FORM -2
THE PATENT ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION
(See Section 10)
COMPLETE GENE SEQUENCE OF COAT PROTEIN OF CHILLI LEAF CURL VIRUS AND
PLANTS TRANSFORMED THEREWITH THE GENE FOR CONFERRING RESISTANCE
AGAINST GEMINI VIRUS AND OTHER VIRUS DISEASES
RALLIS INDIA LIMITED
21 DS MARG, FORT
MUMBAI 400 001
MAHARASHTRA INDIA

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed.
FIELD OF INVENTION
The present invention relates to cloning and characterization of Coat Protein (CP) gene of Chilli Leaf Curl Virus (ChLCV). This invention particularly relates to employing this gene to produce transgenic plants, which will be resistant to viral infection. This invention more particularly relates to gene as a tool to obtain transgenic plants of the species Capsicum frutescens and Capsicum annuum, which will be resistant to viral infections.
The present invention also relates to plant cells which are transformed with the expression vector containing the coat protein gene, optionally in combination with the other sequences of genome, and to plant cells thus transformed which are regenerated into transgenic plants which express said genes from Geminiviruses.
The invention also relates, to seeds and progeny derived from the transgenic plants thus obtained.
Furthermore, the present invention relates to reproducible structures derived from a transformed plant cell or a transgenic plant resistant to Geminivirus, such as seeds, calli. buds, embryos etc. obtained from the transgenic plant or the progeny derived therefrom.
It would be observed that the present invention relates to the field of molecular biology especially relating to Geminivirus causing leaf curl disease in Capsicum frutescens (Chilli pepper or Hot pepper), and

Capsicum annuum (Bell pepper or Sweet pepper) in India, and most likely in many other countries in the world. The virus is commonly referred as Chilli leaf curl virus and is abbreviated as ChLCV or as CLCV also. Most of the Geminiviruses infecting dicot plants are bipartite and belong to group III of classification, which is also referred to as Begemovirus group, which are characteristically transmitted by whiteflies (Bemisia tabaci). Genome of these viruses is divided into two circular single stranded DNA molecules called DNA A and B, hence, referred to as 'bipartite' virus.
PRIOR ART
Plant viruses are a serious problem for many agricultural crops. Capsicum fnitescens (Chilli pepper or Hot pepper), and Capsicum annuum (Bell pepper or Sweet pepper) are two fruit vegetable crops that are grown in tropical and sub-tropical agricultural areas and under greenhouse conditions in moderate climates. Several viruses belonging to the group of the Geminivirus (Geminiviridae), which are DNA-viruses, and the group of the potyvirus (Potyviridae), which are RNA-viruses can heavily infect these crops. In several tropical countries more than ten different viruses belonging to these two groups can
infect Capsicum species.
Different viruses cause a leaf curling complex disease on chilli pepper in India, and the symptoms are described as "Murda syndrome". Geminiviruses transmitted by whiteflies are important contributing components in "Murda syndrome", which leads to severe yield losses.

One of the methods to control this devastating disease would be to develop transgenic chilli pepper and bell pepper varieties that are resistant to Geminiviruses. However, until now, none of the isolates, especially from India, has been characterized on a molecular level.
Chilli leaf curl Geminivinis has been reported to be one of the most prevalent viruses infecting chilli pepper in South India. Molecular characterization is an important aspect in designing strategies for developing transgenic plants resistant to viruses and it also helps to understand the mechanisms underlying the processes of virus infection. Therefore the objective of the invention was to obtain sequence information for the Geminivinis genes such as coat protein and replicase and use this infonnation either alone or in combination in the development of virus resistant plants.
OBJECTIVES OF THE INVENTION
The main objective of the present invention is to purify the DNA A of ChLCV belonging to Geminiviridae causing leaf and fruit damage in Capsicum fruiescens and Capsicum annuum.
Another objective of the present invention is to determine the partial nvicleotide sequence of the DNA A of the genome, to contain the coat protein gene sequence.
Yet another objective of the present invention is to develop transgenic plants more particularly plants belonging to the species Capsicum fruiescens and Capsicum annuum, which are resistant to viral infection.
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Yet another objective of the present invention is to obtain transgenic plants which are resistant to viral infection, more particularly belonging to the species Capsicum frutescens and Capsicum annuum.
The objective of the invention is to develop virus-resistant plant through pathogen-mediated resistance by expressing the coat protein gene sequence, as such or with some modifications or in combinations. It is observed that expressed viral sequences could interfere with virus life cycle and thus check viral multiplication and spread. Coat protein-mediated resistance has been demonstrated in several crop plants for a number of viruses. Particularly, resistance to a Geminivirus is demonstrated through expression of coat protein for Tomato yellow leaf curl virus (Kunik el al., 1994).
The above information provides the feasibility to produce transgenic plant cells and plants harbouring one or more DNA sequences at least 80% homologous to the coat protein gene, optionally in combination with other sequences of the genome.
In the present invention, DNA A of ChLCV virus is isolated; nucleotide sequence is partially determined and characterized. The invention more specifically relates to the use of the above information for obtaining plants resistant to the virus.
DISCLOSURE OF THE INVENTION
The present invention relates to development of a recombinant DNA molecule comprising one or more DNA sequences showing at least 80% homology to the coat protein gene, optionally in combination with other regions of Geminivirus genome, functionally linked with regulatory sequences needed for gene expression in plant cells.

The gene or genes comprised in the recombinant DNA molecule are in general derived from a Geminivinis affecting chilli, more in particular from ChLCV. However, similar genes with a high level of homology (> 80%) but originating from other Geminiviruses are expected to give the same results in plants belonging to Capsicum sp. On the other hand, introduction of the recombinant DNA molecule according to this invention into other plants, in particular, plants belonging to the Solanaceae family, and Malvaceae family, is expected to result in plants that are resistant to Geminiviruses with at least 80% homology with ChLCV DNA sequence Preferably, the recombinant DNA molecule is based on a binary vector thus facilitating Agmhacterinm-mediated transformation. For Agrohacterium- mediated transformation, the recombinant DNA molecule is engineered into a plant-expressible gene cassette, containing regulatory elements such as a plant-expressible promoter, a translation initiation codon (ATG) and a plant functional poly A addition signal (AATAAA). The gene cassette can be constructed in a suitable transformation vector containing the necessary sequences for obtaining expression of the recombinant molecule in plant tissue. A suitable transformation vector is, for example, pGA643.
"The invention employs the recombinant DNA molecule for transformation, preferably Agrobacteriumrmediatexi, of plant cells and plants in order to produce plants which are resistant to the particular Geminivinis from which the gene or genes is are derived, or to other Geminiviruses. However, the use of DNA molecule is not restricted to Agrobacterium-mediated transformation only.
In a preferred embodiment of the invention, the recombinant DNA molecule employed in the present invention, comprises a DNA sequence that is at least 80% homologous to the coat protein sequence of ChLCV as shown in Figure 1 (As shown in the drawing sheet No. 1). Preferably, the homology is at least 90%, more preferably at least 95%.

The process of producing transgenic plants which are carrying the DNA sequences that are at least 80% homologous to coat protein gene, optionally in combination with the other sequence of genome,'which comprises preparation of a recombinant DNA molecule carrying the above DNA sequences, transforming plant cells with the said molecule in order to obtain transgenic plant cells and regenerating complete plants out of the transgenic plant cells.
Plants that can be transformed according to the invention belong for example to the family Solanaceae and more specifically, the species Capsicum fniiescens (hot pepper). Capsicum annuum (bell or sweet pepper), Solamim tuberosum L. (potato), I.ycopersicon esculentum L. (tomato) and Solamim melongena L. (eggplant), and members of the family Malvaceae like Gossypium Sp. (cotton), Ahelmoschus esculentus (Okra/Bhendi/Lady's finger) and also Caricaceae: Carica papaya L (Papaya).
Accordingly the invention provides:
1. A recombinant DNA molecule characterized in that the sequences in ChLCV were different from the conserved sequences of Begomoviruses.
2. A recombinant DNA molecule wherein the CP (Coat Protein) 2 region was different from the Begomovirus sequences.
3. A recombinant DNA molecule wherein the coat protein gene sequence of ChLCV shows maximum homology of89% with Tomato leaf curl virus followed by Papaya leaf curl virus (86%), and Cotton leaf curl virus (85%).
4. A recombinant DNA molecule wherein the DNA sequences show at least 80% homology to the sequences as shown in Fig. 1 (As shown in the drawing sheet No. 1).

5. A recombinant DNA molecule wherein the DNA sequences show at least 90% homology to the sequences as shown in Fig. 1 (As shown in the drawing sheet No. I).
6. A recombinant DNA molecule, wherein the DNA sequences show at least 95% homology to the sequences as shown in Fig. 1 (As shown in the drawing sheet No. 1).
7. A transformed plant cell comprising a recombinant DNA molecule as described above.
8. Transgenic plants belonging to the family Solanaceae and more specifically the species Capsicum frutescens, C. annum, So/anum tuberosum, S. melongena and Lycopersicon esculentum comprising transformed plant cells as defined above.
9. Seeds of transgenic plant comprising transformed plant cells as defined above.
10 Seeds of Transgenic plants belonging to the family Solanaceae and more
specifically the species Capsicum frutescens, C. annum, Solanum tuberosum, Solamim melongena and Lycopersicon esculentum comprising transformed plant cells as defined above.
The details of the invention will be described in the following Examples, which are provided only to illustrate the invention and there/fore should not be construed to limit the scope of the invention.
Example 1
Isolation of double stranded Replicative form (RF) of DNA of ChLCV
Virus infected leaf material ( Murda symptoms, ChLCV) was collected for isolation of viral nucleic acid. Viral nucleic acid was isolated by following one or more methods like ultra centrifugation method as described by Hamilotn et al. (1982). or Dellaporta (Dellaporta et al. 1983) or Sinaloa (Doyle and Doyle 1987) or TES method by alkaline lysis.

Example 2
Generating a ChLCV specific probe
Using a set of primers (as shown below) based on the conserved region of the coat
protein (CP) gene of a number of whitefty-transmitted Geminiviruses, PCR
(Polymerase Chain Reaction) was carried out on the isolated DNA from infected
chilli leaves.
Forward Primer 5' ACA GGC CCA TGT ATA GGA AGC C 3' Reverse Primer 5' GAG GCA TGT GTA CAT GCC 3'
A predicted (based on literature and similar studies) fragment of about 500 base pair (bp) was amplified, representing the partial CP gene of Geminiviridae family. In this particular case a 550 bp was obtained; which was gel-eluted and used as probe in Southern hybridization. Further confirmation of the viral origin of this DNA was done by hybridizing with DNA from infected and healthy leaves of chilli and also with the CP gene region of cloned ICMV {Indian Cassava mosaic virus), wherein no hybridization was seen for the DNA from healthy chilli leaves.
Example 3
Cloning of 550 bp PCR product from Example 2
The 550 bp fragment was ligated to an lico RV digest of the vector pBS II KS+. Of the several colonies screened the one strongly hybridizing was chosen Authentication of the insert was carried out through PCR using KS and SK primers flanking the insert and restriction digestion. The clone was designated as pOSK 1. An amplification of about 550 bp was obtained, which in turn was used as probe in hybridization with infected and healthy chilli leaf DNA. Hybridization was observed only with the former, indicating that viral fragment has been cloned.

Example 4
Sequencing and Analysis of pOSKl
The sequencing was performed manually as well as by automated methods. Analysis of the sequence revealed that the clone was not complete. The clone lacked beginning and end of CP gene sequences, indicating that the clone was a partial clone.
Example 5
Attempts to Amplify full length of DNA A of ChLCV using abutting primers
Using abutting primers CLCV1 and CLCV2, a 2.7 Kb fragment of DNA was amplified representing the full length of ChLCV DNA A.
CLCV 1 (Forward) 5' AGC TGG AAA ATA TGA AAA TC 3' CLCV 2 (Reverse) 5' TCC TGC TGG TTA TAC ACA AC 3*
The viral nature of 2.7 Kb DNA was confirmed by its hybridization to DNA from infected chilli leaves and also to pOSKl clone. This amplification was dependent on the presence of intact viral DNA, which was possibly only from freshly infected leaf materia) However, this approach was not pursued fiirther due to the non-availability of freshly infected leaf material.

Example 6
Amplification of full lenpth CP gene
Based on sequence alignment of Begomo viruses (e.g. ICMV) two consensus degenerate primers CP1 and CP2 were designed.
Forward Primer CP1 5' ATG K*CS AAG CGW* CCR*GCA G 3'
Reverse Primer CP2 5' TY*A ATT GS*T B*AC H*GA ATC 3'
• As per IUPA code. K:G/T W:A/T R:A/G Y:C/T S:C/G
B: C/G/T H:A/C/T These primers failed to amplify the CP region from ChLCV. However, amplification was obtained with ICMV, giving the clue that the sequences in ChLCV may be different from the conserved sequences of Begomoviruses.
Example 7
Amplification of full length CP gene (contd.)
PCR was done using combinations of CP1 & CLCV2 and also CP2 & CLCVl primers. Combination of CP1 and CLCV2 could amplify the expected 620 bp product, but CP2 and CLCVl could not amplify the expected 250 bp fragment. It was concluded that CP 2 was the region different from other Begomovirus sequences. The amplified 620 bp product was cloned into pBS II KS + and was named as pRDl. Confirmation of the viral origin of this fragment was done by hybridization with pOSKl clone (representing partial coat protein), infected and healthy chilli leaf DNA.

Example 8
Sequencing of 620 bp fragment
Sequencing by both manual and automated methods revealed that this fragment contains the beginning region of CP gene and extends up to 624 bases. The dideoxy chain-terminating method was used for the manual sequencing (Sanger et a!., 1977).
Example 9
Cloning of 3' end region of CP gene
To clone the end region of CP, a primer (GEMREP) was designed based on the homology among Begomoviruses in the replicase gene. The strategy adopted was to amplify full length of CP beginning and extending into the conserved region of the replicase gene. This would ensure amplification of the end region (250 bp) of the CP gene. Using CPl and GEMREP primers, a 1.4 Kb product was amplified. Further the fragment was cloned in pBS II KS + and named as pRMPl.
GEMREP: 5' ATT AAA GGW* GGN* ATT CCC AC 3'
* As per IUPA code, W:AAT N:A/CGT
A 0.8 Kb fragment was obtained using CLCV1 and GEMREP primers. The 0.8 Kb fragment was cloned in pBS II KS + and named as pRMP2.
Example 10
Sequencing of 1.4 Kb fragment.
By packaging the pRMP 1 clone in VCS Ml 3 helper phage, the single stranded DNA was sequenced manually. The 1.4 Kb insert was also cloned into the vector pGEM-T (Promega) and sequenced by automated methods. Using the 1.4 Kb insert as template, PCR was performed with CPl and CP2 primers, under modified

conditions. This resulted in amplification of 0.8 kb fragment representing the CP gene of ChLCV. This fragment was cloned in pGEM-T vector designated as pRMP 3 and was sequenced, (using Ml3 forward and reverse primers). A large difference between sequences of both the strands (about 10% difference) was observed. Hence the clone was sequenced again at different facility using T 7 and SP 6 primers. Although both the strands matched here, the CP2 region was ambiguous when compared to the sequence of pRMPl clone.
To overcome this ambiguity, specific primers ChLCuVCPl and ChLCuVCP2 were designed to amplify true CP of ChLCV. To facilitate cloning of CP gene in the binary vector, restriction sites {Hind III in the forward primer and Bg] II in the reverse primer; which sequences are shown as underlined) were incorporated in the primer sequences.
ChLCuVCPl: 5' CCC AAG CTT ATG GCC AAG CGT CCG GCA G 3'
ChLCuVCP2: 5' GGA AGA TCT TTA ATT CGA TAC TGA ATC 3'
Using these primers and the 1.4 Kb fragment as the template, the expected 0.8 k Kb CP gene was amplified. The gene was cloned in to pGem-T vector and designated as pRMP6.
Example 11
CP gene cloning into Binary vector !l
The 0.8 Kb fragment released by restriction digestion was cloned into Binary vector and named as pRMP 7. However, sequencing of the clone detected a mutation at position 87 (Glycine to Arginine) from the start of the CP. It was assumed that the mutation could have resulted as an error in the PCR.

To overcome the mutation, PCR was performed using DNA from infected chilli leaves as template and ChLCuVCPl & ChLCuVCP2 primers. A 0.8 Kb fragment was obtained and cloned in pGem-T (pRMP 8) vector. On sequencing the pRMP 8 clone the mutation mentioned above was found to be eliminated. Further, the fragment was cloned into a binary vector and designated as pRMP 9 (Fig. 2, As shown in the drawing sheet No. 2). The clone was further mobilized into Agrobacterium tumefaciens for plant transformation.
Advantages of the invention:
1) Transgenic plants resistant to the virus, which can give improved yields.
2) Reduced use of insecticides to eliminate the insect vector. This would bring down the cost of farming and also would reduce the pollution caused by the insecticides.
3) As the sequence of ChLCV CP described here is related to a number of viruses causing diseases on tomato, papaya and cotton, these crops can also be protected from viral diseases by use of this CP gene and approach.
WE CLAIM
1) A recombinant DNA molecule characterized in that the sequences in ChLCV -were different from the conserved sequences of Begomovirus family. 2) A recombinant DNA molecule according to claim 1 characterized in that the CP 2 region was different from other Begomovirus sequences. A recombinant DNA molecule according to claim 1 & 2 characterized in that the coat protein gene sequence of ChLCV shows maximum homology of 89% with Tomato leaf curl virus followed by Papaya leaf curl virus (86%) and Cotton leaf curl virus (85%). ) A recombinant DNA molecule in which ChLCV CP having the sequence as shown in Fig 1 (As shown in the drawing sheet No. 1).

5) A recombinant DNA molecule as claimed in claim 4, wherein the DNA sequences show at least 80% homology to the sequences as shown in Fig. 1 (As shown in the drawing sheet No. 1).
6) A recombinant DNA molecule as claimed in claims 4 & 5, wherein the DNA sequences show at least 90% homology to the sequences as shown in Fig. 1 (As shown in the drawing sheet No. 1).
17) A recombinant DNA molecule as claimed in claim 4 to 6, wherein the DNA sequences show at least 95% homology to the sequences as shown in Fig: 1
(As shown in the drawing sheet No. 1). 8) A transformed plant cell comprising a recombinant DNA molecule as claimed
in claims 4 to 7. f^P A transgenic plant belonging to the family Solanaceae and more specific the
species Capsicum frutescens, C. annum, Solanum tuberosum, S. melongena and Lycopersicon esculentum comprising transformed plant cells as claimed in
claims 4 to 7.
10) Seeds of transgenic plant as claimed in claims 8 & 9.
11) A process for producing transgenic plants which are resistant to viral infection, which comprises transforming plant cells with the recombinant DNA molecule as claimed in claims 4 to 8.
12) A process as claimed in claim 9 wherein, the plant cells used is selected from the species Capsicum frutescens, Capsicum annum, Solanum tuberosum, S melongena and Lycopersicon esculentum.
13) A recombinant DNA molecule substantially as herein described.

14) A transfonned plant cell substantially as herein described.
15) Seeds of transgenic plant substantially as herein described.
16) A process for producing transgenic plants which are resistant to viral infection' substantially as herein described.
Dated this .....'21th day of July, 2003

(M S MITHYANTHA) Vice President (Res. & Dev.)
RALLIS INDIA LTD

(K. DHARMALINGAM) Secretary MKU-ICG
MADURAI KAMARAJ UNIVERSITY SECRETARY MKU-1CC M.K. UNIVERSITY