EP0914436A1

EP0914436A1 - Plant retinoblastoma-associated proteins

Info

Publication number: EP0914436A1
Application number: EP97928187A
Authority: EP
Inventors: C.-Centro de Biologia Molecul. GUTIERREZ-ARMENTA; Qi;-Centro de Biologia Molecular XIE; A.-Centro de Biologia Molecul PELAYO SANZ-BURGOS; Paula-Centro de Biologia Molecular SUAREZ LOPEZ
Original assignee: Consejo Superior de Investigaciones Cientificas CSIC
Current assignee: Consejo Superior de Investigaciones Cientificas CSIC
Priority date: 1996-06-13
Filing date: 1997-06-12
Publication date: 1999-05-12
Also published as: CN1227605A; CA2257828A1; WO1997047647A1; AU3257997A; CA2257972A1; ZA975202B; WO1997047745A1; AU721332B2

Abstract

The present invention is based on the isolation and characterization of a plant cell DNA sequence encoding for a retinoblastoma protein. Such finding is based on the structural and functional properties of the plant retinoblastoma protein as possible regulator of the cellular cycle, of the cellular growth and of the plant cellular differentiation. For this reason, among other aspects, it is claimed the use of retinoblastoma protein or the DNA sequence which encodes for it in the growing control of vegetable cells, plants and/or vegetable virus, as well as the use of vectors, cells, plants or animals, or animal cells modified through the manipulation of the control route based on plant retinoblastoma protein.

Description

PLANT RETTNOBLASTOM A-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 Gl phase. In mammalian cells, Rb regulates Gl/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 Wemberg, R.A Cell 81,323 (1995) ; Nevins, J.R. Science 258,424 (1992) ) . DNA tumor viruses that infect animal cells express oncoproteinε 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 gemimvirus requires the integrity of an LXCXE ammo acid motif in the viral RepA protein and that RepA can interact with members of the human Rb family in yeast (Xie, Q., Suarez-Lόpez, P. and Gutierrez, C. EMBO J. 14, 4073 (1995) . The presence of the LXCXE motif m plant D-type cyclins has also been reported (Soni, R., Carmichael, J. P., Shah, Z. H. and Murray, J. 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 pl30, 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 as well as plant Rb protein such as ZmRbl will be expected to have utility in, in ter alia , plant therapeutics, diagnostics, growth control or investigations and many such plant proteins will have similar utility in animals.

In a first aspect of the present invention there is provided the use of retinoblastoma protein in controlling the growth of plant cells and/or plant viruses. Particularly, the present invention provides control of viral infection and/or growth in plant cells wherein the virus requires the integrity of an LXCXE amino acid motif in one of its proteins, particularly, e. g., m the viral RepA protein, for normal reproduction. Particular plant viruses so controlled are Gemmiviruses .

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 does 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 S-phase. 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) , encoding for expression of Rb protein that is characteristic of plants. This nucleic acid is characterised by one or more characteristic regions that differ from known animal Rb protein nucleic acid and is exemplified herein by SEQ ID No 1, bases 31- 2079. 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 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 ammo 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-doma s having between 30% and 75% homology with human Rb protein, particularly as compared with pl30, more preferably from 50% to 64% homology. Particularly the plant Rb protein so encoded has the C706 ammo 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 utagenic means such as use of mutagenic PCR primers. 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 m the case of proteins or may involve indirect means, such as electroporation of plant seed cells with DNA or by transforma ion 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 N- terminal domain corresponding in sequence to the ammo 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 ammo acids and 900 pairs of more bases.

The present invention will now be illustrated further by reference to the following non-limit g Examples Further embodiments falling within the scope of the claims attached hereto will occur to those skilled 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 amino acids sequences of the Pocket A and Pocket B of the ZmRbl with that of the Xenopuε, chicken, rat and three human protein (Rb, pl07 and pl30) .

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 32P- labelled Pstl internal fragment (1.4 kb) .

A portion of a maize cDNA library (106 pfu) in 1ZAPII (Stratagene) was screened by subsequent hybridization to 5' -labelled oligonucleotides designed to be complementary to a known EST sequence of homologue maize of pl30. 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 ExAssiεt 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 DraI35 (5' -GATTTAAAATCAAGCTCC, nt positions 113-96) . After denaturation at 90°C for 3 min, RNA was eliminated by 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 (50 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, W.-L. 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 (Destree, 0. H. J. et al, Dev. Biol. 153, 141 (1992) ) .

Plasmid pWoriΔΔ 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 pl30 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 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 b . 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 non- conserved N-terminal and C- erminal domains. Overall, ZmRbl shares -28-30% amino acid identity (-50% similarity) with the Rb family members (Hannon, G. J. , Demetrick, D. & Beach, D. Genes Dev. 7, 2378 (1993) ; Cobrinik, D., Whyte, P., Peeper, D.S., Jacks, T. & Weinberg, R. A. ibid., p. 2392 (1993) . Ewen, M. E., 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 (-50-64%) . Interestingly, amino acid C706 in human Rb, critical for its function (Kaye, F. J., Kratzke R. A., Gerster, J. L. and Horowitz, J. M. Proc. Natl. Acad. Sci. USA 87, 6922 (1990) ) , is also conserved in maize p75ZmRbl .

Note: The 561-577 amino acids encompass a proline-rich domain. ZmRbl contains 16 consensus sites, SP or TP or phosphorilation by cyclins dependant kinaseε (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 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 mtact LXCXE motif of the viral RepA protein This motif can mediate interaction with a member of the human Rb family, pl30, m yeasts Therefore, the inventors investigated whether p75ZmRbl could complex with WDV RepA by using the yeast two-hybrid system (Fields, S and Song, 0 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 fuεion could also complex with GAL4BD-RepA to allow growth of the recipient yeast cells in the absence of histidme This interaction was slightly stronger than that seen with the human pl30 protein RepA could also bind to some extent to a N-terminally truncated form of p75ZmRbl The role of the LXCXE motif in RepA-p75ZmRbl interaction was assessed using a point mutation in WDV RepA (E198K) which we previously showed to destroy interaction with human pl30. Co-transformation of ZmRbl with a plasmid encoding the fusion GAL4BD-RepA(E198K) indicated that the interaction between RepA and p75ZmRbl occurred through the LXCXE motif In this respect, the E198K mutant of WDV RepA behaves similarly to analogous point mutants of animal virus oncoprotems (Moran, E , Zerler, B , Harrison, T M and Mathews, M B Mol Cell Biol. 6, 3470 (1986) , Chermgton, V et al ibid , p 1380 (1988) , Lillie, J W , Lowenste , P M , Green, M R and Green, M Cell 50, 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(El98K) fusions and the plasmids expressing the

GAL4AD alone (Vec) or fused to human pl30, maize p75

(p75ZmRbl) or a 69 amino acids N-terminal deletion of p75

(p75ZmRbl-DN) . Cells were streaked on plates with or without hiεtidine 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 fuεion proteins, since this triggers expresεion 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 analysiε are deεcribed in Xie et al (1995) . The GAL4AD-ZmRbl fuεions 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. 5, l ⁿ (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 differentiated cells (Nagar, S., Pedersen, T. J., Carrick, K. M., 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 S- phase. 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 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 wheat cells transfected with plasmids expressing p75ZmRbl or human pl30, when expression of WDV replication protein (s) s 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 p75ZmRbl and human pl30 strongly evidences a role for retinoblastoma protein in the control of the Gl/S transition n 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, H , Pay, A., Bogre, L., 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 concert with plant versions of CDK4 and CDK6 , plant D- type cyclms may regulate passage through Gl phase by controlling the phoεphorylation state of Rb-like proteins . In animal cells, the Rb family has been implicated in tumor suppression and m 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 raiεed by the exiεtence 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 VιrB4 protein encoded by the Ti plasmids of both Agrobac eri uw tumefaciens and A . rhyzogenes contains an LXCXE motif. Although the VιrB4 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 m this context remains to be examined. Geminivirus infection is not accompanied by tumor development in the infected plant, but in some cases an abnormal growth of enactions has been obεerved (G. Dafalla and B. Gronenborn, personal communication)

Inhibition of wheat dwarf geminivirus (WDV) DNA replication by ZmRbl or human pl30 cultured wheat cells was carried out as follows. A. Wheat cells were transfected, as indicated, with pWori (Xie et al . 1995) alone (0.5g) , a replicating WDV-based plasmid which encodes WDV proteins required for viral DNA replication, and with control plasmid pBS (10 g) or p35S.Rbl (10 g) , which encodes ZmRbl sequences under the control of the CaMV 35S promoter. Total DNA was purified one and two days after transfection, equal amounts fractionated in agarose gels and ethidium bromide staining and viral pWori DNA identified by Southern hybridization. Plasmid DNA represents exclusively newly-replicated plasmid DNA since it is fully resistant to Dpnl digestion and sensitive to Mbol . Note that the Mbol-digested samples were run for about half of the length than the undigested samples. B. To test the effect of human pl30 on WDV DNA replication, wheat cells were co-transfected with pWori

(0.5 g) and plasmids pBS (control) , p35S.Rbl or p35S.130

(10 g 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 pl30 on WDV DNA replication when expression of viral proteins was directed by the CaMV 35S promoter, the test plasmid pWoriΔΔ (which does not encode functional WDV replication proteins but replicates when they are provided by a different plasmid, i. e. pWori) was used Wheat cells were co-transfected, as indicated, with pWoriΔΔ (0.25 g) , pWori (0.25 g) , p35S.A+B (6 g) , p35S.Rbl (10 g) and/or p35S.130 (10 g) . Replication of the test plasmid (pWoriΔΔ) was analyzed 36 hours after transfection and was detected as described in part A using ethidium bromide staining; Southern hybridization. Plasmids pWori (Ml) and pWoriΔΔ (M2; Sanz and Gutierrez, 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 m (Soni and Murray. Arnal . Biochem. 218, 474-476 (1995) SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT:

(A) NAME: CRISANTO GUTIERREZ ARMENTA (A) NAME: QI XIE

(A) NAME: ANDRES PELAYO SANZ-BURGOS

(A) NAME: PAULA SUAREZ-LOPEZ

(B) STREET: CSIC-UAM, UNIVERSIDAD AUTONOMA, CANTOBLANCO

(C) CITY: MADRID (E) COUNTRY: SPAIN

(F) POSTAL CODE (ZIP) : 28049

(ii) TITLE OF THE INVENTION: PLANT PROTEINS (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)

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3747 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Zea mayε

(ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 31..2079

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

GAATTCGGCA CGAGCAAAGG TCTGATTGAT ATG GAA TGT TTC CAG TCA AAT TTG 54

Met Glu Cyε Phe Gin Ser Asn Leu 1 5

GAA AAA ATG GAG AAA CTA TGT AAT TCT AAT AGC TGT AAA GGG GAG CTT 102 Glu Lyε Met Glu Lys Leu Cyo Asn Ser Asn Ser Cye Lys Gly Glu Leu

10 15 20

GAT TTT AAA TCA ATT TTG ATC AAT AAT GAT TAT ATT CCC TAT GAT GAG 150 Aεp Phe Lys Ser lie Leu lie Asn Aøπ Aep Tyi lie Pro Tyr Aisp Glu 25 30 35 40

AAC TCG ACG GGG GAT TCC ΛCC AAT TTA GGA CAT TCA AAG TGT GCC TTT 198 Asn Ser Thr Gly Asp Ser Thr Asn Leu Gly His Sei Lys Cyε Ala Phe 45 50 55

GAA ACA TTG GCA TCT CCC ACA AAG ACA ATA AAG AAC ATG CTG ACT GTT 246 Glu Thr Leu Ala Ser Pro Thr Lyε Thr lie Lys Asn Met Leu Thr Val 60 65 70

CCT AGT TCT CCT TTG TCA CCA GCC ACC GGT GGT TCA GTC AAG ATT GTG 294 Pro Ser Ser Pro Leu Ser Pro Ala Thr Gly Gly Ser Val Lye lie Val 75 80 85

CAA ATG ACA CCA GTA ACT TCT GCC ATG ACG ACA GCT AAG TOG CTT CGT 342 Gin Met Thr Pro Val Thr Ser Ala Met Thr Thr Ala Lyε Trp Leu Arg 9U 95 100

GAG GTG ATA TCT TCA TTG CCA GAT AAG CCT TCA TCT AAG CTT CAG CAG I bO Glu Val lie Ser Ser Leu Pro Asp Lys Pro Ser Ser Lyε Leu Gin Gin 105 110 115 120

TTT CTG TCA TCA TGC GAT AGG GAT TTG ACA AAT GCT GTC ACA GAA AGG 438 Phe Leu Ser Ser Cyε Asp Arg Aεp Leu Thr Aεn Ala Val Thr Glu Arg 125 130 135

GTC AGC ATA GTT TTG GAA GCA ATT TTT CCA ACC AAA TCT TCT GCC AAT 436 Val Se He Val Leu Glu Ala He Phe Pro Thr Lyε Ser Ser Ala Aεn 140 145 150

CGG GGT GTA TCG TTA GGT CTC AAT TGT GCA AAT GCC TTT GAC ATT CCG 534 Arg Gly Val Sei eu Gly Leu Asn Cyε Ala Aεn Ala Phe Aεp He Pro

155 160 165

TGG GCA GAA GCC AGA AAA GTG GAG GCT TCC AAG TTG TAC TAT AGG GTA 582 Trp Ala Glu Ala Arg Lyε Val Glu Ala Ser Lyε Leu Tyr Tyr Arg Val 170 175 180 TTA GAG GCA ATC TGC AGA GCG GAG TTA C'AA AAC AGC AAT GTA AAT AAT 630

Leu Glu Ala He Cys Arg Ala Glu eu Gin Aεn Sei Acn Val Aen Asn

185 190 195 200

CTA ACT CCA TTG CTG TCA AAT GAG CGT TTC CAC CGA TGT TTG ATT GCA 678

Leu Thr Pro Leu Leu Ser Asn Glu Arg Phe Hie Arg Cyε Leu He Ala

205 210 215

TGT TCA GCG GAC TTA GTA TTG GCG ACA CAT AAG ACA GTC ATC ATG ATG 726

Cyε Sei Ala Asp Leu Val Leu Ala Thr Hie Lyε Thi Val He Met Met

220 225 2 0

TTT CCT GCT GTT CTT GAG ΛGT ACC GGT CTA ACT GCA TTT GAT TTG AGC 774

Phe Pro Ala Val Leu Glu Sci T i Gly Leu Thr Ala Phe Aεp Leu Ser

235 240 245

AAA ATA ATT GAG AAC TTT GTG AGA CAT GAA GAG ACC CTC CCA AGA GAA 822

Lye lie H Glu Asn Phe Val Arq His Glu Glu Thi Leu Pi a Al q Glu

250 255 260

TTG AAA AGG CAC CTA AAT TCC TTA GAA GAA CAG CTT TTG GAA AGC ATG 8"O

Leu I.ys Arg His 2--_;u Aεn Ser l.eu Glu Glu Gin Leu I.eu Glu Sei Met

265 270 275 280

GCA TGΩ GAG AAA GGT TCA TCA TTG TAT AAC TCA CTG ATT GTT GC AGG 918

Ala Trp Glu Lys Gly Ser Ser Leu Tyi Asn Sei Leu He Val AJ Arg

285 290 295

CCA TCT GTT GCT TCA GAA ATA AAC CGC CTT GGT CTT TTG GCT GAA CCA 966

Pro Ser Val Ala Ser Glu He Acn Arg Leu Gly Leu Leu Ala Glu Pro

300 305 no

ATG CCA TCT CTT -AT GAC TTA GTG TCA AGG CAG AAT GTT CGT ATC GAG 1014

Met Pro Ser Leu Asp Aεp Leu Val Ser Arg Gin Asn Val Arg He Glu

3 320 325

GGC TTG CCT GCT ACA CCA TCT AAA AAA CGT GCT GCT GGT CCA GAT GAC 1062

Gly Leu Pro Ala Thr Pro Ser Lyε Lyε Arg Ala Ala Gly Pro Asp Aεp

HO 335 340

AAC GCT GAT CCT CGA TCA CCA AAG AGA TCG TGC AAT GAA TCT AGG AAC U K¹

Λεn Ala Aεp Pro .Arq Ser Pro Lys Arq Ser Cyε Λεn Glu Ser Arg Aen

345 350 355 360

ACA GTA GTA GAG CGC AAT TTG CAG ACA CCT CCA CCC AAG CAA AGC CAC 1158

Thi Val Val Glu Arg Asn Leu Gin Thr Pro Pro Pro Lyε Gin Ser His

365 370 375

ATG GTG TCA ACT AGT TTG AAA GCA AAA TGC CAT CCA CTC CAG TCC ACA 1206

Met Val Ser Thr ≤er Leu Lys A Lyε C'ys His Pro Leu Gin Ser Thi

380 385 390

TTT GCA AGT CCA ACT GTC TGT AAT CCT GTT GGT GGG AAT GAA AAA TGT 1254 Phe Ala Sei Pio Thi Val Cys Asn Pro Val Gly Gly Aεn Glu ye Cys 395 400 405

GCT GAC GTG ACA ATT CAT ATA TTC TTT TCC AAG ATT CTG AAG TTG GCT 1302

Ala Asp Val Thi He Hie He Phe Phe Ser Lyε He Leu Lyε Leu Ala

410 41S 420

GCT ATT AGA ATA AGA AAC TTG TGC GAA AGG GTT CAA TGT GTG GAA CAC, 1 50

Ala He Aig He Al q Asn Leu Cyε. Glu Arq Val Gin Cyε Val Glu Gin

425 430 435 440

ACA GAG CGT GTC TAT AAT GTC TTC AAG CAG ATT CTT GAG CAA CAG ACA 1198

Thr Glu Aiq Val Tyr Asn Val Phe Lyr Gin He Leu Glu Gin Gin Thr 445 450 455

ACA TTA TTT TTT AAT AGA CAC ATC GAT CAA CTT ATC CTT TGC TGT CTT 1446

Thi Leu Phe Phe A-.n Arq Hir Tie Aεp Gill Leu He Leu Cyε Cyε Leu

4b0 465 47u

TAT GGT GTT GCA AAG GTT TGT CAA TTA GAA CTC ACA TTC AGG GAG ATA 1494

Tyi Gl\ vjl Ala Ly Val Cyε Gin Leu Glu Leu Thi Phe Arq Glu He 475 480 485

CTC AAC AAT TAC AAA AGA GAA GCA CAA TGC AAG CCA GAA GTT TTT TCA 1542

Leu Asn Asn Tyr ys Arg Glu Ala Gin Cye Lyε Pro Glu Val Phe Ser

490 49^ 500

AGT ATC TAT ATT GGG AGT ACG AAC CGT AAT GGG GTA TTA GTA TCG CGC 15^0

Sci lit Tyi He Gl\ Ser Thi Asn Arg Aεn Gly Val Leu Val Ser Arq

505 51ι 51 520

CAT GIT GGT ATC ATT ACT TTT TAC AAT GAG GTA TTT GTT CCA GCA GCG 1638

Hie val Gly He IIf ni Phe Tyr Asn Glu Val Phe Val Pro Ala Ala 52^c 530 535

AAG CCT TTC CTG GTG TCA CTA ATA TCA TCT GGT ACT CAT CCA GAA GAC 1686

Lys Pi o Plie Leu Val Ser Leu He Sei Ser Gly Thr His Pro Glu Asp

540 545 550

AAG AAG AAT GCT AGT GGC CAA ATT CCT GGA TCA CCC AAG CCA TCT CCT 1734

Ly Lvs Ain Ala Ser Gly Gin He Pro Gly Ser Pro Lys Pro Ser Pro 555 560 565

TTC CCA AAT TTA CCA GAT ATG TCC CCG AAG AAA GTT TCA GCΛ TCT CAT 1782

Phe i a ASII Leu Pro Asp Mel Ser Pro Lys Lyr Val Sei Ala Ser Hiε

5 'i 575 580

AAT GTA TAT GTG TCT CCT TTG CGG CAA ACC AAG TTG GAT CTA CTG CTG 1830

Asn val Tvi VaJ Sei Pro Leu Arq Gin Thi Lys Leu Asp Leu Leu Leu

585 590 595 600 TCA CCA AGT TCC AGG AGT TTT TAT GCA TGC ATT GGT GAA GGC ACC CAT 1878

Sei Pro Ser Sei Arg Ser Phe Tyr Ala Cyε He Gly Glu Gly Thr His 605 610 615

GCT TAT CAG AGC CCA TCT AAG GAT TTG GCT GCT ATA AAT AGC CGC CTA 1926

Ala Tyi Gin Sei Pro Sei Lys Asp Leu Ala Ala He Asn Sei Arg Leu

620 625 630

AAT TAT AAT GGC AGG AAA GTA AAC AGT CGA TTA AAT TTC GAC ATG GTG 1974

A=-ιι Tyi Aen Glv Arg Lyε Val Asn Sei Arg Leu Asn Phe Asp Met Val fa35 640 645

AG- GAC TCA GTG GTA GCC GGC AGT CTG GGC CAG ATA AAT GGT GGT TCT 2022

Ser Asp Sei Val Val Ala Gly Ser Leu Gly Gin He Aεn Gly Gly Sei

650 655 660

«Cr TCG GAT CCT GCA GCT GCA TTT AGC CCC CTT TCA AAG AAG AGA GAG 2070

Tin Sei Ac) i i Ala Ala Ala Phe Sei ho Leu Sei Lvf Lyu Arq Glu ■><^•■" 6/0 675 680

ACA GAT ACT TGATCAATTA TAAATGGTGG CCTCTCTCGT ATATAGCTCA 2119

Tin Aεp Thi

CAGATCCGTG CTCCGTAGCA GTCTATTCTT CTGAATAAGT GGATTAACTG GAGCGATTTA 2179

ACTGTACATG TATGTGTTAG TGAGAAGCAG CAGTTTTTAG GCAGCAAACT GTTTCAAGTT 2239

AGCTTTTGAG CTATCACCAT TTCTCTGCTG ATTGAACATA TCCGCTGTGT AGAGTGCTAA 2299

TGAATCTTTA GTTTTCATTG GGCTGACATA ACAAATCTTT ATCCTAGTTG GCTGGTTGTT 2359

GGGAGGCATT CΛTCAGGGTT ATATTTGGTT GTCAAAAAGT ACTGTΛCTTA ATTCACATCT 2419

ΓΓCACATTTT TCACTAGCAA TAGCAGCCCC AAATTGCTTT CCTGACTAGG AACATATTCT 24 9

TTACAGGTAT AAGCATGCCA ACTCTAAACT ATATGAATCC TTTTTATATT CTCATTTTTA 2539

ΛGTACTTCTC TGTTTCTGCT ACTTTTGTAC TGTATATTTC CAGCTTCTCC ATCAGACTGA 2599

TGATCCCATA TTCAGTGTGC TGCAΛGTGAT TTGACCATAT GTGGCTTΛTC CTTCAGGTAT 2659

5TCTCATGTT GTGACTTCAT TGCTGATTGC TTTTGTAATG GTACTGTTGA GTTCATTTCT 2719

GGTTACAATC AGCCTTTΛCT GCTTTATATT GTTCTACTAA TTTTGGCTTG CACAGCCAGG 2779

^"."GATTGGTT TTCTGCATCA ATCAATCTTT TTTAGGACAA GATATTTTTG TATGCTACAC 2839

TTCCCAAATT GCAATTAΛTC CAGAΛGTCTA CCTTGTTTTΛ TTCTATTAGT TCTCAGCAAC 2899

AGTGAATGAA TATGAATCAG TCATGCTGAT AGATGTTCΛT CTGGTTATTC CAAACAATCT 2959

GACATCGCAT CTCTTTCTGC AAGTGAGATG AAGAAAACCT GAAΛTGCTΛT CACCATTTAΛ 3019 AACATTGGCT TCTGGAAGTT CAGGTGATTA GCAGGAGACG TTCTGACATT GCCATTGACA 3079

TGTACGGTAG TGATGGCAGG AGACGTTCTT AAACAGCAGC TGCTCCTTCA GCTTGTAATG 3139

TCTGATTGTA TTGACCAAGA GCATCCACCT TGCCTTATGG TACTAACTGA ATGAGCTGGT 3199

GACGCTGΛCT CATCTGCATA ATGGCAGATG CTTAACCATC TTTAGGAGCT CATGTCATGA 3259

TTCCAGCTGC ACCGTGTCAA ATGTGAAGGC CCTGCAAGGC TTTCCAGGCC GCACCAATCC 3319

TGCTTGCTTC TTGAAGATAC ATATGGTGCC ACCTAAATAA AAGCTGTTTC TGGTTATGTC 3379

TGTCCTTGAC ATGTCAACAG ATTAGTGTTG GGTTGCAGTC ATGTGGTGTT TAAΩTCTTGG 3439

AGAAGGCGAG AAGTCATTGC TGCCAGCATT GTGATCGTCA GGCACAGAAG TACTCAAAAG 3499

TGAGAGCTAC TTGTTGCGAG CAAACGGAGG GCGATATAGG TTGATAGCCA ATTTCAGTTC 3559

TCTATATΛCA ΛGCAGCGGAT TTTGTTTAGA GTTAGCTTTT GAGATGCATC ATTTCTTTCA 3619

CATCTGATTC TGTGTGTTGT AACTCGGAGT CGCGTAGAAG TTAGAATGCT AACTGACCTT 1679

AATTTTCACC GAATAATTTG CTAGCGTTTT TCAGTATGAA ATCCTTGTCT TAAAAAAAAΛ 3739

AAAAAAAΛ 3747

(2) INFORIATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH : 683 amino acids

(B ) TYPE : amino acid (D ) TOPOLOGY : linear

( i i ) MOLECULE TYPE : protein

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

Met Glu Cyr- Phe Gin Sei Aεn Leu Glu Lyε Met Glu Lye Leu Cye Aεn ) 5 10 15

Ser Aen Sei Cyε Lyε Gly Glu Leu Aεp Phe Lyε Ser He Leu He Aεn

20 25 30

Asn Aεp Tyr He Pro Tyr Aεp Glu Asn Ser Thr Gly Asp Ser Thr Aen 35 40 45

Leu Gly His Ser Lys Cys Ala Phe Glu Thr Leu Ala Ser Pro Thr Lys

50 55 60 Thr He Lyε Asn Met Leu Tin Val Pro Ser Ser Pro Leu Ser Pr Ala _b5 70 75 80

Thr Gly Gly Ser Val Lyε He Val Gin Met Thr Pro Val Thr Sei Ala

85 90 95

Met Thr Thr Ala Lys Trp Leu Arg Glu Val He Ser Ser Leu Pro Asp

100 105 HO

Lyε Pro Sei Se Lyε Leu Gin Gin Phe Leu Ser Ser Cyε Aεp Arg Asp

11 120 125

Leu Thr Asn Ala Val Thr Glu Aig Val Ser He Val Leu Glu Ala He lid 135 140

Phe i o Thr l.yε Ser Ser Ala Aεn Arg Gly Val Sei Leu Gly Leu Asn

14^c 150 155 160

Cyi Ala asn Ala Phe Asp He I'ro Trp Al,. Glu Ala A q Lyr, Val Glu

1 5 170 IT^r-

Ala Ser Ly_ Leu Tyr Tyi Arg Val eu Glu Ala He Cys Aig Ala Glu

180 185 110

Leu Gin Aεn Sei Asn Val Asn Asn Leu Thi Pro Leu Leu Sei Aen Glu

195 200 205

Aig Phe His Arq Cyε Leu He Ala Cyc Ser Ala Aεp Leu Val Leu Ala

210 215 220

Tin His Lys Thr Val He Met Met Phe Pro Ala Val Leu Glu Sei Thr

22 '-. 230 235 240

Glv Leu Thr Ala Phe Asp Leu Ser Lys He He Glu Aεn P e Val Arg

245 250 255

HID Glu Glu Thr Leu Pro Arg Glu Leu Lys Ar Hi" Leu Asn Sei Leu

26C 265 270

Glu Glu Gin Leu Leu Glu Ser Met Ala Trp Glu Lyε Gly Sei Sei Leu

275 280 2B^c

Ivi Asn 5eι Leu He Val Ala Arq Pro Ser Val Ala Ser Glu He Asn

Arcr Leu Gly Leu Leu Ala Glu Pro Met Pro Sei Leu Aep Aep Leu V l

305 310 315 320

Sei Λrg Gin Asn Val Arq He Glu Gly Leu Pio Ala Thi Pio Sei Lyπ

125 330 335

Lys Arg Ala Ala Gly Pro Asp Aεp Asn Ala Asp Pro Arq Sei Pro Lys

34C 345 350 Aiq Sei Cyε Asn Glu Ser Arq Asn Thr Val Val Glu Al g Asn Leu Gin

355 360 365

Thr Pro Pro Pro Lye Gin Ser His Met Val Ser Thr Ser Leu Lyε Ala 370 375 380

Lyo Cyε His Pro Leu Gin Ser Thr Phe Ala Ser Pro Thr Val Cys Aεn 385 390 395 400

Pro Val Gly Gly Asn Glu Lys Cys Ala Asp Val Thr He His He Phe 405 410 415

Phe Sei Lyr lie Leu Lyε Leu Ala Ala He Arg He Arg Aεn Leu Cys

420 425 430

OJu Arg Val Gin Cys Val Glu Gin Thr Glu Arg Val Tyr Asn Val Phe

435 440 445

Lvi- Gin i lr- Leu Glu Gin Gin Tin Thr Leu Phe Phe Asn Arq His lie 450 455 460

Aεp Gin Leu He Leu Cye Cyre Leu Tyi Gly Val Ala Lye Val Cys Gin

465 470 475 480

Leu Glu Leu Thr Phe Arg Glu He Leu Aεn Aεn Tyr Lyε Arg Glu Ala 4β5 490 495

Gin Cys Lyo Pro Glu Val Phe Ser Ser He Tyr He Gly Ser Thr Aen

500 505 510

Arg Asn Gly Val Leu Val Ser Arg His Val Gly He He Thr Phe Tyr 51^c 520 525

Asn Glu Val Phe Val Pro Ala Ala Lyε Pro Phe Leu Val Ser Leu He 530 535 540

Sei Sei Glv Thr His Pro Glu Asp Lys Lys Asn Ala Ser G y Gin He 545 550 555 560

Pro Gly Ser Pro Lyε Pro Ser Pro Phe Pro Aεn Leu Pro Asp Met Sei

565 570 575

Pro Lys iΛ's Val Sei Ala Ser Hiε Aεn Val Tyr Val Ser Pro Leu Arg 580 585 590

Gin Thr Lys Leu Asp Leu Leu Leu Sei- Pro Ser Ser Arq Ser Phe Tyr 59^r 600 605

Ala Cys He Gly Glu Gly Thr Hiε Ala Tyr Gin Ser Pro Ser Lyε Asp 61!i 615 620

Leu Ala Ala He Aεn Ser Arg Leu Aεn Tyr Aεn Gly Arq Lyε Val Ann 625 630 635 640 Ser Arg Leu Asn Phe Aεp Met Val Ser Asp Sei Val Val Ala Gly Ser 645 650 655

Leu Gly Gin He Λεn Gly Gly Ser Thr Ser Aep Pro Ala Ala Ala Phe

660 665 670

Ser Pro Leu Ser Lyε Lyε Arg Glu Thr Asp Thr

675 680

INFORMATION RELATIVE TO THE DEPOSIT OF A MICRO-ORGANISM The micro-organism to which reference is made in page 6 of the disclosure has been deposited in the following institution: COLECCION ESPANOLA DE CULTIVOS TIPO (CECT) Departamento de Microbiologia Facultad de Ciencias Biolόgicas 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 .

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule l3b )

For International Bureau use ooly

1 I This sheec was received by the International Bureau or_

Authoiizc officer

Claims

1. Use of a retinoblastoma (Rb) protein for the control of the growth and/or replication of plant cells and plant viruses.

2. Use as claimed in claim 1 characterised in that the virus requires the integrity of an LXCXE amino acid motif in one of its proteins for the normal reproduction.

3. Use as claimed in claim 1 wherein the virus is a Geminivirus.

4. Use in accordance with claim 1 characterised in that the virus binds a retinoblastoma (Rb) protein in order to release a transcription factor.

5. A method of controlling the growth and/or replication of a plant cell or a plant virus within that cell, comprising the increase or decrease of the level and/or activity of a retinoblastoma protein in that plant cell.

6. A method as claimed in claim 5 characterised in that the level of protein is increased by direct application.

7. A method as claimed in claim 5 characterised in that the level of protein is increased by introduction of DNA or RNA encoding for its expression into the plant cell which it is desired to treat.

8. A method as claimed in claim 5, 6 or 7 wherein the protein is overexpressed .

9. A method of controlling the growth and/or replication of a plant cell or a plant virus comprising expressing an Rb protein, or peptide fragment thereof that interacts with the LXCXE motif of the virus but does not affect the normal functioning of the cell, such as to inhibit cell growth or normal viral growth.

10. Recombinant nucleic acid encoding for expression of an Rb protein that has one or more characteristics of plant Rb protein not shared by animal Rb protein.

11. Nucleic acid as claimed in claim 10 characterised in that it comprises one or more characteristic regions that differ from known animal Rb protein nucleic acid.

12. Recombinant nucleic acid in the form of DNA or cRNA which encodes for a plant Rb protein having A and B pocket subdomains having a sequence with between 30% and 75% homology with human Rb protein.

13. Nucleic acid as claimed in claim 12 having a sequence with between 30% and 75% homology with p130 Rb retinoblastoma protein.

14. Nucleic acid as claimed in claim 12 or 13 characterised in that it has from 50% to 64% homology with animal or p133 Rb retinoblastoma protein.

15. Nucleic acid as claimed in any one of claims 12 to 14 encoding for the C706 amino acid of human Rb .

16. Nucleic acid as claimed in any one of claims 12 to 15 wherein the spacer sequence between the A and B pockets is not conserved with respect to animal Rb proteins.

17. Nucleic acid as claimed in claim 16 wherein the spacer sequence has less than 50% homology to the same region found in animal retinoblastoma proteins.

18. Nucleic acid as claimed in any one of claims 12 to 17 having 80% or more homology with that of SEQ NO 2.

19. Nucleic acid as claimed in claim 18 wherein the homology is 90% or more.

20. Recombinant DNA comprising a sequence corresponding to SEQ ID No 1 bases 31 to 2079.

21. Recombinant DNA comprising a sequence corresponding to SEQ ID No 1 or corresponding RNA encoding for maize cDNA clone encoding ZmRb1 of SQ ID No 2.

22. Protein encoded by the recombinant DNA or RNA as claimed in any one of claims 12 to 21 or novel proteins derived from such DNA or RNA, and protein derived from naturally occurring DNA or RNA altered by mutagenic means.

23. Protein as claimed in claim 22 wherein the mutagenic means comprises mutagenesis using mutagenic PCR primers.

24. Anti-sense DNA or RNA of a gene encoding for a plant retinoblastoma protein, a gene which possesses the nucleic acid sequence as the one which is claimed in any one of the claims 10 to 21.

25. Vectors, cells, plants or animals comprising the DNA or RNA as claimed in any one of claims 12 to 22.

26. A method to control the growth and/or the proliferation of a vegetable cell or of a plant virus comprising the decrease of plant retinoblastoma protein levels in the cell by incorporation to this cell of anti- sense DNA or RNA to the retinoblastoma protein.

27. cDNA encoding a protein as it is claimed in the claim 22.

28. A nucleic acid encoding a protein in which one or more of these sites are altered or deleted, making the protein more resistant to the phosphorilation and thus, to its functionality, for example, linking to E2F or similar.

29. An encoded protein by the nucleic acid which is described in claim 28.