BRPI0611815B1 - METHOD TO INCREASE SUGAR MOSAIC VIRUS RESISTANCE AND SUGAR MOSAIC VIRUS RESISTANT PLANTS - Google Patents

Abstract

method for increasing plant resistance to sugarcane mosaic virus and plants resistant to sugarcane mosaic virus. The present invention relates to a method of increasing plant resistance to sugarcane mosaic virus (SCMV) infection. such a method comprises transforming or transfecting said plant with an expression vector comprising a promoter operably linked to a nucleic acid molecule encoding (i) a protein having properties of said scmv protein coat, or (ii) an rna molecule antisense complementing the positive rna strand of the protein coat gene or (iii) a dsrna hairpin molecule for the protein coat gene of said scmv, wherein expression of said expression cassette inhibits scmv infection. The present invention also relates to a transgenic plant or a part, propagule or progeny thereof that is SCMV tolerant, resistant or immune. the genome of such a plant comprises one or more copies of a sequence of nucleic acid molecules encoding (i) a protein having protein coat properties of said scmv or (ii) an antisense mRNA molecule complementing the mRNA positive strand of the protein coat gene or (iii) a dsrna hairpin molecule for the protein coat gene of said scmv.

Description

(54) Title: METHOD FOR INCREASING THE RESISTANCE OF THE PLANT TO THE SUGAR CANE MOSAIC VIRUS AND PLANTS RESISTANT TO THE SUGAR CANE MOSAIC VIRUS (51) Int.CI .: A01H 5/00; C12N 15/82; C12N 15/33; C12N 15/113 (30) Unionist Priority: 06/30/2005 US 60 / 695,935 (73) Holder (s): MONSANTO DO BRASIL LTDA.

(72) Inventor (s): JESUS APARECIDO FERRO; PATRÍCIA BRANT MONTEIRO

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Invention Patent Descriptive Report for: METHOD

TO INCREASE THE PLANT'S RESISTANCE TO MOSAIC VIRUS

OF SUGAR CANE AND PLANTS RESISTANT TO MOSAIC VIRUS

SUGAR CANE.

RELATED ORDER

This order claims priority of order serial number US 60 / 695,935 filed on June 30, 2005, and incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is generally related to the field of molecular biology, biochemistry, phytopathology and agriculture. More specifically, the invention relates to the genetic engineering of monocotyledonous plants to resist strains of Sugarcane Mosaic Virus (SCMV) through overexpression of the SCMV protein layer (CP) or the expression of its inhibitory transcript.

BACKGROUND OF THE INVENTION

Resistance to viruses introduced into plants through genetic engineering began with the development of resistance to tobacco mosaic virus (TMV)

Tobacco Mosaic Virus) in Nicotiana tabacum transformed with nucleic acid molecules that encode the protein layer (CP) of TMV (Powell-Abel et al., Science 232: 738743, 1986). In this particular case, called

2/34 resistance mediated by CP (CP-MR), it is believed that the protein layer encoded by the transgene inhibits TMV infection by preventing the disassembly of the protein layer units ..... from virion at the entrance of the virus (Lu , B, et al., Virology 248: 188-198, 1998; Beauchy, RN, Philosophical

Transactions of the Royal Society of London Biological

Sciences 354: 659-664, 1999). CP-MR is moderately specific and experiments show a correlation between resistance and the degree of similarity of the CP amino acid sequence in transgenic plants and the CP of virus that infects the plant. It has been found in some cases that a 60% identity between the protective CP sequences expressed in transgenic plants and a virus CP can provide sufficient conformational similarity for the protective CP to block the layer removal process (Nejidat,

A. and Beachy, R. N., Mol. Plant-Microbe Interact. 3: 247251, 1990). Resistance to infection by several viruses in a number of plant species has been achieved by CP-MR (Lawson C. et al., Bio / Technology 8, 127-134, 1990;

Fítchen, J. H. and beachy, R. N. Annu. See. Microbiol. , 47:

739-763, 1993; Hackland, A. F. et al., Arch. Virol., 139:

1-22, 1994; Lomonosossoff, G. P. Annual Review of Plan

Pathology 33, 323-343, 1995; Wilson Τ. Μ. A., Proc. Natl.

Acad. Sci. USA, 90: 3134-3141, 1993).

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However, it was found that in several tobacco plants transformed with TMV, higher levels of resistance were associated with low or undetectable levels of CP protein production (Lawson C. et al., Bio / Technology 8, 127-134 , nineteen ninety). Resistance to TMV could even be achieved using antisense or non-translating constructs from the CP gene (Lindbo, J.A.

et al. Molecular Plant Microbe Interactions, 5: 144-153,

1992; Lindbo, J. A. et al. , Virology, 189: 725-733, 1992;

Lindbo, J. A. et al, The Plant Cell 5: 1749-1759, 1993;

Smith, Η. A., The Plant Cell, 6: 1441-1453, 1994), suggesting that resistance could be mediated by RNA. The evidence for RNA-mediated resistance leads to a discovery in which the resistance of the transgenic plant to viruses could result from homology-based gene silencing, a surveillance mechanism induced by cellular RNA against viruses containing sequences homologous to the transgene (for reviews see Baulcombe, CD Current Opinion in Plant Biology 2: 109-113, 1999; Marathe, R. et al., Plant Molecular Biology, 43: 295-306, 2000;

Waterhouse, P.M. et al., Nature 411: 834-842, 2001; Lu R. et al., Methods 30: 296-303, 2003; Burch-Smith, T.M. et al.,

The Plant Journal 39: 734-746, 2004).

Since RNA-mediated resistance is sequence 0 * X \ J

4/34, viruses not very similar to the transgenic sequence may not be targets for silencing. In addition, they can cause reversal of silencing, as revealed by the recovery of marker expression in transgenic plants supposedly silenced after virus infection (Anandalakshmi, R. et al., Proc. Natl.

Acad. Sci USA 95: 13079-13084, 1998; Brigneti G. et al.,

EMBO J., 1Ί ·. 6739-6Ί46, 1998); Kasschau, K. D. and

Carrington, J. C., Cell, 95: 461-470, 1998; Voinnet, O., et al., Proc. Natl. Acad. Sci. USA, 96: 14147-14152, 1999). The defense of the virus against silencing is mediated by proteins that suppress viral RNA silencing (reviewed in Carrington, J. C. et al., Virology 281 (1): 15, 2001).

The level of expression, number and configuration of the integrated transgenes, as well as other environmental and development factors that are much less understood, may influence the occurrence of gene silencing based on homology. Resistance in transgenic plants to viruses has often been shown to be based on this mechanism (for reviews see Baulcombe, D.C. Plant Cell,

8: 1833-1844, 1996; Ratcliff, F.R. et al., The Plant Cell,

11: 1207-1215, 1999; Ratcliff, R. et al., Plant Molecular

Biology, 43: 295-306, 2000).

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RNA silencing essentially acts to destroy transcripts of exogenous genomic material, so as to inhibit the replication and spread of viral particles and the development of viral disease (Vance, V. and Vaucheret, H.,

Science, 292: 2277-2280, 2001; Vaucheret, H. et al., J.

Cell.Sei, 114: 3083-3091, 2001; Waterhouse, P. M. et al. ,

Nature, 411: 834-842, 2001). The key characteristic of RNA silencing is that it is caused by the formation of the double RNA strand (dsRNA), which is cleaved into small interference RNAs (siRNAs), 21 to 25 nucleotides in length, which subsequently direct sequence degradation homologous (ie, RNA sequences encoded by the viral genome or in case of silencing of the transgene encoded by the transgene and / or endogenous genomic DNA).

It is evident that RNA viruses are capable of generating siRNA, which can trigger the development of RNA silencing within a host plant (Voinnet, O. et al., Proc. Natl. Acad. Sci. USA 96: 1414714152, 1999 ). Although the presence of an exogenous dsRNA is a strong trigger for RNA silencing, plant cells also appear to have the ability to recognize aberrant single-stranded RNA molecules from different sources and then direct the synthesis of dsRNA homologues

6/34 or siRNA, which serve as triggers for RNA silencing.

In some transgenic plants, the virus can be both an inducer and a target for gene silencing. The lower leaves of these plants show normal viral symptoms, however the upper leaves that emerge after systemic infection are asymptomatic and virus-free. These upper leaves are resistant to secondary infection by virus induction and are considered 10 recovered (Lindbo et al., The Plant Cell, 5: 1749-1759,

1993; Guo H. S. et al., Gene, 206 (2): 263-72, 1997).

The expression of viral protein coat transcripts, which have been modified to be unable to be translated into a (untranslated) polypeptide, have shown the ability to confer resistance to several viruses. The example of this, but by no means absolute, is Potato Virus Y (PVY) in potatoes (Van der Vlugt, R. A. et al., Plant Mol. Biol. 17: 431-439, 1991); Tobacco Etch

Virus (TEV) in tobacco (Lindbo, JA, et al., Mol. Plant 20 Microbe Int. 5 (2): 144-153, 1992; Publication of PCT application No. WO 93/17098 to Dougherty, WG et al. , September 2, 1993); Tomato Spotted Wilt Virus in tomatoes (Pang, S et al., Bio / Technology 11: 819-824, 1993; DeHaan et al.,

Bio / Technology 10: 1133-1137, 1992); Sorghum Mosaic Virus (Q>

7/34 (SMVr) in sugar-ethanol (Ingelbrecht, I.L. et al. Plant Physiology 119: 1187-1197, 1999); Maize Dwarf Mosaic Virus (MDMV) on maize (US Patent No. 6,040,496); and

Papaya Ringspot Virus on papaya (US Patent 6,750,382).

Cane Mosaic Virus (SCMV) is a member of the genus

Potyvirus in the family Potyviridae. The genus Potyvirus is the largest and possibly the most economically important group of plant pathogenic viruses. Potyvirus includes Papaya Ringspot Virus, Watermelon Mosaic Virus II (WMVII) and Watermelon Mosaic Virus I (PRV-p and PRV-w, two different strains of the same virus), Zucchini Yellow Mosaic Virus (ZYMV), Potato Virus Y, Tobacco Etch Virus , Take to Etch

Virus, Plum Pox Virus and others. These viruses have a long, flexible, rod morphology, approximately 780x12 nanometers in size and are transmitted by aphids non-persistently (see Hollings, M. and Brunt, A. pages 732-807 of Handbook of Plant Virus Infection and Comparative Diagnosis, 'id. By E. Kurstak, Elsevier / North Holland Biomeduical Press, Amsterdam, 1981). Potyviruses have a genome composed of a single strand of positive sense messenger RNA of approximately 10 kb. The Potyvirus genome is translated into a single large polyprotein of about 330 kDa which is subsequently cleaved into its components by plant and virus proteases.

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One of the proteins contained within this polyprotein is a 35 kDa capsid or protein layer that coats and protects viral DNA from degeneration. The protein layer is also involved in the transmission by the vector insect, in the cell-cell movement of the virus particles and in the regulation of the formation of the virus genome replication complex.

SCMV is an important pathogen of sugarcane (Saccharum L. interspecific hybrids), occurring in many parts of the world where susceptible host species are cultivated. SCMV causes mosaic disease and loss of yield in sugar cane, corn, sorghum, and other plants of the Poaceae family. The SCMV complex consists of four distinct Potyvirus (Shukla DD, et al., The sugarcane mosaic virus subgroup; In The Potyviridae. CAB International, Wallingford, ÜK, pp360-371, 1994) including strains of Johnsongrass Mosaic Virus (JGMV ), Maize Dwarf Mosaic Virus (MDMV), Sorghum Mosaic Virus (SrMV) Sugarcane Mosaic Virus (SCMV) (Yamg, ZN and Mirkov, TE Phytopathology 87: 932-939, 1997). SCMV has a monopartite positive sense single-stranded RNA genome, which is polyadenylated in the final 3 'portion (Shukla DD et al., The sugarcane mosaic virus subgroup; In The Potyviridae. CAB International, Wallingford, UK, pp360- 371, 1994).

In sugar cane, strains of SCMV and SrMV are the majority

9/34 of the widespread viruses, and can significantly reduce sugar cane production and sugar yield. In the mid-1920s, epidemics caused by members of the SCMV subgroup were responsible for the near collapse of the industry

Brazilian, Argentina and Louisiana (USA). Although these epidemics were controlled by the introduction of resistant sugarcane cultivars, strains of SCMV and SrMV remain a potential threat to the industry (Koike, H. and Gillaspie, AG Jr In: Diseases of sugarcane. Major diseases. Ricaud, C., Egan BT, Gillaspie Jr, AC and Hugues, CG (eds), p.301-322. Amsterdam, The Netherlands,

Elsevier Science Publishers B.V., 1989).

The generation of transformed plants expressing both the MDMV protein coat and the untranslated form of the messenger RNA 15 of the MDMV protein coat has shown the ability to confer resistance to A, B and SC strains from

MDMV in corn (US patent 5,428,144; US 5,530,193 and ÜS 6,040,496). In addition, SrMV-resistant sugarcane plants transformed with an untranslated form of the SrMV strain SCH CP gene depends on the homology-dependent mechanism mediated by RNA (Ingelbracht, IL et al., Plant Physiology 119: 1187-1197 , 1999). This transgenic sugarcane plant is resistant to SrMV, but not to SCMV. Notably, the

10/34 sequence of identity among their corresponding CP genes is only 69%, which could impair the homology-dependent RNA silencing mechanism.

In the present invention, constructs expressing the SCMV CP gene 5 in the positive sense and negative sense orientations were used to genetically engineer SCMV-resistant sugarcane plants.

SUMMARY OF THE INVENTION

The present invention relates to a method to increase the resistance of plants, preferably in monocotyledonous plants, more preferably still in sugar cane, to infection by the Sugarcane Mosaic Virus (SCMV). Such a method comprises transforming or transfecting said plant with an expression vector comprising a promoter operatively linked to a nucleic acid molecule that encodes (i) a protein having properties of the protein cover of said SCMV, (ii) an antisense RNA molecule complementary to the positive RNA strand of the protein coat gene or (iii) a hairpin dsRNA molecule for the protein coat gene of said SCMV, where the expression of said expression cassette inhibits infection by

SCMV.

The present invention also relates to a transgenic plant or a part, propagule or progeny thereof

11/34 that is tolerant, resistant or immune to SCMV. The genome of such a plant comprises one or more copies of the sequence of a nucleic acid molecule that encodes (i) a protein having properties of the protein cover of said SCMV, (ii) an antisense RNA molecule complementary to the positive RNA strand of the gene of the protein coat or (iii) a dsRNA hairpin molecule for the protein coat gene of said SCMV, where said plant is tolerant or immune to SCMV.

Still within the scope of this invention are 10 expression vectors designed to express, respectively, the antisense RNA molecules or sense of the virus protein coat gene that infects the plant, as well as plants containing such antisense or sense constructs permanently integrated into its genome. Such plants and their progeny are protected from virus infection that produces sense messenger RNA strand (which shares sequence identity with the modified RNA molecule expressed by the integrated stable antisense sequence), or from virus infection that expresses a protein coat with at least 60% if the amino acid sequence is similar to the protein layer encoded by the transgene overexpression.

Methods to increase the plant's resistance to infection with SCMV by transforming it with constructs containing the nucleic acid molecules of the cover «2 /

X

12/34 SCMV proteins are part of the present invention, according to the transgenic plants and their resulting progeny.

Such methods include introducing expression vectors containing at least one nucleic acid molecule from SCMV CP into plant parts, as well as into susceptible commercial varieties of sugarcane or any other germplasm and parental strains used in breeding programs. conventional that can be used to create new varieties that carry and express the buildings that give resistance.

The invention additionally relates to transgenic plants, such as plants of the genus Sacharum, in which SCMV CP nucleic acid molecules have been introduced by different constructs. Examples of these plants are all hybrid cultivars of Sacharum sp. Plants can be a parental line. The transformed plants comprise a selection marker such as a herbicide resistance gene.

BRIEF DESCRIPTION OF THE FIGURES

The invention can be readily understood by reference to the drawings:

Figure 1 shows a map of the plant expression cassette (pCANA) containing the corn promoter Ubiquitina-1,

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13/34 a multiple cloning site (MCS) and the NOSPolyA transcription terminator. The multiple cloning site has the following enzyme restriction sites

PacI-AscI-AvrlI-Pmel-Sgfl-BstEII-BamHI (Seq ID No. 8).

Figure 2 shows a map of the plant expression cassette (pCANA) carrying the sense sequence of the SCMV CP cDNA under the control of the UBI-1 promoter.

Figure 3 shows a map of the plant expression cassette (pCANA) carrying the antisense sequence of the SCMV CP cDNA under the control of the UBI-1 promoter.

Figure 4 shows a DNA gel analysis of patterns of integration of the CP transgene and number of copies in transgenic sugarcane (samples 1,2 and 3). The DNA gel with total DNA digested with Barriül and hybridized with a fragment of the 875 bp SCMV-CP gene marked with P ³² .

Figure 5A shows an RT-PCR agarose gel analysis of the level of expression of transgenic antisense CP transcribed in transgenic sugarcane plants. Figure 5 B shows an RT-PCR analysis of the expression of the endogenous sugarcane actin gene. Control reactions include RT-PCR from an unprocessed and uninoculated plant (NT) and as a positive control (+ C), the expression cassette pUBI :: CPas was used as a template.

DETAILED DESCRIPTION OF THE INVENTION £

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In the description that follows, several terms are used extensively. The following definitions are provided to facilitate understanding of the invention.

A gene is a region of DNA, a sequence of polynucleotides, having a sequence that is transcribed into messenger RNA (mRNA). The polynucleotide sequence can be cloned in the sense or antisense orientation. In the present invention, the coding sequence component comprises a sequence that, when transcribed, produces an RNA molecule corresponding to a target viral sequence.

As used here, a promoter includes reference to a region of DNA upstream from the start of transcription and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A plant promoter is a promoter capable of initiating transcription in plant cells. The promoter component can be any promoter that is capable of regulating and directing the expression of an operably linked gene in the target monocot plant.

The term expression refers to the biosynthesis of a gene product. For example, in this case, expression involves transcribing the gene into mRNA or translating it into the protein that has properties of an SCMV protein coat.

A vector is a DNA molecule, such as a plasmid, <2Η

15/34 cosmid or bacteriophage, which has the ability to replicate autonomously in the host cell. Vectors typically contain one or more restriction endonuclease recognition sites in which exogenous DNA sequences can be inserted in a determined way without loss of an essential biological function of the vector, as well as a marker gene that is appropriate for use in identifying and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline, ampicillin or kanamycin.

An expression vector is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory regions, including constitutive or inducible promoters, · tissue-specific regulatory regions and enhancers. Such a gene is said to be operationally linked to regulatory regions.

A transgenic plant is a plant that comprises a region of DNA or a modification of the DNA introduced as a result of the transformation process.

The term introduced, in the context of inserting a nucleic acid molecule into the cell, means transfection, transformation or transduction and includes

16/34 reference the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated within the cell's genome (eg, chromosome, plasmid, plastid, mitochondrial DNA) converted into an autonomous replicon or transiently expressed (for example, transfected mRNA).

As used here, the term plant includes reference to whole plants, plant organs (for example, leaves, stem, roots, etc.), seeds, plant cells and their progeny. Plant cell, as used here, includes, but is not limited to, seeds, suspension cultures, embryos, meristemic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.

For the purpose of the present invention, a pathogen-tolerant plant is one that is capable of tolerating the attack of a pathogen better than the wild-type plant, but will generally succumb to infection or die under conditions other than a mild disease. The resistant plant is a plant that has the ability to exclude or overcome the growth or effects of the pathogen, except under extremely high pressure from the disease. An immune plant is one that is able to be completely resistant to disease, without reacting plant tissue to a potential pathogen.

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The present invention relates to methods for increasing the resistance of the plant, preferably a monocot, by using isolated nucleic acid molecules and nucleic acid constructs comprising a SCMV CP polynucleotide sequence. Included within this invention are expression vectors designed to express antisense or sense RNA molecules of the SCMV CP gene in the plant, as well as plants containing such stable antisense or sense constructs integrated into its genome.

The present invention relates to an expression vector in plants. In the present invention, an expression vector comprises a promoter operatively linked to SCMV CP polynucleotide sequences in antisense or sense orientation. The promoter may be a specific organ or tissue promoter, and may be an inducible or constitutive promoter. The expression vector and the selection marker gene (which confers resistance to a selective agent under the control of a promoter expressed in plants) are introduced into the cells of the plant. Finally, resistant plants are regenerated from cells or tissues resistant to the selective agent. Candidates can also be obtained without using a selection marker.

As an example, the expression vector is introduced into the

18/34 να plant cells without the accompanying selection marker gene.

The invention thus relates to methods of transforming plants, such as monocots, with constructions containing SCMV CP polynucleotide sequences in antisense or sense orientation, to produce plants that are more resistant to plant viruses.

Numerous methods for introducing exogenous genes into plants are known and can be used to insert a gene into a host plant, including physical and biological plant transformation protocols. The methods chosen vary according to the host plant and include chemical transfection methods such as phosphate and calcium, microorganism-mediated gene transfer, such as Agrobacterium (Horsh, et al., Science, 277: 1229-31, 1985), electroporation, micro-injection and biobalistic bombardment.

A plant transformation method generally applicable is microprojectile-mediated transformation, where DNA is deposited on the surface of microprojectiles that measure about 1 to 4 μτη. The expression vector is introduced into the tissues of the plant with a biobalistic device that accelerates the microprojectiles at a speed of 300 to 600 m / s, which is sufficient to penetrate the $

19/34 plant cell walls and membranes (Sanford et al.,

Part Sci. Technol., 5: 27-37, 1987; Sanford Trends Biotech,

6: 299-302, 1998; Sanford, Physiol. Plant 79: 206-209,

nineteen ninety; Klein et al., Biotechnology, 10: 286-291, 1992).

The most used method to introduce an expression vector into plants is based on the natural transformation system of Agorbacterium. A. tumefaciens and A. rhizogenes, plant-pathogenic soil bacteria that genetically transform plant cells. The plasmids Ti and Ri of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for the genetic transformation of plants. Descriptions of the Agrobacterium vector system and methods for transferring Agrobacterium-mediated genes are provided in Gruber et al.,

1993 Vector for Plant Transformation In: Methods in Plant

Molecular Biology and Biotechnology. Glick and Thompson, eds. CRC Press, Inc., Boca Raton, pages 89-119.

Methods of the present invention include introducing constructs containing SCMV CP polynucleotide sequences into any or all parts of the plant.

Alternatively, since a single transformed plant has been obtained by recombinant DNA method, conventional methods of producing plants can be used to transfer the gene and associate regulatory sequences

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20/34 via crossing and retro-crossing. Such intermediate methods will comprise the additional steps of (1) sexually crossing the disease-resistant plant with the disease-susceptible taxon plant; (2) recover the reproductive material from the progeny of the crossing; and (3) to develop disease-resistant plants from reproductive material.

For the construction of the expression vector, polyA-RNA extracted from leaves of different varieties of sugarcane, collected in different areas of mosaic epidemic in Brazil, was used for RT-PCR together with a pair of primers SCMV-specific, SCMV-f4 (5'gttttycaccaagctggaacagtc 3 ') (SEQ ID No: 1), and SCMV-r3 (5' agctgtgtgtctctctgtattctc 3 ') (SEQ ID No; 2) (Joy,

O.M. et al. Arch Virol. 148: 357-72, 2003). SCMV-Í4 hybridizes at the junction of the open reading windows (ORFS) Nlbs and CP of the SMCV which encodes, respectively, an RNA dependent on RNA polymerase (RdRp) and the protein coat, while SCMV-r3 hybridizes in the 3 'region of CP gene .

875 bp fragments of the protein coat (CP) gene from different isolated sugarcane mosaic viruses were amplified. The PCR products from the region that encode the protein coat of different isolated SCMVs were cloned into pGEM-T vector and sequenced, and then via BLASTX

TO

21/34 and BIASTN, these exhibited high similarity with the SCMV protein cover gene.

SEQ ID No: 1, referred to above (Alegria, O.M. et ..... al.

Arch Virol. 148: 357-72,2003), was additionally used in conjunction with an oligo dT (23 nt) for cloning the complete SCMV CP gene by RT-PCR from polyA RNA from leaves of sugarcane plants sick sugar showing mosaic symptoms. The sequenced cDNA clones contained the complete SCMV protein coat gene and a 3'-UTR region of the SCMV genome (nucleotide sequences 14-943 and 944-1135 of SEQ ID No: 3, respectively). The amplified fragment containing the complete CP cDNA was cloned both in the antisense and sense orientation into a plant expression vector for expression of the SCMV CP derived from the antisense RNA or for overexpression of the protein coat protein, respectively. Sugarcane plants transformed with both vectors were regenerated and after virus inoculation tests, plants resistant to SCMV were selected.

All technical terms used here are terms commonly used in biochemistry, molecular biology, phytopathology and agriculture, and will be understood by a technician in the subject to which this invention belongs. Technical terms can be found in, for example,

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Molecular Cloning: A Laboratory Manual, 3rd ed. , vol. 1-3, ed. Sambrook and Russel, Cold spring Harbor Laboratory Press,

Cold Spring Harbor, NY, 2001; Current Protocols i-n

Molecular Biology, ed. Ausubel et al., Greene Publishing Associates and Wiley-Interscience, NY, 1988 (with periodic updates); Short protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology. 5 * ed., Vol. 1-2, ed. Ausubel et al. , John Wiley & Sons, Inc., 2002. Methods involving plant molecular biology techniques are described here and are described in detail in methodology treaties such as: Methods in Plant Molecular Biology: A Laboratory Course Manual, ed. Maliga et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995. Various techniques using PCR are described, for example, in Innis et al., PCR

Protocols: A Guide to Methods and Applications, Academic Press, San Diego, 1990 and in Dieffenbach and Dveksler, PCR Primer: A laboratory Manual, 2 'ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003. Peers of PCR primers can be derived from sequences known by known techniques such as using computer programs intended for this purpose {eg Primer, Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge,>

% -V

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MA). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Caruthers (1981)

Tetra. Letts. 22: 1859-1862 and Matteucci and Caruthers (1981)

J. Am. Chem. Soe. 103: 3185.

EXAMPLES

The present invention is further illustrated by the following specific examples. The examples are provided by way of illustration and should not be construed to limit the scope or content of the invention in any way.

EXAMPLE 1

Synthesis of cDNA and cloning procedures of the SCMV protein coat gene.

This example describes the identification and cloning of the nucleic acid molecule encoding the Protein Cover and the 3'UTR region of SCMV by RT-PCR. mRNA was extracted from sugar cane leaves expressing symptoms of

SCMV. The first cDNA strand was synthesized, after denaturing 500 ng of mRNA at 94 ° C for 5 min. In a final reaction volume of 12 μΐ, using 500 ng of oligo dT, and 1.2 mM dNTP mix. The solution was then incubated on ice and 4 μΐ of 5x First-Strand buffer (250 mM Tris-HCl, pH

8.3, 375 mM KC1, 15 mM MgCl ₂ ), 2 μΐ of 0.1 M dtt and

5.3

24/34 μΐ of RNaseOUT (40 units / μΐ) were added to the tube. The mixture was incubated at 42 ° C for 2 min., 1 μΐ (200

U) of the SuperScript II reverse transcriptase enzyme was added and the tube was incubated again for 90 min. The

42 ° C. The reaction was inactivated by heating at 70 ° C for 15 min. Two units of RNase H were added and the mixture was incubated at 37 ° C for 20 min. For PCR, 1 μΐ of the first cDNA strand was used as a sample, 0.5 μΜ of each primer (oligo dT primer and SEQ ID No: 1, which is specific), 0.2 mM of each dNTP, 0.2 mg / ml BSA, 2 mM MgCl ₂ ), IX of the reaction buffer (20 mM Tris-HCl, pH 8.4, 50 mM KC1), and 1 U of Taq polymerase. The reaction was heated for 3 min at 94 ° C and 40 cycles of amplification (60 s to 94 ° C, 60 s to 42 ° C, 90 s to 72 ° C) and a final incubation at 72 ° C for 10 min. DNA fragments were separated on a 1% agarose gel, stained with 100 ng / ml ethidium bromide and analyzed under UV light. The 1,135 bp band generated by RT-PCR was cloned into a pGEM-T vector and sequenced using an ABI 3700 sequencer.

BLASTX and BLASTN against a local NCBI NR database showed similarity with the SCMV protein coat sequence (nucleotide sequence 14-943 of SEQ ID No.: 3) and the 3'UTR region of SCMV (nucleotide sequence 944-1135 of

Λ

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SEQ ID NO .: 3).

EXAMPLE 2

Construction of plant transformation vector

Numerous transformation vectors are available for plant transformation, and the gene in the antisense or sense orientation used in this invention can be used in conjunction with any of such vectors. The selection of vector for use will depend on the preferred transformation technique and the target species for transformation. For certain target species, different herbicide or antibiotic selection markers can be used. Selection markers used routinely in the transformation including the nptll gene that confers resistance to kanamycin and related antibiotics (Messing and Vieira, Gene 19: 259268, 1982; Bevan et al., Nature 304: 184-187, 1983), the hph gene that confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), the bar gene that confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res. 18: 1062,

nineteen ninety; Spencer et al., Theor. Appl. Gener 79: 625-631, 1990) and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2 (7): 1099-1104, 1983).

The SCMV protein cover gene does not contain the necessary translation and transcription signals for its expression

26/34 when transferred and integrated within the plant genome, since it is part of the polyprotein encoded by the viral genome. For the expression of the SCMV protein cover in the plant cell, in addition to the need for a promoter that confers expression in plants, a SCMV CP gene must be engineered to contain the ATG initiation codon followed by a eukaryotic translation initiation signal, for allow a stronger ribosomal bond for efficient transduction and a higher level of expression of the transgenic protein in plant cells (see Kozak, M. Cell 44: 283-292,1986 incorporated by reference). To engineer the SCMV CP gene, specific primers derived from SVMC CP cDNA, isolated and cloned, were designed. The following fCPXKM {5 'gctctagaccaccatggctggaacagtcgatgcag 3') primer (SEQ ID No .: 4) contains (i) an Xbal restriction site (3-8 nucleotides), (ii) a Kozak consensus sequence (8-17 nucleotides) and (iii) an ATG initiation codon (14-16 nucleotides) in the first position of the codon of the CP coding sequence. The reverse primer rCPX (5'gctctagactagtggtgctgctgcactc 3 ') (SEQ ID No.: 5) contains an Xbal restriction site (3-8 nucleotides) immediately after the TAG termination codon (9-11 nucleotides).

The 0.939 kb Cp cDNA fragment was amplified

27/34 by PCR using SEQ ID Nos. : 4 and 5, subcloned into a pGEM-T vector and sequenced. This is determined in SEQ ID N: 6. The 310 encoded amino acid sequence of the transgenic CP protein is determined in SEQ ID No .: 7. To engineer the plant expression vector, the modified complete CP cDNA was digested with Xbal and cloned in pCANA treated with Avrll (Fig. 1).

Constructions containing the CP cDNA cloned in sense and antisense orientations in relation to the OBI-1 promoter were selected. The resulting cassettes DBI1:: CPaS:: NOSpolyA (Fig. 3) and UBI-1:: CPs:: NOSpolyA (Fig. 2) cloned in pCANA were completely sequenced. The expression cassettes were purified by electrophoresis on 0.8% agarose gel digested Notl vector carrying pCANA.

For selection, sugarcane plants were cotransformed with UBI1 :: Bar :: NOSPolyA expression cassette, a ubiquitin-based cassette for high level of marker gene expression in monocot plants containing the Bar coding region (Biolaphos-resistance gene) under the control of the corn ubiquitin promoter, first exon and first intron, followed by a NOS-polyA terminator (nopaline synthase gene). The Bar gene encodes a phosphonitricin28 / 34 acetyl transferase (PAT) enzyme that detoxifies phosphonitricin (Biolaphos) by acetylation. Cassette UBI-1 :: Bar:.-NOSpolyA has been completely sequenced. For the transformation of the plant, the UBI-1 :: Bar:.-NOSpolyA cassette was purified by electrophoresis in 0.8% agarose gel restricted Earl carrying pBLUESCRIPT (SK-) (Christensen, AH and Quail, PH Transgenic research 5: 213-218, 1996).

EXAMPLE 3

Sugar cane processing

Cultures of embryonic sugarcane corns were established from apical meristem and primordial leaves of sugarcane (Saccharum spp. Hybrid).

Eight-week-old calluses were co-bombarded with an equimolar mixture of the expression cassettes

UBI:: Bar:: NOSpolyA and UBI-1 :: CPas:: NOSpolyA or UBI1 :: Bar :: NOSpolyA and UBI-1:: CPs :: NOSpolyA (10 gg of DNA / 3 mg of particle) per bombardment particle as previously described (Sanford, JC Plant Physiol. 79: 206-209,

nineteen ninety). After the bombardment, calluses were transferred to the MS medium containing 1 mg / L of PPT and 1 mg / L of BAP to promote bud regeneration and inhibit the development of non-transgenic tissue. After two weeks, calluses were transfected into a medium containing 1 mg / L of PPT and 1 mg / L of BAP to elongate the sprout and to induce the formation of

29/34 ‘7c \ J root. After two weeks, seedlings were placed in magenta boxes for acclimatization and two weeks later, sprouts (10-15 cm) with well-developed roots were transferred to pots with soil and placed in the greenhouse.

The selected transgenic plants containing the Ubi :: CPs construct were genotyped by PCR using a combination of SCMV-f4 primers (SEQ ID No.: 1) and

SCMV-r3 (SEQ ID No .: 2) from SCMV CP. Southern Blot analysis of the total DNA of transgenic plants, digested with BamH I and hybridized with a SCMV-CP probe, revealed the presence of the transgene in different numbers of copies in transgenic plants as shown in Figure 4. In transgenic plants containing ÜBI constructions :: CPas, detectable levels of antisense CP mRNA were observed in some of the transgenic plants by RT-PCR using specific primers SCMV-f4 (SEQ ID No .: 1) and SCMV-r3 (SEQ ID No .: 2) of SCMV (figure 5).

EXAMPLE 4

Inoculation test of SCMV in sugarcane plants

A transgenic plant is considered to have increased tolerance or resistance to SCMV when it exhibits significantly less or no symptoms of disease infection caused by SCMV compared to the same variety as

30/34 non-transgenic plant when challenged with the same strain of

SCMV.

The SMCV inoculants were propagated in sugar cane. Leaves of sugarcane plants (cultivar Co740) in the development stage from four to eight leaves (6-7 weeks of age) conducted in the greenhouse were injured and mechanically inoculated with sap extract from symptomatic sugarcane leaves de-sugarinfected with SCMV virus (25 mL of a 0.1 M potassium phosphate buffer containing 0.25 g carhorundum / g tissue). Plants were observed for a period of 2-8 weeks after inoculation. Symptoms were assessed visually and, in some cases, the level of the virus in leaves was quantified by ELISA, according to standard procedures (Converse, R. H and Martin, R. R., 1990; APS Press, St. Paul, MN, pp.

179-196) using 1: 500 diluted SCMV alkaline-conjugated IgG. Observations included a visual assessment of the severity of the SCMV viral infection as susceptible (S;

most tillers had severe symptoms), tolerant (T; develops symptoms in a significant time and statistically later and significantly less severe than those in control plants, and / or mosaic symptoms restricted in the minority of tillers) and resistant (R; all tillers show no symptoms). An Index ίω

ÕC7V

31/34 disease severity (DSI) was recorded as percentage of infected plants in the clump DSI = Σ symptomatic plants / total number of plants in the clump x 100). The presence of leaf mottle symptoms induced by SCMV in any clump results in an SCMV score of 1 for this access or control. A DSI score of 0 indicates that there is no SCMV infection and a score of 100 indicates that all plants have been infected. As with the control experiments, non-transformed plants of the same cultivar and plants transformed only with the Bar gene (übi :: Bar) were also mechanically infected with the same amount of virus and examined.

Thirty-six independent transgenic plants containing the übi :: Cpas transgenic cassette were examined for resistance to SCMV over mechanical virus inoculation. In this experiment, 4 replicates of each plant were used. The plants were inspected for leaf mottle symptoms induced by SCMV at 10, 22, 35, 57, 81 and 111 days after inoculation and the disease severity index was calculated. As controls, replicates of the unprocessed parent cultivar Co740 were used. SCMV was detected in control plants by ELISA assays. Although the majority of transgenic plants showed the same values as the parental cultivar,

Ο

Over there}-'

32/34 transgenic (Table 1), nine übi :: CPas plants had significantly lower DSI scores (DSI <55%) than the unprocessed parent cultivar

Co740, with two Ubi plants :: CPas showing scores

DSI of 18.2% and 21.7%. These two events were also evaluated under field conditions for a period of one year and remained less susceptible to SCMV infection than their unprocessed counterparts.

In an additional trial, 13 independent events containing the transgenic cassette übi :: CPs were examined for resistance to SCMV. As controls, replicates of the unprocessed parental cultivar Co740, as well as their counterpart transformed only with übi :: Bar cassette, were evaluated (Table 2). Four übi :: CPs events had significantly lower DSI scores (<60%) than unprocessed Co740 and übi :: Bar control events, with one übi :: CPs event having a DSI score of zero. These events will be evaluated under field conditions. Similarly, the observations made for the transgenic events Ubi :: CPas, most transgenic events übi :: Cps showed the same DSI values as the control events.

Table l

33/34

Disease Severity Index in inoculation of SCMV of Co740 transgenic events (Ubi :: CPs) and Co740 non-transgenic controls. Four replicates were evaluated for each event and non-transgenic control.

DSI Genotype

Ubi :: CPs 14 21, 7 Ubi :: CPs 21 54.5 Ubi :: CPs 45 100 Ubi :: CPs 47 100 Ubi :: CPs 48 100 Ubi :: CPs 49 100 Ubi :: CPs 51 100 Ubi :: CPs 53 46.2 Ubi :: CPs 54 100 Ubi :: CPs 55 31.9 Ubi :: CPs 60 18.2 Ubi :: CPs 62 100 Ubi :: CPs 63 100 Ubi :: CPs 65 100 Ubi :: CPs 66 85.7 Ubi :: CPs 71 100 Ubi :: CPs 76 46.2 Ubi :: CPs 78 100 Ubi :: CPs 85 78.3 Ubi :: CPs 86 36.8 Ubi :: CPs 88 77.8 Ubi :: CPs 92 47.1 Ubi :: CPs 93 100 Ubi :: CPs 96 80 Ubi :: CPs 97 100 Ubi : CPs 101 100 Ubi : CPs 102 45.5 Ubi : CPs 103 94.4 Ubi : CPs 105 100 Ubi : CPs 107 100 Ubi : CPs 109 88.9 Ubi : CPs 110 80 Ubi : CPs 113 66.7 Ubi : CPs 115 62.5 Ubi : CPs 119 100 Ubi : CPs 121 100

Co740 100

Petition 870170035878, of 05/29/2017, p. 31/33

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Table 2

Disease Severity Index in inoculation of SCMV of transgenic events Ubi :: CPs, transgenic controls Ubi :: BAR and non-transgenic controls Co740. Four replicates were evaluated for each event and non-transgenic control.

DSI Genotype

Ubi : CPs 19.1 100, 0 Ubi : CPs 19.3 100, 0 Ubi : CPs 19.4 40.0 Ubi : CPs 33.2 100.0 Ubi : CPs 37.1 0.0 Ubi : CPs 38.1 100.0 Ubi : CPs 38.4 100.0 Ubi : CPs 38. 6 40.0 Ubi : CPs 39.1 100.0 Ubi : CPs 40.3 60, 0 Ubi : CPs 43, 1 100.0 Ubi : CPs 43, 2 100.0 Ubi : CPs 43, 3 100.0

Co740 100.0 Co740 0.0 Co740 80, 0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100.0 Ubi: :Pub 100, 0 Ubi :Pub 100, 0

Petition 870170035878, of 05/29/2017, p. 32/33

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Claims (2)

1. Method to increase the resistance of plants to infection by the Sugarcane Mosaic Virus (SCMV) characterized by the fact that it comprises the transformation or transfection of said plant with an expression vector comprising a promoter operationally linked to a molecule of nucleic acid as defined by SEQ ID NO: 6 which encodes (i) a protein having protein kappa properties, (ii) an antisense RNA for the protein kappa gene of said Sugarcane Mosaic Virus (SCMV), where the expression of said expression cassette inhibits infection by the Sugarcane Mosaic Virus (SCMV). 2. Method, according to claim 1, characterized by the fact that said plant is sugarcane. Petition 870170035878, of 05/29/2017, p. 33/33 1/2 Ay 4y N // < TOI-1