTAGTGA GGACTGAACC TTGTGTCATC AAGATTACTG GAGAATCAAT 300

CACAGTGTTG GCTTGCAAAT TAGATGCAGA AACCATCAGA GCCATTGCCG ATCTCAAGCC 360

ACTCTCCGTT GAACGGTTAA GTTCCATTGA TACTCGAAAG ATGCAGCACC AGCTAGCACA 420 ACACAGGCCA CAGGGTCAAC TACCTCAACT ACCACAAAAA CTGCAG 466

Claims

A DNA construct comprising sequentially a promoter which is operable in plants, a polynucleotide comprising a sequence encoding an RNA capable of binding to an RNA dependent RNA polymerase of a plant virus, said RNA being the promoter region of a gene which when present in the viral genome is sub-genomic, and a terminator sequence, characterised in that the said sequence is heterologous with respect to the plant operable promoter.

2. A DNA costruct according to claim 1 in which the polynucleotide is selected from the group of sequences as depicted in SEQ ID Nos. 1 or 2, or is a polynucleotide which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequences as depicted in SEQ ID Nos. lor 2.

3. A DNA construct according to claim 2 wherein the said polynucleotides is/are repeated at least once and arranged in tandem.

4. A DNA costruct according to any one of claims 1-3, wherein the plant operable promoter is constitutive, developmentally regulated or inducible.

5. A DNA costruct according to claim 4, wherein the promoter is tissue specific and/or inducible.

6. A method of imparting to or improving the ability of a plant to inhibit the replication of an infecting virus comprising inserting into the genome of plant material, a construct according to any of claims 1 -5 , regenerating said plant material, and selecting from the progeny those regenerants which have an improved ability to inhibit the infecting virus replication.

7. A method according to claim 6 wherein the transformation is achieved via the Agrobaterium, particle mediated, fibre mediated or direct insertion methods.

8. A plant resulting from the method according to claims 6 or 7, characterised in that the said plant exhibits an increased inhibition of viral replication in said plant.

9. A plant according to claim 8, selected from potato, tomato, letuce and tobacco.

10. A polynucleotide and/or method substantially as herein described.