AU721332B2 - Plant retinoblastoma-associated proteins - Google Patents

Description

WO 97/47745 PCT/EP97/03070 1 PLANT RETINOBLASTOMA-ASSOCIATED PROTEINS

DESCRIPTION

The present invention relates the proteins having biological activity in plant and animal systems, to polynucleotides encoding for the expression of such proteins, to oligonucleotides for use in identifying and synthesizing these proteins and polynucleotides, to vectors and cells containing the polynucleotides in recombinant form and to plants and animals comprising these, and to the use of the proteins and polynucleotides and fragments thereof in the control of plant growth and plant vulnerability to viruses.

Cell cycle progression is regulated by positive and negative effectors. Among the latter, the product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. In mammalian cells, Rb regulates G1/S transit by inhibiting the function of the E2F family of transcription factors, known to interact with sequences in the promoter region of genes required for cellular DNA replication (see eg Weinberg, R.A. Cell 81,323 (1995); Nevins, J.R. Science 258,424 (1992)). DNA tumor viruses that infect animal cells express oncoproteins that interact with the Rb protein via a LXCXE motif, disrupting Rb-E2F complexes and driving cells into S-phase (Weinberg ibid; Ludlow, J.

W. FASEB J. 7, 866 (1993); Moran, E. FASEB J. 7, 880 (1993); Vousden, K. FASEB J. 7, 872 (1993)).

The present inventors have shown that efficient replication of a plant geminivirus requires the integrity of an LXCXE amino acid motif in the viral RepA protein and that RepA can interact with members of the human Rb family in yeast (Xie, Suarez-L6pez, P. and Guti6rrez, C. EMBO J. 14, 4073 (1995) The presence of the LXCXE motif in plant D-type cyclins has also been reported (Soni, Carmichael, J. Shah, Z. H. and Murray, J.

CONFIRMATION COPY A. H. Plant Cell 7, 85-103 (1995)).

The present inventors have now identified characteristic sequences of plant Rb proteins and corresponding encoding polynucleotides for the first time, isolated such a protein and polynucleotide, and particularly have identified sequences that distinguish it from known animal Rb protein sequences. The inventors have determined that a known DNA sequence from the maize encoding a vegetable Rb plant protein and is hereinafter called ZmRbl. ZmRbl has been demonstrated by the inventors to interact in yeasts with RepA, a plant geminivirus protein containing LXCXE motif essential for its function. The inventors have further determined that geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRbl or human p130, a member of the human Rb family.

Significantly the inventors work suggests that plant and animal cells may share fundamentally similar strategies for growth control, and thus human 15 as well as plant Rb protein such as ZmRbl will be expected to have utility in, inter alia, plant therapeutics, diagnostics, growth control or investigations and many such plant proteins will have similar utility in animals.

e: In a first aspect of the present invention there is provided a method of controlling viral infection in a plant cell characterised in that it inhibits passage 20 of the cell from the G1 phase to the S phase of the cell cycle by increasing the level and/or activity of retinoblastoma protein in that plant cell by incorporation of a recombinant nucleic acid into the cell which encodes for a protein of amino acid sequence having 80% or more homology with that of SEQ ID No. 2 and which is capable of binding to plant E2F or (ii) a peptide fragment of protein which is capable of interacting with geminivirus Rep A LXCXE motif.

A preferred method of control using such proteins involves applying these to the plant cell, either directly or by introduction of DNA or RNA encoding for their expression into the plant cell which it is desired to treat. By over expressing the retinoblastoma protein, or expressing an Rb protein or peptide fragment thereof that interacts with the LXCXE motif of the virus but K. oes not affect the normal functioning of the cell, it is possible to inhibit normal virus growth and thus also to produce infection spreading from that cell to its neighbours.

Alternatively, by means of introducing anti-sense DNA or RNA in plant cells in vectors form that contain the necessary promoters for the DNA or RNA transcription, it will be possible to exploit the well known anti-sense mechanism in order to inhibit the expression of the Rb protein, and thus the Sphase. Such plants will be of use, among other aspects to replicate DNA or RNA until high levels, e.g. in yeasts. The methods to introduce anti-sense DNA in cells are very well known for those skilled in the art: see for example "Principles of gene manipulation An introduction to Genetic Engineering (1994) R.W. Old S.B. Primrose; Oxford-Blackwell Scientific Publications Fifth Edition p398.

In a second aspect of the present invention there is provided recombinant nucleic acid, particularly in the form of DNA or cRNA (mRNA), a recombinant nucleic acid includinga promoter capable of functioning in plant cells and a sequence encoding for expression of a retinoblastoma protein S9 which has an amino acid sequence having 80% or more homology with that of SEQ N02. This nucleic acid may be characterised by one or more 9 characteristic regions that differ from known animal Rb protein nucleic acid 20 and is exemplified herein by SEQ ID No. 1, bases 31-2079.

lO99 The DNA or RNA can have a sequence that contains the degenerated substitution in the nucleotides of the codons in SEQ ID No. 1, and in where the RNA the T is U. The most preferred DNA or RNA are capable of hybridate with the polynucleotide of the SEQ ID No. 1 in conditions of low stringency, preferably being the hybridization WO 97/47745 PCT/EP97/03070 4 produced in conditions of high stringency.

The expressions "conditions of low stringency" and "conditions of high stringency" are understood by those skilled, but are conveniently exemplified in US 5202257, Col-9-Col 10. If some modifications were made to lead to the expression of a protein with different amino acids, preferably of the same kind of the corresponding amino acids to the SEQ ID No 1; that is, are conservative substitutions. Such substitutions are known by those skilled, for example, see US 5380712, and it is only contemplated when the protein has activity with retinoblastoma protein.

Preferred DNA or cRNA encodes for a plant Rb protein having A and B pocket sub-domains having between 30% and 75% homology with human Rb protein, particularly as compared with p130, more preferably from 50% to 64% homology. Particularly the plant Rb protein so encoded has the C706 amino acid of human Rb conserved. Preferably the spacer sequence between the A and B pockets is not conserved with respect to animal Rb proteins, preferably being less than 50% homologous to the same region as found in such animal proteins. Most preferably the protein so encoded has 80% or more homology with that of SEQ NO 2 of the sequence listing attached hereto, still more preferably 90% or more and most preferably 95% or more. Particularly provided is recombinant DNA of SEQ ID No 1 bases 31 to 2079, or the entire SEQ ID No 1, or corresponding RNAs, encoding for maize cDNA clone encoding ZmRbl of SQ ID No 2.

In a third aspect of the present invention there is provided the protein expressed by the recombinant DNA or RNA of the second aspect, novel proteins derived from such DNA or RNA, and protein derived from naturally occurring DNA or RNA by mutagenic means such as use of mutagenic PCR primers.

WO 97/47745 PCT/EP97/03070 In a fourth aspect there are provided vectors, cells and plants and animals comprising the recombinant DNA or RNA of correct sense or anti-sense, of the invention.

In a particularly preferred use of the first aspect there is provided a method of controlling cell or viral growth comprising administering the DNA, RNA or protein of the second or third aspects to the cell. Such administration may be direct in the case of proteins or may involve indirect means, such as electroporation of plant seed cells with DNA or by transformation of cells with expression vectors capable of expressing or over expressing the proteins of the invention or fragments thereof that are capable of inhibiting cell or viral growth.

Alternatively, the method uses an expression vector capable of producing anti-sense RNA of the cDNA of the invention.

Another one of the specific characteristics of the plants protein and of the nucleic acids includes a Nterminal domain corresponding in sequence to the amino acids 1 to 90 of the SEQ ID No. 2 and a nucleotides sequence corresponding to the basis 31 to 300 of the SEQ ID No. 1. These sequences are characterized by possessing less than 150 and less than 450 units that the animal sequences which possess more than 300 amino acids and 900 pairs of more bases.

The present invention will now be illustrated further by reference to the following non-limiting Examples.

Further embodiments falling within the scope of the claims attached hereto will occur to those skilled in the light of these.

Figures.

Fig. 1. The sub-figure A shows the relative lengths of the present ZmRbl protein and the human retinoblastoma proteins. The sub-figure B shows the alignment of the WO 97/47745 PCT/EP97/03070 amino acids sequences of the Pocket A and Pocket B of the ZmRbl with that of the Xenopus, chicken, rat and three human protein (Rb, p107 and p130).

Fig. 2. This figure is a map of the main characteristics of the WDV virus and the pWori vector derived from WDV and the positions of the deletions and mutations used in order to establish that the LXCXE motif is required for its replication in plants cells.

EXAMPLE 1.

Isolation of DNA and protein expressing clones.

Total RNA was isolated from maize root and mature leaves by grinding the material previously frozen in liquid nitrogen essentially as described in Soni et al (1995). The major and minor p75ZmRbl mRNAs were identified by hybridization to a random-primed 32Plabelled PstI internal fragment (1.4 kb).

A portion of a maize cDNA library (106 pfu) in 1ZAPII (Stratagene) was screened by subsequent hybridization to oligonucleotides designed to be complementary to a known EST sequence of homologue maize of p130. These oligonucleotides were 5'-AATAGACACATCGATCAA/G (M.5m, nt positions 1411-1438) and 5'-GTAATGATACCAACATGG (M.3c, nt positions 1606-1590)(Isogen Biosciences).

After the second round of screening, pBluescript SK- (pBS) phagemids from positive clones were isolated by in vivo excision with ExAssist helper phage (Stratagene) according to protocols recommended by the manufacturer.

DNA sequencing was carried out using a SequenaseTM Kit

(USB).

The 5'-end of the mRNAs encoding p75ZmRbl was determined by RACE-PCR. Poly-A+mRNA was purified by chromatography on oligo-dT-cellulose (Amersham). The first strand was synthesized using oligonucleotide nt positions 113-96). After denaturation at 90 0 C for 3 min, RNA was eliminated by WO 97/47745 PCT/EP97/03070 7 RNase treatment, the cDNA recovered and 5'-tailed with terminal transferase and dATP. Then a PCR fragment was amplified using primer DraI35 and the linker-primer bp) of the Stratagene cDNA synthesis kit.

One of the positive clones so produced contained a -4 kb insert that, according to restriction analysis, extended both 5' and 3' of the region contained in the Expressed Sequence Tag used. The nucleotide sequence corresponding to the longest cDNA insert (3747 bp) is shown in SEQ ID No. 1. This ZmRbl cDNA contains a single open reading frame capable of encoding a protein of 683 amino acids (predicted Mr 75247, p75ZmRbl) followed by a 1646 bp 3'-untranslated region. Untranslated regions of similar length have been also found in mammalian Rb cDNAs (Lee, et al, Science 235, 1394 (1987); Bernards, R.

et al, Proc. Natl. Acad. Sci. USA 86, 6474 (1989)).

Northern analysis indicates that maize cells derived from both root meristems and mature leaves contain a major message, ~2.7+0.2 kb in length. In addition, a minor ~3.7+0.2 kb message also appears. Heterogeneous transcripts have been detected in other species (Destrie, O. H. J. et al, Dev. Biol. 153, 141 (1992)).

Plasmid pWoriAA was constructed by deleting in pWori most of the sequences encoding WDV proteins (Sanz and Gutierrez, unpublished) Plasmid p35S.Rbl was constructed by inserting the CaMV 35S promoter (obtained from pWDV3:35SGUS) upstream of the ZmRbl cDNA in the pBS vector. Plasmid p35S.130 was constructed by introducing the complete coding sequence of human p130 instead of ZmRbl sequences into p35S.Rbl. Plasmid p35.A+B was constructed by substituting sequences encoding the WDV RepA and RepB ORFs instead of ZmRbl in p35S.Rbl plasmid.

(See Soni, R. and Murray, J. A. H. Anal. Biochem. 218, 474-476 (1994)).

The sequence around the methionine codon at nucleotide WO 97/47745 PCT/EP97/03070 8 position 31 contains a consensus translation start (Kozak, M. J. Mol. Biol. 196, 947 (1987)). To determine whether the cDNA contained the full-length ZmRbl coding region, the 5'-end of the mRNAs was amplified by RACE-PCR using an oligonucleotide derived from a region close to the putative initiator AUG, which would produce a fragment of -150 bp. The results are consistent with the ZmRbl cDNA clone containing the complete coding region.

The ZmRbl protein contains segments homologous to the A and B subdomains of the "pocket" that is present in all members of the Rb family. These subdomains are separated by a non-conserved spacer. ZmRbl also contains nonconserved N-terminal and C-terminal domains. Overall, ZmRbl shares -28-30% amino acid identity similarity) with the Rb family members (Hannon, G. J., Demetrick, D. Beach, D. Genes Dev. 7, 2378 (1993); Cobrinik, Whyte, Peeper, Jacks, T. Weinberg, R. A. ibid., p. 2392 (1993). Ewen, M. Xing, Y. Lawrence, J. B. and Livingston, D. M. Cell 66, 1155 (1991)) (Lee W. L. et al, Science 235, 1394 (1987); Bernards et al, Proc. Natl. Acad. Sci. USA 86, 6974 (1989)), with the A and B subdomains exhibiting the highest homology Interestingly, amino acid C706 in human Rb, critical for its function (Kaye, F. J., Kratzke R. Gerster, J. L. and Horowitz, J. M. Proc.

Natl. Acad. Sci. USA 87, 6922 (1990)), is also conserved in maize Note: The 561-577 amino acids encompass a proline-rich domain.

ZmRbl contains 16 consensus sites, SP or TP for phosphorilation by cyclins dependant kinases (CDKs) with one of the 5'-tail of the sub-domain A and several in the C-terminal area which are potential sites of phosphorilation. A nucleic acid preferred group which encodes proteins in which one or more of these sites are WO 97/47745 PCT/EP97/03070 9 changed or deleted, making the protein more resistant to the phosphorilation and thus, to its functionality, for example linking to E2F or similar. This can be easily carried out by means of mutagenesis conducted by means of

PCR.

EXAMPLE 2 In vivo activity.

Replication of wheat dwarf geminivirus (WDV) is dependent upon an intact LXCXE motif of the viral RepA protein. This motif can mediate interaction with a member of the human Rb family, p130, in yeasts. Therefore, the inventors investigated whether p75ZmRbl could complex with WDV RepA by using the yeast two-hybrid system (Fields, S. and Song, O. Nature 340, 245-246 (1989)).

Yeast cells were co-transformed with a plasmid encoding the fusion GAL4BD-RepA protein and with plasmids encoding different GAL4AD fusion protein. The GAL4AD-p75ZmRbl fusion could also complex with GAL4BD-RepA to allow growth of the recipient yeast cells in the absence of histidine. This interaction was slightly stronger than that seen with the human p130 protein. RepA could also bind to some extent to a N-terminally truncated form of The role of the LXCXE motif in interaction was assessed using a point mutation in WDV RepA (E198K) which we previously showed to destroy interaction with human p130. Co-transformation of ZmRbl with a plasmid encoding the fusion GAL4BD-RepA(E198K) indicated that the interaction between RepA and occurred through the LXCXE motif.

In this respect, the E198K mutant of WDV RepA behaves similarly to analogous point mutants of animal virus oncoproteins (Moran, Zerler, Harrison, T. M. and Mathews, M.B. Mol. Cell Biol. 6, 3470 (1986); Cherington, V. et al., ibid., p. 1380 (1988); Lillie, J. W., Lowenstein, P. Green, M. R. and Green, M. Cell WO 97/47745 PCT/EP97/03070 10 1091 (1987); DeCarpio, J. A. et al., ibid., p. 275 (1988)).

Specific interaction between maize p75ZmRbl and WDV RepA in the yeast two-hybrid system (Fields et al) relied on the ability to reconstitute a functional GAL4 activity from two separated GAL4 fusion proteins containing the DNA binding domain (GAL4BD) and the activation domain (GAL4AD). Yeast HF7c cells were co-transformed with a plasmid expressing the GAL4BD-RepA or the GAL4BD- RepA(E198K) fusions and the plasmids expressing the GAL4AD alone (Vec) or fused to human p130, maize or a 69 amino acids N-terminal deletion of Cells were streaked on plates with or without histidine according to the distribution shown in the upper left corner. The ability to grow in the absence of histidine depends on the functional reconstitution of a GAL4 activity upon interaction of the fusion proteins, since this triggers expression of the HIS3 gene which is under the control of a GAL4 responsive element. The growth characteristics of these yeast co-transformants correlate with the levels of b-galactosidase activity.

Procedures for two-hybrid analysis are described in Xie et al (1995). The GAL4AD-ZmRbl fusions were construed in the pGAD424 vector.

EXAMPLE 3 In vivo activity.

Geminivirus DNA replication requires the cellular DNA replication machinery as well as other S-phase specific factors (Davies, J. W. and Stanley, J. Trends Genet, 77 (1989); Lazarowitz, S. Crit. Rev. Plant Sci. 11, 327 (1992)). Consistent with this requirement, geminivirus infection appears to drive non-proliferating cells into S-phase, as indicated by the accumulation of the proliferating cell nuclear antigen (PCNA), a protein which is not normally present in the nuclei of WO 97/47745 PCT/EP97/03070 11 differentiated cells (Nagar, Pedersen, T. J., Carrick, K. Hanley-Bowdoin, L. and Robertson, D.

Plant Cell 7, 705 (1995)). The inventors finding that efficient WDV DNA replication requires an intact LXCXE motif in RepA coupled with the discovery of a plant homolog of Rb supports the model that, as in animal cells, sequestration of plant Rb by viral RepA protein promotes inappropriate entry of infected cells into Sphase. Therefore, one way to investigate the function of p75ZmRbl was to measure geminivirus DNA replication in cells transfected with a plasmid bearing the ZmRbl sequences under a promoter functional in plant cells, an approach analogous to that previously used in human cells (Uzvolgi, E. et al., Cell Growth Diff 2, 297 (1991)).

Accumulation of newly replicated viral plasmid DNA was impaired in wheat cells transfected with plasmids expressing p75ZmRbl or human p130, when expression of WDV replication protein(s) is directed wither by the WDV promoter or by the CaMV 35S promoter.

Since WDV DNA replication requires an S-phase cellular environment, interference with viral DNA replication by and human p130 strongly evidences a role for retinoblastoma protein in the control of the Gl/S transition in plants. The existence of a plant Rb homolog implies that despite their ancient divergence, plant and animal cells use, at least in part, similar regulatory proteins and pathways for cell cycle control.

Two lines of evidences reinforce this model. First, a gene encoding a protein that complements specifically the Gl/S, but not the G2/M transition of the budding yeast cdc28 mutant has been identified in alfalfa cells (Hirt, Pay, B6gre, Meskiene, I. and Heberle-Bors, E.

Plant J. 4, 61 (1993)). Second, plant homologs of D-type cyclins have been isolated from Arabidopsis and these, like their mammalian relatives, contain LXCXE motifs. In i a.

concert with plant versions of CDK4 and CDK6, olant Dtype cyclins may regulate passage through G. phase by controlling the phosphorylation state of Rb-like proteins.

In animal cells, the Rb family has been implicated in tumor suppression and in the control of differentiation and development. Thus, p75ZmRbl could also play key regulatory roles at other levels during the plant cell life. One key question that is raised by the existence of Rb homologs in plant cells in whether, as in animals disruption of the Rb pathway leads to a tumor-prone condition. In this regard, the inventors have noted that the VirB4 protein encoded by the Ti plasmids of boch Agrobacterium tumefaciens and A. rhyvogenes contains an LXCXE motif. Although the VirB4 protein is required for tumor induction (Hooykas, P. J. J. and Beijersbergen,

A.

G. M. Annu. Rev. Phytopathol. 32, 157 (1994) the function of its LXCXE motif in this contexc remains to be examined. Geminivirus infection is not accomoanied by 20 tumor development in the infected plant, but in some cases an abnormal growth of enactions has been observed Dafalla and B. Gronenborn, personal communication) Inhibition of wheat dwarf geminivirus (WDV) DNA replication by ZmRbl or human p130 in cultured wheat 25 cells was carried out as follows. A. Wheat cells were transfected, as indicated, with pWori (Xie et al. 1995) alone (0.5 pg) a replicating WDV-based plasmid which encodes WDV proteins required for viral DNA reolication, and with control plasmid poBS (10 g) or p35S.Rb1 (1049) which encodes ZmRb1 sequences under the control of the CaMV 35S promoter. Total DNA was purified one and two days after ctransfeccion, equal amounts fractionated in agarose gels and ethidium bromide scaining and viral pWori DNA idencified by Southern hvbridizaiorn. Plasmid DNA represents exclusively newly-replicated plasmid DNA a 13 since it is fully resistant to DpnI digestion and sensitive to Mbol. Note that the MboI-digested samples were run for about half of the length than the undigested samples. B. To test the effect of human p130 on WDV DNA replication, wheat cells were co-transfected with pWori ig) and plasmids pBS (control), p35S.Rbl or p35S.130 jg in each case). Replication of the test plasmid (pWori) was analyzed two days after transfection and was detected as described in part A using ethidium bromide staining; and Southern hybridization. C. To test the effect of ZmRbl or human p130 on WDV DNA replication when expression of viral proteins was directed by the CaMV promoter, the test plasmid pWoriAA (which does not encode functional WDV replication oroteins but replicates when they are provided by a different plasmid, i. e. pWori) was used. Wheat cells were co-transfected, as indicated, with pWoriAA (0.25pg) pwori (0.25 g) p35S.A+B (6 g), p35S.Rbl (10 g) and/or p35S.130 (10 Replication of the test plasmid (pWoria6) was analyzed 36 hours after S 20 transfection and was detected as described in part A using ethidium bromide staining; Southern hybridization.

*Plasmids pWori (Ml) and pWoriAA (M2; Sanz and Guti6rrez, unpublished), 100 pg in each case, were used as markers.

Suspension cultures of wheat cells, transfection by particle bombardment and analysis of viral DNA replication were carried out as described in (Xie et al.

1995), except that DNA extraction was modified as in (Soni and Murray. Arnal. Biochem. 218, 474-476 (1995).

WO 97/47745 PCT/EP97/03070 14 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: CRISANTO GUTIERREZ ARMENTA NAME: QI XIE NAME: ANDRES PELAYO SANZ-BURGOS NAME: PAULA SUAREZ-LOPEZ STREET: CSIC-UAM, UNIVERSIDAD AUTONOMA, CANTOBLANCO CITY: MADRID COUNTRY: SPAIN POSTAL CODE (ZIP): 28049 (ii) TITLE OF THE INVENTION: PLANT PROTEINS (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30

(EPO)

INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 3747 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA.(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Zea mays (ix) FEATURE: NAME/KEY: CDS WO 97/47745 15 LOCATION: 31. .2079 PCT/EP97/03070 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCGGCA CGAGCAAAGG TCTGATTGAT ATG GAA TGT TTC CAG TCA AAT TTG Met Glu Cys Phe Gin Ser Asn Leu 1 GAA AAA ATO GAG AAA CTA TGT AAT TCT AAT AGC TGT AAA GGG GAG CTT Giu Lye Met Glu Lye Leu Cys Asn Ser Asn Ser Cys Lys Gly Glu Leu 15 GAT TTT AAA TCA ATT TTG ATC AAT AAT GAT TAT ATT CCC TAT GAT GAG Asp Phe Lys Ser Ile Leu Ile Asn Asn Asp Tyr Ilie Pro Tyr Asp Giu 30 35 AAC TCG ACG GOO GAT TCC ACC AAT TTA GGA CAT TCA AAG TGT GCC TTT Asn Ser Thr Gly Asp Ser Thr Asn Leu Gly His Ser Lys Cys Ala Phe 102 150 GAA ACA TTG GCA TCT CCC ACA AAG ACA ATA AAG AAC ATG Giu Thr Leu Ala Ser Pro Thr Lys Thr Ile Lys Asn Met CTG ACT GTT Leu Thr Val 246 CCT AGT TCT CCT TTG TCA CCA Pro Ser Ser Pro Leu 3cr Pro GCC ACC GGT GOT TCA GTC AAG ATT GTG Ala Thr Gly Gly Ser Val Lye Ile Val 294 CAA ATG ACA CCA GTA ACT TCT GCC ATG ACG ACA GCT AAG TOG CTT CGT Gin Met Thr Pro Val Thr Ser Ala Met Thr Thr Ala Lye Trp Leu Arg 95 100 GAG GTG ATA TCT TCA TTG CCA GAT ALAG CCT TCA TCT AAG CTT CAG CAG Giu Val Ilie Ser Ser Leu Pro Asp Lye Pro Ser Ser Lye Leu Gin Gin 342 110 115 TTT CTG TCA TCA TGC GAT AGOGOAT TTO ACA AAT OCT OTC ACA Phe Leu Ser Ser Cye Asp Arg Asp Leu Thr Aen Aia Val Thr 125 130 GTC AGC ATA GTT TTG GAA GCA ATT TTT CCA ACC AAA Val Ser Ile Vai Leu Giu Aia Ile Phe Pro Thr Lye 140 145 TCT TCT GCC AAT Scr 3cr Ala Asn 150 486 CGG GGT GTA TCO TTA GGT CTC AAT Arg Gly Val Ser Leu Giy Leu Asn 155 160 TOG OCA OAA GCC AGA AAA GTG GAG Trp Ala Giu Ala Arg Lye Vai Olu 170 175 TOT GCA AAT GCC Cys Ala Asn Ale GCT TCC AAG TTG Ala Ser Lys Leu 180 GAC ATT CCG Asp le Pro TAC TAT AGO GTA Tyr Tyr Arg Val WO 97/47745 PCT/EP97/03070 16 TTA GAG GCA ATC TGC AGA GCG GAG TTA Leu Glu Ala Ile Cys Arg Ala Glu Leu 185 190 CTA ACT CCA TTG CTG TCA AAT GAG CGT Leu Thr Pro Leu Leu Ser Asn Giu Arg 205 TGT TCA GCG GAC TTA GTA TTG GCG ACA CAA AAC AGC AAT OTA AAT AAT 630 Gin Arn Ser Arn 195 Val Asn Asn 200 TTG ATT GCA Leu Ile Ala 215 CAC CGA TOT His Arg Cys Cys Ser Ala TTT CCT GCT Plie Pro Ala 235 Leo Val Leu Ala CAT AAG ACA GTC ATC ATG ATG His Lys Thr Val Ile Met Met 230 OTT CTT GAG AGT ACC GOT CTA ACT GCA Val Leu Olu Ser Thr Oly Leu Thr Ala 240 GAT TTG AGC Asp Leo Ser AAA ATA ATT GAG C TTT GTG AGA CAT GAA GAG Lye Ile Ile Giu Asn Phe Val Arg His Glu Glu

ACC

Thr 250 255 TTO AAA AGO CAC TA AAT TCC TTA GAA GAA Leu Lye Arg 1-is Leu Asn Scr Leo Glu Glu 260 CAG CTT Glil Leo 275 CTC CCA AGA OAA Leu Pro Arq Glu TTG OAA AGC ATG Leu Glu Ser Met 265 27 0 OCA TGO GAG AAA GGT TCA TCA TTG TAT Ala Trp Glu Lys G.-y Ser Ser Leo Tyr TCA CTO ATT GTT Ser Leo Ile Val 918 CCA TCT OTT Pro Ser Val ATO CCA TCT Met Pro Ser 315 CA OAA ATA AAC CGC CTT GOT CTT TTO OCT OAA CCA .er Gu Ile Asn Arg Leo Gly Leo Leo Ala Glu Pro 966 CTr CA- T GAC TTA Leo iep Asp Leo TCA AGO CAG AAT Ser Arg Gin Asn CGT ATC GAG Arg Ile Glu 1014 GOC TTO Gly Leo 330 CCT OCT CCA TCT AAA AAA Pro Ala -r Pro Ser Lys Lye 335 COT OCT OCT Arg Ala Ala 340 GOT CCA GAT GAC Gly Pro Asp Asp 1062 AAC OCT GAT Asn Ala Asp 345 CCT CSA TCA CCA AAO AGA TCG Pro A-rg Ser Pro Lye Arg Ser 350 TOC AAT OAA Cys Asn Glu 355 TCT AGO AAC Ser Arg Asn 360 1110 ACA OTA OTA GAO CGC AAT TTG CAG ACA CCT CCA CCC AAG CAA Thr Val Val 010 ;rYg Asn Leo Gin Thr Pro Pro Pro Lye Gin AOC CAC Ser His 375 1158 ATO GTG TCA Met Val Ser ACT :ZT TTG AAA OCA AAA TOC Thr Ser Leo Lys Ala Lye Cys IRO 38 S CAT CCA CTC CAG TCC ACA His Pro Leo Gin Ser Thr 390 1206 'rr GCA AGT CCA AC-T GTC TGT AAT CCT GTT GOT 000 AAT GAA AAA TOT 1254 WO 97/47745 PCTIEP97/03070 17 Phe Ala Ser Pro Thr Val Cys Asn Pro Val Gly Gly Asn Glu Lys Cye 395 400 OCT GAC Ala Asp 410 GTG ACA ATT CAT ATA TTC TTT TCC AAG ATT CTG AAG TTG GICT Val Thr Ile His Ile Plie Phe Ser Lys Ile Leu Lys Leu Ala 1302 GCT ATT AGA Ala Ile Arg 425 ATA AGA AAC TTG Ile Arg Asn Leu TGC GAA AGG Cys Giu Arg CAA TGT GTG GAA CAG Gin Cys Val Giu Gin 440 1350 ACA GAG CGT GTC TAT AAT GTC TTC AAG CAG ATT CTT GAG CAA Thr Giu Arg Vai Tyr Aen Vai Phe Lys Gin Ile Leu Giu Gin CAG ACA Gin Thr 455 445 450 ACA TTA TTT TTT AAT AGA CAC ATC Thr Leu Phe Phe Aen Arq His Ile 460 CAA CTT ATC CTT TGC TGT CTT Gin Leu Ile Leu Cys Cye Leu 470 1446 TAT GGT GTT GCA Tyr Giy Val Ala 475 AAG OTT TGT CAA TTA GAA CTC ACA Lys Val Cys Gin Leu Glu L.eu Thr 480 AGG GAG ATA Arg Giu Ile 1494 CTC AAC Leu Asn 490 AAT TAC AAA AGA GAA OCA CAP TGC Asn Tyr Lye Arg Glu Ala Gin Cye 495 AAG CCA Lys Pro 500 GGG GTA Gly Val 515 GAA GTT TTT TCA Glu Val Phe Ser 1542 1590 AGT ATC TAT ATT G AGT ACG AAC CGT AAT Ser Ile Tyr Ile Oly Ser Thr Aen Arg Aen 505 510 TTA GTA TCG Leu Val Ser CAT GTT GOT ATC AT'r ACT TTT TAC His Val Gly Ilie Ile Thr Phe Tyr 525 AAT GAG Asn Glu 530 GTA TTT GTT Val Phe Val CCA GCA GCG Pro Ala Ala 535 CCA GAA GAC Pro Giu Asp 550 1638 AAG CCT TTC CTG GTG TCA CTA ATA TCA TCT GGT ACT CAT Lys Pro Phe Leu Val Ser Leu Ile Ser Ser Gly Thr His 540 545 1686 AAG AAG AAT Lys Lye Asn 555 GCT AGT GGC CAA ATT COT GGA TCA CCC AAG CCA TCT OCT Ala Ser Gly Gin Ile Pro Gly Ser Pro Lys Pro Ser Pro 1734 560 565 TTC CCA AAT TTA Phe Pro Aen Leu 570 CCA GAT ATG Pro Asp Met 575 TCC COG AAG AAA GTT TCA GCA TOT CAT Ser Pro Lye Lye Val Ser Ala Ser His 580 1782 AAT GTA TAT Aen Val Tyr 585 OTG TCT CCT TTG CGG CAA ACC Val Ser Pro Leu Arg Gin Thr 590 AAG rro GAT CTA CTG Lys LeoA Asp Leu Lou 595 1830 WO 97/47745 PCTIEP97/03070 18 TCA CCA AGT TCC AGG AGT TTT TAT GCA TGC ATT GGT GAA GGC ACC CAT Ser Pro Ser Ser Arg Ser Phe Tyr Ala 605 Ile Gly Glu GCT TAT CAG Ala Tyr Gin AAT TAT AAT Asn Tyr Asn 635 AGT GAC TCA Ser Asp Ser 650 CCA TCT AAG GAT Pro Ser Lys Asp GCT GCT ATA AAT Ala Ala Ile Asn Gly Thr His 615 AGC CGC CTA Ser Arg Leu 630 GAC ATG GTG Asp Met Vai 1878 1926 1974 2022 AGG AAA GTA Arg Lys Val TTA AAT TTC Leu Asn Phe 645 GTG GTA GCC Val Val Ala GGC AGT Gly Ser 655 GCA TTT Ala Phe CTG GGC CAG ATA AAT GGT GGT TCT Leu Gly Gin Ile Asn Gly Gly Ser 660 AGC CCC CTT TCA AAG AAG AGA GAG Ser Pro Leu Ser Lys Lye Arg Giu ACC TCG GAT CCT GCA GCT Thr Ser Asp Prc Ala Ala 2070 675 680 ACA GAT ACT TGATCAATTA TAAATGGTGG CCTCTCTCGT ATATAGCTCA Thr Asp Thr CAGATCCGTG CTCCGTAGCA GTCTATTCTT CTGAATAAGT GGATTAACTG GAGCGATTTA ACTGTACATG TATGTGTTAG TGAGAAGCAG CAGTTTTTAG GCAGCAAACT GTTTCAAGTT AGCTTTTGAG CTATCACCAT TTCTCTGCTG TGAATCTTTA GTTTTCATTG GGCTGACATA GGGAGGCATT CATCAGGGTT ATATTTGGTT TTACATTTT TCACTAGCAA TAGCAGCCCC TTACAGGTAT AAGCATGCCA ACTCTAAACT AGTACITCTC TGTTTCTGCT ACT'FrTGTAC TGATCCCATA TTCAGTGTGC TGCAAGTGAT GTCTCATGTT GTGACTTCAT TGCTGATTGC GGTTACAATC AGCCFI'TACT GCTTTATATT .ZCGArTGGTT TTCTGCATCA ATCAATCTTT TTCCCAAA VT GCAATTAATC CAGAAGTCTA AGTGAATGAA TATGAATCAG TCATGCTGAT GACATCGCAT CTCTTrCTGC AAGTGAGATG ATTGAACATA. TCCGCTGTGT ACAAATCTTT ATCCTAGTTG GTCAAAAAGT ACTGTACTTA AAATTGCTTT CCTGACTAGG ATATGAATCC TTTTTATATT TGTATATTTC CAGCTTCTCC TTGACCATAT GTGGCTTATC

AGAGTGCTAA

GCTGGTTGTT

ATTCACATCT

AACATATTC7

CTCATTTTTA

ATCAGACTGA

CTTCAGGTAT

2119 2179 2239 2299 2359 2419 2479 2539 2599 2659 2719 2779 2839 2899 2959 3019 TTTTGTA.ATG GTACTGTTGA GTTCATTTCT GTTCTACTAA ETTTGGCTTG CACAGCCAGG TTTAGGACAA GATATTTTTG TATGCTACAC CCTTGT'rTrA TTCTATTAGT TCTCAGCAAc AGATGTTCAT CTGGTTATTC CAAACAATCT AAGAAAACCT GAAATGCTAT CACCATTTAA WO 97147745 AACATTGOCT TCTGGAAGTT TGTACGGTAG Tc3ATGGCAGG TCTGATTGTA TTGACCAAGA GACGCTGACT CATCTGCATA TTCCAGCTGC ACCGTGTCAA TGCTTGCTTC TTGAAGATAC TGTCCTTGAC ATGTCAACAG 19 CAGGTGATTA GCAGGAGACG AGACGTTCTT AAACAGCAGC GCATCCACCT TGCCTTATGG ATGGCAGATG CTTAACCATC ATGTGAAGGC CCTGCAAGGC ATATGGTGCC ACCTAAATALA .ATTAGTGTTG GGTTGCAGTC TGCCAGCATT GTGATCGTCA CAAACGGAGG GCGATATAGG TTTG TTTAGA GTTAGCTTTT AACTCGGAGT CGCGTAGAAG CTAGCGTTTT TCAGTATGAA

TTCTGACATT

TGCTCCTTCA

TACTAACTGA

TTAGGAGCT

TTTCCAGGCC

AAGCTGTTTC

ATGTGGTGTT

GGCACAGAAG

TTGATAGCCA

GAGATGCATC

TTAGAATGCT

ATCCTTGTCT

GCCATTGACA

GCTTGTAATG

ATGAGCTGGT

CATGTCATGA

GCACCAATCC

TGGTTATGTC

TAAGTCTTGG

TACTCAAAAG

AT'rTCAGTTC

ATTTCTTTCA

AACTGACCTT

TAAAAAAAAA

PCTIEP97/03070 3079 3139 3199 3259 3319 3379 3439 3499 3559 3619 3679 3739 3747

AGAAGGCGAG

TGAGAGCTAC

TCTATATACA

CATCTGATTC

AATTTTCACC

AAGTCATTGC

TTGTTGCGAG

AGCAGCGGAT

TGTGTGTTGT

GAATAATTTG

INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 683 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Glu Cvs Phe Gin Ser Asn Leu Giu Lys Met Glu Lys Leu Cys Asn 1 5 10 Ser Aen Ser Cys Lye Gly Giu Leu Asp Phe Lys Ser Ile Leu Ile Asn 2G 25 Aen Asp Tyr Ile Pro Tyr Aep Glu Aen Ser Thr Gly Aep Ser Thr Aen 40 Leu Gly Hie Ocr Lye Cye Ala Phe Giu Thr Leu Ala Ser Pro Thr Lye 55 WO 97/47745 PCT/EP97/03070 20 Thr Ile Lys Asn Met Leu Thr Val Pro Ser Ser Thr Gly Met Thr Lys Pro Leo Thr 130 Phe Pro 145 Cys Ala Ala Ser Leu Gin Arg Phe 210 Thr His G1ly Levi His Glu Glu Giu Tvr Asia 290' Arg Leu 305 Ser Arg Gin Met Thr Glu Vai Ile 105 Phe Leu Ser Val Ser Ile Arg Giy Vai 155 Trp Ala Glu 170 Leu Giu Aia 185 Leu Thr Pro Cys Ser Ala Phe Pro Ala 235 Lys Ile Ile 250 Leu Lye Arg 265 Ala Trp Glu Pro Ser Val >Met Pro 6Cr 315 iGly Leu Pro 330 Pro Leu Ser Pro Ala Pro Val Thr Ser Ala Ser 6cr Leu Pro Asp 110 Ser Cys Asp Arg Asp 125 Val Leu Glu Ala Ilie 140 6cr Leo Gly Leu Aen 160 Ala Arg Lys Val Glu 175 Ile Cys Arg Ala Giu 190 Leu Leu Ser Aen Glu 205 Asp Leu Val Leo Ala 220 Val Leu G10 Ser Thr 240 Glu Aun Phe Val Arg 255 His Leu Asn 6cr Leo 270 Lye Giy Ser Set Leu 285 Ala Set 10 Ile Aen 300 Leu Asp Asp Leo Val 320 Ala Thr Pro Ser Lye 335 325 Lys Arg Ala Ala 340 Gly Pro Asp Asp Aen Ala Asp Pro Arg 6cr Pro Lye 345 350 WO 97/47745 PCTIEP97/03070 r V Arg Ser Cys Asia Giu Ser Arq Asia Th 355 Thir Pro Pro Pro 370 Lys Cys His Pro 385 Pro Val Gly Giy Phe Ser Lys :le 420 Giu Arg Val Glin 435 Gin Ile Leu 450 Asp Gin Leo lie 465 Leu Giu Leu Thr Gin Cys Lye Pro S00 Arg Asia Gly *Jai 515 Asia Giu Val Phe 530 Ser Ser Giy Thr 545 Pro Giy Ser Pro Pro Lys Lys Val 580 360 Ser His MeL 375 Ser Thr Phe I Lys Cys Aia 1 Leu Aia Aia 425 Gio Gin Thr 440 Gin Thr Thr 455 Cys Leo Tyr Glu Iie Leu Phe Ser Ser 505 Ser Arg His 520 Ala Aia Lys 535 Giu Asp Lye Ser Pro Phe Ser His Asia 5851 Leu Leo ger 600 Thr His Aia 615 Arg Leo Acan 21 rai Val Tai Ser la Ser 395 sep Vai 110 Ise Arg G1o Arg Lexi Phe Giy Vai 475 Asia Asia 490 Ile Tyr Vai Giy Pro Phs Lys Asti 555 Pro Asr 570 Val Tyi.

Pro Sei Tyr Gli Tyr Asi 6 3' Gio Arg Asia Leu Gin 365 Thr Ser Leu Lys Aia 380 Pro Thr Vai Cys Asna 400 Thr Ile His Ile Phe 415 Ile Arg Asia Leo Cys 430 Val Tyr Asia Vai Phe 445 Phe Asia Arg His Ile 460 Aia Lye Vai Cys Gin 480 Tyr Lye Arg Gio Ala 495- Ile Giy Ser Thr Asia 510 Ile Ile Thr Phe Tyr 525 Leu Vai Ser Leo le 540 LAia Ser Gly Gin Ilie 560 kLeo Pro Asp Met Ser 575 7Vai Ser Pro Leo Arg 590 Ser Arg Ser Phe Tyr 605 a Ser Pro Ser Lye Asp 620 ai Giy Arg Lys Vai Asia 640 Asp Leo Gio Giy Asia Ser 630 WO 97/47745 PCT/EP97/03070 -22- Ser Arg Leu Asn Phe Asp Met Val Ser Asp Ser Val Val Ala Gly Ser 645 650 655 Leu Gly Gln Ile Asn Gly Gly Ser Thr Ser Asp Pro Ala Ala Ala Phe 660 665 670 Ser Pro Leu Ser Lys Lys Arg Glu Thr Asp Thr 675 680 23 INFORMATION RELATIVE TO THE DEPOSIT OF A MICRO-ORGANISM The micro-organism to which reference is made in page 6 of che disclosure has been deposited in the following institution: COLECCION ESPANOLA DE CULTIVOS TIPO (CECT) Departamento de Microbiologia Facultad de Ciencias Biol6gicas 46100 BURJASOT (Valencia) Spain Deposit identification: pBS.Rbl Deposit date: June 12, 1996 Order No.: 4699 This information appears reflected in the form PCE/RO/134 enclosed to the request.

"Comprises/comprising" when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

*i J

*F

Claims (18)

1. A method of controlling viral infection in a plant cell characterised in that it inhibits passage of the cell from the G1 phase to the S phase of the cell cycle by increasing the level and/or activity of retinoblastoma protein in that plant cell by incorporation of a recombinant nucleic acid into the cell which encodes for a protein of amino acid sequence having 80% or more homology with that of SEQ ID No. 2 and which is capable of binding plant E2F or (ii) a peptide fragment of protein which is capable of interacting with geminivirus Rep A LXCXE motif. S2. A method as claimed in claim 1 characterised in that the level or activity of retinoblastoma protein is increased sufficient to inhibit replication of plant 99 virus.

3. A method as claimed in claim 1 or claim 2 characterised in that the 9 nucleic acid is such as to increase expression of a retinoblastoma protein in the cell.

4. A method as claimed in claim 3 characterised in that the recombinant nucleic acid is such that it expresses protein resistant to phosphorylation by cyclin dependent kinases by change or deletion of one or more consensus SP or TP sites found in the SEQ ID No.2. Recombinant nucleic acid including a promoter capable of functioning in plant cells and a sequence encoding for expression of a retinoblastoma protein which has an amino acid sequence having 80% or more homology with that of SEQ NO 2.

6. Recombinant nucleic acid as claimed in claim 5 characterised in that it comprises SEQ ID No. 1, bases 31-207, sequences only having degenerate substitutions thereof or sequences capable of hybridizing with a polynucleotide of SEQ ID No. 1 under conditions of high stringency.

7. Recombinant nucleic acid as claimed in claim 5 or 6 characterised in that it encodes for a retinoblastoma protein conservatively substituted with respect to SEQ ID No. 2.

8. Recombinant nucleic acid as claimed in any one of claims 5 to 7 characterised in that it encodes for a retinoblastoma protein or a peptide fragment of a retinoblastoma protein that interacts with viral LXCXE motif without affecting the normal functioning of a plant cell and further comprising a promoter capable of functioning in plant cells. 4:

9. Recombinant nucleic acid as claimed in claim 8 characterised in that it encodes for a plant retinoblastoma protein in which one or more consensus SP or TP sites found in the SEQ ID No. 2 have been changed or deleted.

10. Recombinant nucleic acid as claimed in any one of claims 5 to 9 characterised in that the promoter is WDV promoter or a a.

11. A protein produced by the expression of a recombinant DNA or RNA as a claimed in any one of claims 5 to

12. A protein as claimed in claim 11 characterised in that one or more consensus SP or TP sites found in the SEQ ID No. 2 have been changed or deleted.

13. A recombinant vector characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 5 to 11.

14. A plant cell characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 5 to 13. A plant cell as claimed in claim 14 characterised in that it expresses a retinoblastoma protein from said nucleic acid.

16. A transgenic plant characterised in that it comprises a cell as claimed in claim 14 or

17. A method of replicating plant DNA or RNA at increased level characterised in that it comprises control of passage of that plant cell from the G1 phase to the S phase of the cell cycle by decreasing the level and/or activity of retinoblastoma protein in that plant cell, and thus inhibiting cell entry into the S phase wherein the method comprises incorporation of a recombinant nucleic acid into the cell which encodes for anti-sense nucleic acid to that encoding for a protein of amino acid sequence having homology with that of SEQ ID No. 2 and which is capable of binding to plant E2F. I*O*

18. A method as claimed in claim 17 characterised in that the DNA or RNA is anti-sense to retinoblastoma protein encoding DNA or RNA and inhibits retinoblastoma protein expression.

19. Recombinant nucleic acid including a promoter capable of functioning in plant cells and a sequence comprising anti-sense DNA or RNA to a plant retinoblastoma protein, that protein having 80% or more homology with that of SEQ NO 2. Recombinant nucleic acid as claimed in claim 19 characterised in that it comprises anti-sense DNA or RNA to that of SEQ ID No. 1 or a sequence 1tving at least 80% homology thereto.

21. A plant cell characterised in that it comprises a recombinant nucleic acid as claimed in any claims 19 or

22. A transgenic plant characterised in that it comprises a cell as claimed in claim 21. DATED this 181h day of April 2000 CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS WATERMARK PATENT AND TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA LCG:KMH:VRH P5353AU00.DOC a. S. S. S e.~ S* S. S S SS S 5. S 04 55 S 5.-S 0 *5S~ S 0 *SOS S Odes 0 0 S. Sq S.. S. S *5 S S