MXPA00008207A - Banana streak virus promoter and detection - Google Patents

Abstract

Banana Streak Virus (BSV) genome has been cloned and the promoter identified. Operable linkage of the promoter to a transcribable sequence allows for transgenic expression in plants, including non-graminaceous monocots particularly Musaceae (Musa and Ensete) and graminaceous monocots such as rice and sugar cane. Methods for detection of BSV infection in plants are also provided.

Description

DETECTION AND PROMOTER OF BANANA OR BANANA STRETCHER VIRUS.

The present invention relates to the Banana Screener Virus, in particular, to the cloning and identification of its genome and promoter, useful in the transgenic strategies in Musaceae. { Musa and Ensete) and in the identification of antiviral agents, and is also related to the detection of the virus in plants, in particular, through the use of PCR primers, designed based on the genomic sequence.

The banana . { Musa) is the fourth most important global trade item and is a subsistence and cash crop for small farmers, particularly in western and central Africa. At present, improvements are being investigated for disease resistance, fruit production and other agronomic qualities of bananas and plantains, by the International Institute for Tropical Agriculture (UTA), Nigeria and other institutes. These improvements have been hampered by the recent diagnosis of Banana Strip Virus (BSV) (1) in banana producing supplies, and quarantine regulations have prevented movement of infected plant materials, including improved varieties.

The BSV is a member of the badnavirus group (2) which has uncoated basiliform particles of the size of 30 x 130-150 nm, and which contain a double-stranded circular DNA genome of 7.4 - 8.0 kbp. The complete sequences of the genomes of four members of this group have been reported, the variegated yellow virus of Commelina (CoYMV; 3), the basiliform virus tungro of rice (RTBV; 4, 5), the bacilliform virus of the sugar cane. sugar (ScBV; 6), the cocoa root shoot virus (CSSV; 7), and are known to at least two other members. This information reveals common characteristics of the group. All have similar genomic organizations with three open reading blocks (ORF) (except in the RTBV, which has four) encoded in a thread. The first two ORFs potentially code for two small proteins (ORF 1 ~ 22 kD, ORF ~ 14 kD) with an unknown function. The third ORF (~ 210 kD) codes for a polyprotein, which is proteolytically cleaved to produce the viral coat protein containing a region with homology to a DNA agglutination domain, together with the regions with aspartic protease homology ( AP), reverse transcriptase (RT) and an RNase H (RH). In the RTBV, the extra ORF, the ORF 4, is in the 3 'direction in relation to the ORF 3 and has an unknown function. These characteristics, together with a potential binding site for the tRNA, suggest that the badnaviruses are pararetrovirus.

Following the entry of the virus into the cell and the nucleus, the genome is transcribed into a copy with a length greater than that of the genome, which (presumably) is both a polycistronic ANRm, and a template for replication. The negative strand of the DNA, which is primed by an ANRtmet, is synthesized by a virally encoded reverse transcriptase, and the positive strand, by the same enzyme and the virally encoded ribonuclease H.

The use of RT, in its replication, can potentially lead to a high degree of variation between isolates and different members of the group, and this variation has already been reported for the BSV (8).

The present inventors have succeeded in cloning the genome of a Nigerian isolation of the BSV and in identifying its promoter, which provides an aspect of the present invention. The promoter is useful for the expression of transgenes in plants, including non-gramineous monocotyledons, particularly Musaceae. { Musa and Ense) and gram-rich monocotyledons, such as rice and sugarcane. At least one bacilliform strain of sugarcane has been shown to infect plantain (Bouhida et al., 1993 J. Gen Virol. 74: 15-22).

The growing importance of BSV highlights the need for the determination of the specific properties of BSV that can be used for its diagnosis. The symptoms of BSV can be easily confused with those of the cucumber mosaic virus (CMV). The BSV is serologically heterogeneous (8) and is present at a low titration in the host. Serological methods in use, particularly the ISEM, require a sophisticated method and are relatively insensitive. In another aspect of the present invention, the inventors who have cloned the BSV genome have designed PCR-based diagnostic systems.

PCR has been used in numerous studies due to its rapid, sensitive and reliable detection of viruses from a wide variety of sources. A direct protocol for PCR for a sensitive detection of BSV of Musa plants is reported in Harper et al. , 1996 (In (Ed) Marshall, Di agnosti cs for Crop Protecti on, BCPC Proccedings 65n, BCPC Surrey, UK, pp 47-51. The provisional results when using this method indicate a widespread presence, if not universal, of the BSV sequences in Musa, in contrast to the results obtained using other methods, which indicate a much lower incidence of the virus.These findings and other reports provide an indication of the BSV sequences that are integrated into the Musa genome The integrated sequences do not necessarily lead directly to observable symptoms of the disease, as there are plants that have been documented to be and have been apparently free of disease.

The present invention now provides a method that can be used for specific, reliable and sensitive detections of the episomal BSV.

According to the first aspect of the present invention, there is provided an isolated polynucleotide that includes the promoter of the plantain virus (BSV). The nucleic acid may consist essentially of promoter sequences. The promoter sequence may be part of a larger molecule that includes, for example, a heterologous coding sequence operably linked to the promoter.

A preferred embodiment of a promoter according to the present invention has the sequence shown in NO SEC ID: 2, -350 to + 100 regarding the start of the transmission sequence.

In addition, a part (fragment), allele, mutation, variant or derivative of the promoter sequence that is shown (such as the part that goes in the 5 'direction of the NO SEC ID: 2 or a fragment thereof) should be sufficient for the promoter activity, to promote the transcription of a heterologous sequence linked in an operational manner, i.e. under the control of the variant part or derivative of the sequence shown . One or more fragments of the sequence may be included in a promoter according to the present invention, for example, one or more portions may be coupled to a "minimal" promoter. These -proportions can give the Banana Streamer Virus a promoter function in a promoter, such as a fitness for, or an improved performance in non-gramineous monocotyledons.

In another aspect, the present invention provides an isolated polynucleotide that includes a promoter, the promoter includes a nucleotide sequence that is shown in SEQ ID NO: 2 and confers the BSV promoter function on a sequence operably linked to the promoter. The restriction enzymes o-nucleases can be used to digest the total length nucleic acid shown, followed by an assay to determine the minimum sequence required for this function. A preferred embodiment of the present invention provides an isolated nucleic acid + with the minimal nucleotide sequence that is shown in SEQ ID NO: 2 required for the function of the BSV as a promoter.

The promoter may include one or more sequence portions or elements that give the BSV promoter a regulatory control of expression.

Other regulatory sequences may include, for example, how they are identified by a mutation or assessment of digestion in an appropriate expression system or by a sequence comparison with available information, for example, by using the computer to investigate bases of data through the Internet.

The word "promoter" means a sequence of nucleotides by which the transcription of the DNA bound operably in the 3 'direction can be initiated (ie, in the 3' direction in the sense strand in the double-stranded DNA).

The words "operably linked" means that it is linked as part of the same nucleic acid molecule, placed and oriented appropriately so that transcription starts from the promoter. The DNA operably linked to a promoter is "under a transcriptional initiation regulation" of the promoter.

The present invention extends to a promoter having a sequence of nucleotides that are alleles, mutants, variants or derivatives, in the manner of addition, insertion, substitution or deletion of nucleotides in a promoter sequence as provided herein. The systematic or random mutagenesis of nucleic acids to make an alteration to the nucleotide sequence can be carried out using a technique known to those skilled in the art. One or more alterations to a promoter sequence according to the present invention can increase or decrease the promoter activity, or increase or decrease the magnitude of the effect of a substance capable of modulating the promoter activity.

The "promoter activity" is used to refer to the ability to initiate transcription. The level of the promoter activity is quantifiable, for example, by assessing the amount of ANRm produced by the transcription of the promoter, or by assessing the amount of protein product produced by the translation of the ANRm that is produced by the transcription of the promoter. The amount of a specific ANRm that is present in an expression system can be determined for example by using specific oligonucleotides that are capable of hybridizing with the ANRm and that are labeled or can be used in a specific amplification reaction such as the chain reaction of the polymerase. The use of a reporter gene facilitates the determination of promoter activity by reference to protein production.

In various embodiments of the present invention, a promoter having a sequence which is a fragment, mutant, allele, variant or derivative by way of addition, insertion, substitution or deletion of one or more nucleotides, of the promoter sequence that is shown in SEQ ID NO: 2, has a homology / similarity to the sequence shown, which is at least 5% greater than the homology that any of the promoter sequences of the other badnaviruses have the sequence shown which is shown herein, preferably at least about 10% more homology, more preferably at least about 20% homology, more preferably at least about 25% more homology. These badnaviruses are Commelina yellow spot virus, rice tundra basiliform virus, cocoa bloated sucker virus, sugarcane basiliform virus and Dioscorea alata basiliform virus (data not published), such as it is noted elsewhere in the present. The sequence in accordance with one embodiment of the invention can hybridize to the sequence shown in SEQ ID NO: 2, but with none of the other promoter sequences of these other badnaviruses under the appropriately stringent conditions of selective hybridization. A promoter according to the invention may include one or more portions that appear in SEQ ID NO: 2 and are capable of conferring a BSV promoter function on a promoter that contains them.

Similarly, the nucleic acid according to certain embodiments of the present invention may have a homology to all or parts of the nucleotide sequences shown in SEQ ID NO: 2, whose homology is greater than the length of the relevant part (ie, the fragment) that the homology that is shared between the part of SEQ ID NO: 2 and a respective part of the nucleotide sequence of any of these badnaviruses, and that may be greater than about 5% more, and more preferably greater than about 10% more, and more preferably greater than about 20% more, and more preferably even greater than about 30% more.

The homology can be taken over the entire length of the sequence or over a part, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200 continuous nucleotides. The fact that two nucleotide sequences share a "homology" or that are "homologous" is based on the comparison of frequencies. Any phlogogenetic relationship is irrelevant to this. For those skilled in the art they usually refer to the homology between nucleotide sequences without any implication for their evolutionary origin. Two homologous nucleotide sequences may also be "similar" or may have a certain percentage of similarity or a certain percentage of identity.

In general, it is not critical which of the various conventional algorithms is used to determine how homologous two nucleotide sequences are relative to one another. A preferred algorithm may be GAP, which uses the Needleman and unsch alignment method ("Mol. Biol. (1970) 48: 443-453) and is included in the Program Manual or the Wisconsin Package, Version 8 , September 1994, or the Genetics Computation Group, 575 Science Drive, Madison, Wisconsin, USA.) In the absence of instructions to do otherwise, the right-handed person will understand that the predetermined parameters should be used in order to maximize the maximized alignment, with a penalty for the creation of a gap = 12 and a penalty for the creation of a gap = 4.

The similarity or homology (the terms are used interchangeably) or the identity can be defined and determined by the program TBLASTIN, of Altschul et al. (1990) J. Mol. Bi ol. 215: 403-10, or BestFit, which is part of the Wisconsin Package Version 8, September 1994 (Computer Group for Genetics, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711). Preferably, sequence comparisons are made using FASTA and FASTP (see Pearson &Lipman, 1988. Methods in Enzimology 183: 63-98). Preferably, the parameters are arranged, using the predetermined matrix, as follows: Gapopen (penalty for the first residue in a separation): -12 for proteins / -16 for DNA; Gapext (penalty for additional residues in a separation): -2 for proteins / -4 for DNA / KTUP word length: 2 for proteins / 6 for DNA.

The homology between the nucleic acid sequences can be determined by selective hybridization between the molecules under stringent conditions.

Preliminary experiments can be carried out by hybridizing under conditions of low severity. For the survey, the preferred conditions are those strict enough for there to be a simple pattern with a small number of hybridizations identified as positive, which can be further investigated.

For example, hybridizations can be carried out according to the method of Sambrook et al. (below) using the hybridization solution comprising: 5X SSC (where SSC = 0.15 M sodium chloride, 0.15 M sodium citrate, pH7), 5X Denhardt's reagent, 0.5-1.0% SDS, 100 μg / ml denatured, DNA fragmented from salmon sperm, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried to 37-42 ° C for at least six hours. Following hybridization, the filters are washed as follows: (1) for 5 minutes at room temperature in 2X SSC and 1% SDS; (2) for 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) for 30 minutes - 1 hour at 37 ° C in IX SSC and 1% SDS; (4) for 2 hours at 42-65 ° C in IX SSC and 1% SDS, and changing the solution every 30 minutes.

A formula that is common for calculating the stringent conditions that are required to activate hydrolyzation between nucleic acid molecules of a specified sequence homology is (Sambrook - et al., 1989): Tp. = 81.5 ° C + 16.6Log [Na +] + 0.41 (% G + C) - 0.63 (% formamide) - 600 / # bp in duplex.

By way of illustration of the above formula, when using [Na +] = [0.368] and 50% formamide, with a GC content of 42% and an average probe size of 200 bases, the Tm is 57 ° C. The Tm of a duplex DNA decreases by 1 - 1.5 ° C with each 1% decrease in homology. Therefore, targets with more than about 75% sequence identity are observed when using a hybridization temperature of 42 ° C. This sequence can be considered substantially homologous to that of the nucleic acid sequence of the present invention.

It is well known in the art to increase the stringency of hybridization gradually until only some positive clones remain. Other suitable conditions, include, for example for sequence detection are approximately 80-90% identical, hybridization overnight at 42 ° C in 0.25M Na2HP04, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55 ° C in 0. IX SSC, 0.1% SDS. For the detection of sequences that are greater than about 90% identical, suitable conditions include overnight hybridization at 65 ° C in 0.25M Na2HP0., PH 7.2, 6.5% SDS, 10% dextran sulfate and one wash final at 60 ° C in 0. IX SSC, 0.1% SDS. An alternative, which may be particularly appropriate with plant nucleic acid preparations, is a 5x SSPE solution (final 0.9M NaCl, 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt's solution , 0.5% SDS at 65 ° C during the night, (for a high rigor, and highly similar sequences) or 50 ° C (for a low rigor, and less similar sequences). Washes in 0.2x SSC / 0.1% SDS at 65 ° C for high rigor, alternatively at 50-60 ° C in Ix SSC / 0.1% SDS for low stringency.

In another embodiment, the hybridization of a nucleic acid molecule to a variant can be determined and identified directly, for example by using an amplification reaction of the nucleic acids, particularly the polymerase chain reaction (PCR). PCR requires the use of two primers to specifically amplify the target nucleic acid, so two molecules of nucleic acids with characteristic sequences for BSV are preferably used. When using the RACE PCR, only one of these primers can be necessary (see "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990)).

Therefore, a method involving the use of PCR to obtain the nucleic acid according to the present invention can be included: (a) providing a preparation of nucleic acids, for example a plant cell; (b) providing a pair of primers of nucleic acid molecules useful in (i.e., suitable for) PCR, at least one of these primers is a primer specific for a nucleic acid according to the present invention; (c) contacting this nucleic acid in this preparation with these primers under conditions for PCR performance; (d) carrying out the PCR and determining the presence or absence of an amplified PCR product.

The presence of an amplified PCR product can indicate the identification of an allele or another variant.

Table 1 shows the percentage of nucleotide homologies for various Badnaviruses compared to the BSV sequence shown in SEQ ID NO: 1 (during total length) as determined using the GCC GAP algorithm (see below) .

The present invention also includes promoters that are homologous to the BSV promoter, in particular the sequence of SEQ ID NO: 2. A homologous promoter may show more than 50% homology to the sequence of SEQ ID NO: 2, more than 65% homology, more than 75% homology, more than 85% homology or greater than 95% homology. This homology can be shown for a sequence of at least 20 nucleotide bases, in at least 50 nucleotide bases, in at least 100 nucleotide bases, in at least 300 nucleotide bases or in at least 500 bases of nucleotides. nucleotides.

It should be noted that because the Banana Screener Virus is a retrovirus that uses reverse transcriptase (RT) that is known to be relatively prone to error, a certain tendency must be expected in the sequences that occur in nature. In fact, the sequence described in NO SEC ID :. 2 is obtained from a Nigerian isolation of BSV but other isolates may have sequences that vary from this specific sequence to different degrees. These are encompassed by aspects and modalities of the present invention.

An allelic variant of the BSV genome sequence of SEQ ID NO: 1 may in accordance with the present invention include one or more, preferably all, of the following changes with respect to NO SEQ ID: 1: insertion of C as the nucleotide 6779, insertion of G as nucleotide 7087 (which is numbered 7088 if C is inserted as nucleotide 6779), substitution of T for A in nucleotide 4218, substitution of C for G in nucleotide 6606, substitution of G for A at nucleotide 7118 (numbered 7119 if C is inserted as nucleotide 6779 or G is inserted as nucleotide 7087, and number 7120 is numbered if C is inserted as nucleotide 6779 and G is inserted as nucleotide 7088), substitution of C by G at nucleotide 7185 (number 7186 is numbered if C is inserted as nucleotide 6779 or G is inserted as nucleotide 7087, and 7187 is numbered if C is inserted as nucleotide 6779 and G is inserted as nucleotide 7088), substitution of T by C in nucleotide 7207 ( 7208 is numbered if C is inserted as nucleotide 6779 or G is inserted as nucleotide 7087, and 7209 is numbered if C is inserted as nucleotide 6779 and G is inserted as nucleotide 7088). An allelic variant of a promoter according to the invention may include one or more or preferably all of the aforementioned changes that fall within the promoter sequence.

Further provided by the present invention is a nucleic acid structure which includes a promoter region or a fragment, mutant, allele, derivative or variant thereof as discussed, and which is capable of promoting transcription in a plant, particularly in Musaceae. or in monocotyledons, which are operably linked to a heterologous nucleic acid sequence, preferably a gene, for example a coding sequence. By "heterologous" in this context, a gene is denoted apart from any coding sequence that occurs naturally in the Banana Screener Virus. The modified forms of the BSV coding sequences can be excluded. Generally, the gene can be transcribed into mRNA that can be translated into a peptide or a polypeptide product that can be detected and preferably quantified after expression. A gene whose encoded product can be titrated after expression denotes an "indicator gene", ie, a gene that "indicates" the promoter activity.

The present invention also provides a nucleic acid vector that includes a promoter as disclosed herein. This vector may include a restriction site that is appropriately placed or any other means for insertion into the vector of a sequence that is heterologous to the promoter to which it is to be operably linked. Suitable vectors may be chosen or structured, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes of other sequences as appropriate. For more details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. The procedures for introducing DNA into cells depend on the host used, but they are well known. Several known techniques and protocols for nucleic acid manipulation, for example in the preparation of nucleic acid structures, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & amp;; Sons, 1992. The descriptions of Sambrook et al. and Ausubel et al. are incorporated herein for your reference. The specific procedures and vectors that are previously used with extensive success in plants are described by Bevan (Nucí.

Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors) In: Plant Molecular Biology Labfax (Croyd RRD ed) Oxford, BIOS Scientific Publishers, pp. 121-148).

Selectable genetic markers consisting of chimeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinothricin, chlorsulfuron, methotrexate, gentamicin, spectinomycin, imidazolinones and glyphosate can be used.

Another aspect of the present invention provides a host cell (which may be microbial or plant) that contains a nucleic acid structure that includes a promoter element, as described herein, operably linked to a gene or a heterologous sequence of nucleic acids. Yet another aspect provides a method that includes introducing this structure into a host cell. The introduction may employ any available technique that is well known to the skilled person in the art.

The introduction can be followed by causing or allowing the expression of the gene or the heterologous nucleic acid sequence under the control of the promoter.

In one embodiment, the structure that includes the promoter and the gene or nucleic acid sequence integrates the genome (for example the chromosome) of the host cell. The integration can be promoted by including in the structure, sequences that promote recombination with the genome, in accordance with conventional techniques.

The vectors and nucleic acid molecules according to the present invention can be provided in isolated and / or purified form of their natural environment, in a homogeneous or substantially pure form, or free or substantially free of genes or nucleic acids of the species of interest or of the different origin of the sequence encoding a polypeptide with the required function. The nucleic acid according to the present invention can include cDNA, RNA, genomic DNA which can be totally or partially synthetic. The term "isolated" encompasses all these possibilities. Where a DNA sequence is specified, for example with reference to a figure, 'unless the context otherwise requires the RNA equivalent, with the U substituted for T in- where it occurs, this is encompassed. One aspect of the present invention is the use of the nucleic acid according to the invention for the production of transgenic plants.

When a structure of a selected gene is introduced into a cell, certain considerations, which are well known to those skilled in the art, should be taken into consideration. The nucleic acid to be inserted must be assembled within a structure that contains effective regulatory elements that drive transcription. A method must be available to transport the structure to the cell. Once the structure is inside the cell membrane, integration into the endogenous chromosomal material will occur or it will not occur. Finally, as far as the plants are concerned, the type of target cell must be such that the cells can regenerate into whole plants. Plants that are transformed with a segment of DNA that contains a sequence can be produced by conventional techniques that are already known for the genetic manipulation of plants. DNA can be transformed into plant cells by using any suitable technology, such as a Ti vector - disarmed plasmid transported by the Agrobacterium, thus exploiting its natural ability for gene transfer (EP-A-270355, EP- A-0116718, NAR 12 (22) 8711-87215 1984), bombardment of particles or microprojectiles (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Pl ant Tissue and Cell Cul ture, Academic Press), electroporation (EP 290395, WO 8706614), other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome-mediated DNA uptake (eg Freeman et al., Plant CPhysiol., 29: 1353 (1984)), or the vortex method (eg, Kindle, PNAS USA 87: 1228 (1990d). Physical methods for transforming plant c are reviewed in Oard, 1991 , Bi otech, Adv. 9: 1-11.

The transformation of Agrobacterium is widely used by those skilled in the art to transform dicotyledonous and monocotyledonous species. For examples of the production of stable transgenic plants, and fertile in economically relevant monocotyledonous plants see inter alia Toriyama, et al. (1988) Bi o / Technology 6, 1072-1074; Zhang, et al. (1988) Plant CRep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet 76, 835-840; Shi amoto, et al. (1989) Na ture 338, 274-276; Datta, et al. (1990) Bio / Technology 8, 736-740; Chistou, et al. (1991) Bi o / Technology 9, 957-962; Peng, et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992) Plant CRep. 11, 585-591; Li, et al. (1993) Plant CRep. 12, 250-255; Rathore, et al. (1993) Plant Mol ecular Biology 21, 871-884; Fro, et al. (1990) Bio / Technolgy 8, 833-839; Gordon-Kamm, et al. . { 1990) Plant C2, 603-618; D'Halluin, et al. (1992) Plant C4, 1495-1505; Walters, et al. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somers, et al. (1992) Bio / Technology 10, 1589-1594; W092 / 14828. In particular, Agrobacterium-mediated transformation now also emerges as a highly efficient alternative for a transformation method in monocotyledons (Hiei et al. (1994) Tyhe Plant Journal 6, 271-282).

Generations of fertile transgenic plants have been achieved in the cereals of rice, corn, wheat, oats, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162; Vasil, et al. (1992) Bio / Technology 10, 667-674; Vain, et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).

The banana has been transformed: for example see Sagi et al. , 1995, Bi oTechnol ogy 13 481-485; and May, et al. , 1995, BioTechnology 13 486-492.

Microprojectile bombardment, electroporation and direct DNA uptake are preferred when the Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques can be used to enhance the efficiency of the transformation process, for example bombardment with microparticles coated by Agrobacterium (EP-A-486234) or bombardment of microprojectiles to induce a wound followed by simultaneous cultivation with Agrobacterium. (EP-A-486233).

After the transformation, a plant can be regenerated, for example from single c, from the callous tissue or from discs of the leaves, as is usual in the art. Almost any plant can be regenerated in its entirety from the c, tissues and organs of the plant. The available techniques are reviewed in Vasil et al. , CCul ture and Somatic CGenetics of Plants, Vol I, II and III Laboratory Procedures and Their Applicants, Academic • Press, 1984, and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

The selection in particular for transformation technology is determined by its efficiency to transform certain plant species as was the experience and preference of the person practicing the invention with a particular methodology of their selection. It is apparent to the skilled person that the particular selection for a transformation system for introducing a nucleic acid into plant c is neither essential for the limitation of the invention, nor is it the selection of the technique for plant regeneration.

Also according to the invention a plant cto which a nucleic acid has been incorporated into its genome is provided., in particular a heterologous nucleic acid, as provided by the present invention. Another aspect of the present invention provides a method for making this plant cell which involves the introduction of a vector that includes the nucleotide sequence in a plant cell and causing or allowing recombination between the vector and the genome of the plant cell to introduce the nucleotide sequence in its genome. The invention extends to plant cells containing nucleic acids according to the invention as a result of the introduction of a nucleic acid into the ancestral cell.

Another aspect of the present invention provides a method for making this plant which involves the introduction of a nucleic acid or a suitable vector that includes the nucleotide sequence in a plant cell and causing or allowing recombination between the vector and the genome of the plant cell to introduce the nucleotide sequence in its genome. The invention extends to plant cells containing nucleic acids according to the invention as a result of the introduction of a nucleic acid into the ancestral cell.

In the context of a host cell containing a "heterologous" nucleic acid, the term "heterologous" can be used to indicate that the gene / nucleotide sequence in question has been introduced into these cells of the plant or into an ancestor thereof, using genetic engineering, that is, through human intervention A transgenic plant cell, ie transgenic by the nucleic acid in question, can be provided.The transgene can be in an extra genomic vector or can be incorporated, preferably stably, in the A heterologous gene can replace an endogenous gene equivalent, that is, one that normally performs an equal or similar function, or the inserted sequence can be additional to the endogenous gene or another sequence.An advantage of the introduction of a heterologous gene is the ability to place the expression of a sequence under the control of a promoter of choice, in order to be able to influence the expr sion according to your preference. In addition, mutants, variants and derivatives of the wild-type gene, for example with greater or less activity than the wild-type, can be used in place of the endogenous gene. Heterologists of nucleic acids, or exogenous or foreign, to a plant cell can occur unnaturally in cells of this type, variety or species. Therefore, the nucleic acid may include a coding sequence of or derived from a particular type of plant cell or species or plant variety, which is placed within the context of a plant cell of a different type or species or variety of plant. Another possibility is that a nucleic acid sequence is placed within the cell in which a homologue is naturally found, but where the nucleic acid sequence is linked and / or adjacent to the nucleic acid which does not occur in a natural within the cell, or cells of that type or species or plant variety, such that it is operably linked to one or more regulatory sequences, such as a promoter sequence, for the control of expression. A sequence within the plant or other host cell can be identifiably heterologous, exogenous or foreign.

Plants that include a plant cell according to the invention are also provided, along with any part or propagule, seed, offspring and single or hybrid descendants thereof. A plant according to the present invention may be one which does not actually reproduce in one or more properties. Plant varieties can be excluded, in particular the varieties of vegetables that can be registered under the Rights of Plant Breeders. It is noted that a plant does not need to be considered a 'plant variety' simply because it contains stably within its genome a transgene, which is introduced into a cell of a plant or an ancestor of this plant. the present invention provides any clone of this plant, seed, offspring and single or hybrid progeny, and any part of any of these, such as cuttings, seeds, etc. The invention provides any plant propagule, which is any part that can be used for reproduction or propagation, sexual or asexual, which includes cuttings, seeds and so on.It is also encompassed by the present invention a plant which is a stem that has been sexually or asexually propagated, a clone or descendant of this plant, or any part or propagule of this plant, stem, clone or descendant.

The invention further provides a method for influencing or affecting a physical characteristic of a plant, which includes causing or allowing expression for a heterologous nucleic acid sequence as discussed in the cells of the plant. Characteristics that can be influenced include resistance, immunity, tolerance, hypersensitivity to pathogens such as viruses, fungi and bacteria, pests such as nematodes and weevils, agronomic characters such as plant dwarfism, seed production or other product, fertility or sterility and fruit quality.

The invention further provides a method for influencing a physical characteristic of a plant that includes the expression of nucleic acids according to the present invention above, within cells of a plant, followed by an earlier step of introducing nucleic acid into the cell of the plant or an ancestor of this. This method can influence or affect a characteristic of the plant, as noted previously. This can be used in combination with any other gene, such as the transgenes involved in the determination or modification of any observed feature or other phenotypic trait or desirable property.

Nucleic acid structures that include a promoter (as described herein) and a heterologous gene (indicator) can be used for the detection of a substance capable of modulating the activity of the promoter. For antiviral purposes, for example, one can search for the treatment of BSV in banana or another disease, a substance capable of regulating the expression of the promoter in the 3 'direction. In other contexts, for example for the expression of a product useful in modifying a plant characteristic as indicated, it may be desirable to obtain a substance capable of regulating the expression of the promoter upstream. A method for detecting the ability of a substance to modulate the activity of a promoter may include contacting an expression system, such as a host cell, containing a nucleic acid structure as described herein with a test substance. or candidate and determine the expression for the heterologous gene.

The level of expression in the presence of the test substance can be compared to the level of expression in the absence of the test substance. A difference in expression in the presence of the test substance indicates the ability of the substance to modulate gene expression. An increase in expression for the heterologous gene compared to the expression of another gene that is not linked to a promoter as described herein indicates the specificity of the substance for the modulation of the promoter.

A structure of a promoter can be introduced into a cell line using a previously described technique to produce a stable cell line that contains the indicator structure and is integrated into the genome. The cells can be cultured and incubated with the test compounds for various times. The cells can be regenerated in plants and the methods of titration or detection carried out in accordance with the present invention in the plant or part thereof, such as a leaf or fruit.

The cells and / or plants can be Musaceae, Musa, Teach, banana or banana. After the identification of a substance that modulates or affects the promoter activity, the substance can be investigated in greater detail. In addition, it can be manufactured and / or used in the preparation, ie the manufacture or formulation, of a composition that can contain at least one additional component, such as a diluent or solvent. These can be administered to the cells or plants to modulate the activity of the promoter.

Therefore, the present invention extends in various aspects not only to the substance that is identified by using a nucleic acid molecule in the manner of a modulator of promoter activity, in accordance with what is described herein, but also a composition that includes this substance as a method that includes the administration of this composition to the plant, for example to decrease expression for example in the treatment (which may include preventive treatment) of BSV or other diseases in a plant, such as in Musaceae (Musa or Ensete) or monocotyledons such as those mentioned above, the use of these substances in the manufacture of this composition and a method for making the composition which includes mixing this substance with an acceptable diluent or carrier, and optionally other ingredients.

Other aspects of the present invention relate to "diagnostic" methods and means for determining the presence in a plant, particularly Musaceae, of episomal banana striator virus.

According to another aspect of the present invention there is provided a method for determining the presence or absence of the Banana Screener Virus in a plant, the method includes contacting the specific agglutination molecules for the Banana Screener Virus and a test sample that it includes the extract of a plant to be analyzed under the conditions where the specific agglutination molecules agglutinate the Banana Streamer Virus particles that are present in the sample of the analysis; carry out a PCR in the sample of analysis using the specific primers for the Banana Screener Virus; determine the presence or absence of a PCR product characteristic of the Banana Streak Virus.

The plant extract to be analyzed can be provided by using a carbonate buffer in the manner of a milling medium, as is well known in the art.

The specific agglutination molecules can be immobilized on a solid support such as a column, which allows easy washing of the binder material (Clark et al., (1986) Methods in Enzymol ogy 118: 742-751). One or more washing steps are preferably included before carrying out the PCR. Specific agglutination molecules may include antibodies specific agglutination fragments thereof.

Methods for producing the antibodies include immunizing a mammal (e.g., human, mouse, rat, rabbit, horse, goat, sheep or primate) with the Banana Screener Virus or one or more proteins with protein fragments thereof. Antibodies can be obtained from animals immunized using any of the varieties of methods known in the art, and can be detected, preferably by using the agglutination of the antibody with the antigen of interest. For example, Western blotting techniques or immunoprecipitation can be used (Armitage et al, 1992, Nature 357: 80-82). The antibodies can be polyclonal or monoclonal.

As an alternative or supplement to immunize a mammal, antibodies with an appropriate agglutination specificity can be obtained from a library that is produced recombinantly from immunoglobulin variable domains expressed, for example by using the bacteriophage lambda or the filamentous bacteriophage demonstrating functional agglutination domains of immunoglobulins on their surfaces; for example, see WO92 / 01047.

Various antibody fragments are known in the art to have the ability to bind specifically to the target antigen, including Fab, scFv, Fd, Fv, diabodies and so on. Since the role of a specific agglutination member in the context of this aspect of the present invention is that of agglutination, the secretory functions that are provided by the complete antibody are not relevant, any specific agglutination molecule including any binder fragment of a antibody, in principle, can be used.

Suitable PCR primers include those that are directed to regions of the genome that are likely to be conserved among the isolated BSVs, such as sequences that are identified for amino acid sequences that are conserved between the plant pararetroviruses in the aspartate protease and the regions of the reverse transcriptase. Due to the redundancy of the genetic code, these sequences are not found in other pararetroviruses. Preferred primers include the following sequences: Progressive V3012, 5 'GGA ATG AAA GAC CAG GCC Inverse V1573, 5' AGT CAT TGG GTC AAC CTC TGT CCC.

PCR techniques for nucleic acid amplification are described in the E. U. A. No. 4,683,195. In general, these techniques require that the sequence information of the effective sequence ends be known to allow direct and indirect oligonucleotide primers to be designed to be identical or similar to the polynucleotide sequence that is the target for the sequence. amplification. PCR comprises the steps of denaturation of the nucleic acid template (if it is double-stranded), the binding of the primer to the target, and the polymerization. The nucleic acid sequence information that is provided herein, particularly in SEQ ID NO:. 1, easily allows the right person to design the PCR primers. References for general use of PCR techniques include Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51: 263, (1987), Ehrlich (ed), PCR technology, Stockton Press, NY, 1989, Ehrlich et al, Science, 252: 1643-1650, (1991), "PCR protocols; A Guide to Methods and Applications ", Eds. Innis et al, Academic Press, New York, (1990).

Based on the sequence and other information provided herein, the oligonucleotide primers can be designed by those skilled in the art. An oligonucleotide for use in nucleic acid amplification may have about 10 or fewer triplets (eg, 6, 7 or 8), ie it is about 30 or less nucleotides in length (e.g., 18, 21 or 24). ). The specific primers are usually higher than 14 nucleotides in length, but do not need to be more than 18-20.

The presence of a PCR product indicative of the characteristic of the Banana Screener Virus can be determined by means of any of the detection methods available to those skilled in the art, including the detection of PCR products by size, hybridization with a BSV probe, restriction endonuclease, restriction patterns and / or sequencing.

Other aspects and modalities will be apparent to those of ordinary skill in the art. The embodiments of the present invention are now illustrated by way of example.

Figure 1 shows a map of the BSV genome, the complete sequence for which it is shown as in the NO SEC ID: '. 1.

The NO ID SEC :. 2 shows a sequence of a preferred promoter according to an embodiment of the present invention.

All documents mentioned in any part of this are incorporated for your reference.

EXAMPLE 1 - BSV cloning Isolation and purification of the virus Banana plants infected with the BSV are supplied (cultivar TMP4698 which is a tetraploid hybrid of Obino l'Ewai x Calcutta 4) in the manner of seedlings in vi tro or vegetable shoots that are grown in greenhouses, by UTA, Onne Field Station, Nigeria. The external tissue of the vegetable shoots is removed and the bulbs are sterilized in 1% sodium hypochlorite for one hour and quarantined for one month, before moving to the greenhouse and kept at 28 ° C during the day, and at 25 ° C during the night.

The material of the leaves is finely milled in liquid nitrogen and mixed in two volumes of buffer A (50 mM sodium phosphate at a pH 6.1, 5 mM dithiothreitol, 5 mM diethyldithiocarbamate, 0.5% polyethylene glycol (PEG 6000) Celluclast (Novo Nordisk) is added at 2%, incubated with shaking at 37 ° C for 2 hours and then overnight at room temperature, Triton X-100 is added at 1% and incubation is carried out during 30 additional minutes All subsequent steps are carried out 4 C. The supernatant of a low speed centrifugation at 10, OOOg for 10 minutes, is further centrifuged at 120, OOOg for 90 minutes.The pellet is resuspended in 100 minutes. ml of shock absorber A, centrifuged through a 5% sucrose mattress at 120, OOOg for 2.5 hours and the pellet is resuspended in 5 ml of buffer A. The virus is re-purified according to Lockhart (2) by centrifugation in a gradient from 0-40% of Cs2S0 in 10% sucrose [steps 40, 30, 20, 10, 0%] to 120, OOOg for 2.5 hours. The viral band is identified by ISEM, which is carried out according to Lockhart (2) using a mixed antiserum of BSV. The virus is diluted 4 times in buffer A, and pelleted at 150, OOOg for 90 minutes and resuspended in 100 μl of 50 mM sodium phosphate at pH 6.1.

Cloning and sequence analysis Virion DNA is purified by digestion of viral particles with proteinase K at 1 mg / ml in 100 mM TrisCl pH 8.0, 2mM CaCl2, 2% SDS, for 2 hours at 65 ° C. After the extraction with phenol, the DNA is precipitated, washed with 70% ethanol, dried and resuspended in 50 μl TE [10 mM TrisCl, 1 mM EDTA pH 8.0 Sambrook et al. (13)]. The DNA is digested with Eco R1 and the resulting fragments are cloned into pBluescript II SK + (Stratagene). One clone has sequence homology for ScBV and other badnaviruses. The primers are designed using the clone sequence information to allow PCR amplification of the complete virion DNA. The primers are contiguous, and face in opposite directions (nucleotide numbering for the complete BSV sequence, see below): V1514 5 'TGCGGGTGCTTCTTCACCC (antisense @ 2778), V1517 5' TATGCACCAGCTACAAGTGC (homosentido @ 2779). The The Expand ™ Long Template (Boehringer) PCR system is used, following the manufacturer's protocol for a 0.5-12 kb template (system 1). The template is 0.1-1.0 μl of the virus DNA isolated and the primers are used at a final concentration of 300 nM. The conditions of the amplification cycle were 94 ° C x 1 min, [92 ° C x 20 s, 50 ° C x 30 s, 68 ° C x 6 min] x 10, [92 ° C x 20 s, 50 ° C x 30 s, 68 ° C x 6 min with 20 s increment / cycle] x 15 and a final extension for 68 ° C x 7 min.

The 7.39 kb PCR product and the subclones derived from it were sequenced manually using the Sequenasa version 2.0 (USB, Unitet States Biochemicals) and by using the Prisma system (Applied Biosystems) and an ABI 373 sequencer automatically. It is analyzed using the GCG sequence pack (14). All DNA manipulations were carried out according to Sambrook et al. (13) For RNA isolation, 2 g of symptomatic banana leaves are ground in a powder in N2 liquid, and added to 24 ml of 100 mM Tris-Cl at a pH of 8.0, 20 mM EDTA, 500 mM NaCl, 20 mM mercaproethanol, 2% SDS and incubate at 65 ° C for 10 minutes. After addition of 8 ml of 3M sodium acetate and incubation on ice for 10 minutes, the solution is centrifuged at 10,000 x g for 10 minutes and the supernatant is filtered through two layers of Miracloth.

The nucleic acid is precipitated by the addition of 0.7 volumes of isopropanol, incubation at -20 ° C for 30 minutes and centrifugation. The sediment is resuspended in 2 ml of TE, extracted with phenol / chloroform then with chloroform and the RNA is precipitated by the addition of lithium acetate to give 2 m and incubation at 4 ° C overnight. The RNA pellet is washed twice in 80% ethanol and resuspended in 40 μl of water.

A gel electrophoresis containing formaldehyde and a Northern blot of RNA as described in Sambrook et al. (13) The 5 'end of the transcript is correlated by an extension of the primer using the method of Medberry et al. (3) . The primer used is 5-ATCTTGCGCTCTACTCGC at 7361 bp in the BSV sequence.

Primer and PCR design The PCR primer pairs are selected from aligned sequences of amino acids corresponding to the aspartic protease and the reverse transcriptase regions of the BSV-derived sequences. PCR is carried out on a DNA isolated from the banana leaves using the method of Li et al. (15), using the basic protocol that is described by the manufacturers of Tag DNA polymerase (Gibco BRL). The conditions were 94 ° C for 2 minutes, [94 ° C for 1 minute, 40-50 ° C for 1 minute, 72 ° C for 1 minute] x 30 cycles, followed by an extension of 5 minutes at 72 ° C . The products of the reaction are separated by electrophoresis through a 1.5% agarose gel and detected by fluorescence of the ethidium bromide under ultraviolet light. After photography, the products of the reaction are transferred onto a nylon membrane (Hybond-N, Amersham). A 32p labeled probe is synthesized by randomly priming the full length of the BSV PCR product in the manner of a template (16). Hybridization and washing were carried out at 65 ° C using the solutions and protocols described by the membrane supplier (Amersham).

Virus purification The purification of the virus provides a low production of bacilliform particles of the size of ~ 30 x 130 nm, which can be trapped in carbon grids coated with BSV antiserum but not in the grates coated with CSSV antiserum. Viruses can be detected by ISEM in a crude extract of infected leaf, but not in leaf immersion preparations.

Analysis of the virus sequence The complete sequence of 7388bp nucleotides of both strands of the isolated Nigerian BSV is determined as shown in SEQ ID NO:. 1. As with other badnaviruses and caulimoviruses, sequence numbering begins in the putative primer sequence without strand in the 5"direction and this sequence for the agglutination site tRNA metinit is found in the BSV.

Overall, the sequence shows a low but recognizable similarity to other badnaviruses, with the greatest similarity to ScBV and less in the RTBV (Table 1). The sequence is sufficiently different from those of other badnaviruses for the BSV to be considered a different virus.

The + strand contains three large ORFs (Figure 1). This number, its size and order are similar to other badnavirus sequences, with the exception of RTBV that has an additional ORF (4, 5). The BSV ORF III shows similarity to the ORF III sequences of other badnaviruses, in particular other regions that code for the conserved functions of the viral putative replicase, AP, RT and RH.

Correlation of the exact copy of the transcript The Northern blot of the total RNA of banana tissue infected with BSV reveals only a specific band for BSV of 7.5 kb. From this expected size for the exact copy of the transcript of the largest size of a pararetrovirus the size of the BSV.

To correlate the 5 'end of the exact copy of the larger transcript, the RNA that is subtracted from the infected banana tissue is used for an extension of the primer. The primer is 5'-ATCTTGCGCTCTACTCGC-3 '. Two strong interruptions are found, indicating that the transcription initiator sequence is at 7260 or 7261bp, 25 nt downstream of a potential TATA block sequence (TATATAA) (see below). Attempts to correlate the 3 'end of the exact copy of the transcript by using the methodology of Medberry et al. (3) prove to be a failure.

Detection by PCR of the BSV The primer sequences for the PCR are selected within the sequences that are conserved, and that putatively code for the RT and the AP. This pair of primers provides, in a consistent manner, a strong amplification product, of an expected size of 644 bp. Hybridization with a BSV probe and sequencing of this product confirm its origin as a BSV. Amplification of this band can also be achieved from crude preparations of infected plants. All the Musa plants that are analyzed provide this same bandwidth, and all the plants of the Onne station give the same pattern when the band is split with Al u 1.

Discussion The BSV genome contains three large ORFs (Figure 2) that potentially code for the 20.8, 14.5 and 208 KD proteins. The size and order of the ORF are very similar to those of most badnaviruses. Characteristically, the ORF overlap, and that between the ORF 1 and 2 with ATGA (the ATG is the beginning of the ORF2 and the TGA is the completion of the ORF 1) and therefore resemble the RTBV (Hay et al) and that between ORF 2 and 3 is TAATG. Badnaviruses are characterized by having very few AUG codons for the start of translation in the first two ORFs (see reference 16). Fütterer et al. (17) propose that ORFs 2 and 3 of the RTBV (and other badnaviruses) are translated by a permeable scan due to the pause or poor context of the initializing codons in the ORFs 1 and 2. It is also suggested that the rich leader sequence in AUG it is ignored, at least in the RTBV, by means of a mechanism of "deviation of ribosomes" (18). The leader sequence of the BSV is rich in AUG codons. ORF 1 has four AUG codons which is more than other badnaviruses but all are in a poor context (19); the only AUG codon in the ORF 2, which in its beginning, is also in a poor context. Therefore, although there are more AUG codons than in other badnaviruses in the ORF 1 it is very likely that the strategy for expression is similar to that described by Fütterer et al. (17) The presence and configuration of the functional domains in the ORF 3, with the domain rich in cysteine agglutinating the RNA similar to a zinc appendage which is common in all the retroelements (20), AP, RT and RH that are found in the ORF III is identical to those found in other badnaviruses (Table 2). This ORF also contains the additional cysteine portion characteristic of all other badnaviruses so far sequenced. The function of this extra sequence of "cys" is unknown but may be involved with translation control (21).

The phylogenetic analysis of the currently available badnavirus sequence shows that the BSV is much more related to the ScBV and the other badnaviruses with "three ORFs" than with the RTBV. The sequence is sufficiently different from those of other badnaviruses to be considered a different virus.

The 5 'end of the exact copy of the transcript correlates for the cytosine / adenosine doublet in 7260/7261, a relatively similar position in the genome as in CoYMV (3) and in the + 25nt with respect to the potential TATA block. There is also a sequence similar to as -1 as described by Medberry et al (3) for CoYMV (23) (Table 2). A potential polyadenylation signal (AAATAAAAA) is found in the 7295 bp that produces an nt terminal redundancy for the total length of the exact copy of the transcript. This terminal redundancy is lower than those of the other plant pararetroviruses (for example CoYMV 109-132 nt, RTBV 215-216 nt, CaMV 176 nt; 3, 23, 23) but of a similar size for some of the terminal redundancies of the retrovirus (mouse mammary tumor virus, 15 nt; avian leukosis virus 20 nt; 24). Although it is not possible to correlate the 3 'end of the exact copy of the BSV transcript, it can not be in the 3' direction of the tRNA binding site since this may affect the mechanism of replication. The 5 'end of the exact copy of the transcript is 128/127 nt from the tRNA binding site but they are not found in portions for the polyadenylation signal in the 3' direction in 7361.

References for Example 1 and cloning of the Banana Streamer virus promoter. 1. Lockhart B.E.L. Phytopathology 76, 995-999. 1986. 2. Lockhart B.B.L. Phytopathology 80,127-131. 1990. 3. Medberry, et al. Nucí Acid Res. 18, 5505-5513. 1990. - 4 . Hay, et al. Nucí Acids Res. 19, 2615-2621. 1991. 5. QU, et al. Virology 185, 354-364. 1991. 6. Bouhida, et al. J. Gen Virol. 74, 15-22. 1993. 7. Hagen, et al. Virology 196, 619-628. 1993. 8. Lockhart B.E.L. and Olszewski N.E. In: Breeding Banana and Plantain for Resistance to Diseases and Pests. J Ganry (ed.). CIRAD / INIBAP, Montpellier, France, pp. 105-113. 1993 9. Bejarano, et al. Proc. Natl Acad. Sci. 93, 759-764. 1996. 10. Richert-Poggeler K.R. and Shepherd R.J. Virology 236, 137-146. 1997 11. LaFleur, et al. Phytopathology 86, S100-S101. nineteen ninety six. 12. Sambrook, et al. Molecular Cloning:: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. 1989. 13. Devereux, et al. Nucí Acid Res. 12, 387-395. 1984. 14. Li, et al. Molec. Biol. Rep. 12, 215-220. 1994. 15. Feinberg A.P. and Vogelstein B. Anal. Biochem. 132, 6-13. 1983. 16. Fütterer, et al. J. Virol. 71, 7984-7989 1997. 17. Fütterer, et al. J. Virol. 70, 2999-3010. 1996. 18. Lutche, et al. EMBO J. 6, 43 -48. 1987. 19. Covey S.N. Nucí Acids Res. 14, 623-633. 1986. 20. Hull R. Ann. Rev. Phytopathol. 34, 275-297. 1996. 21. Lam, et al. Proc. Natl. Acad. Sci. USA 86: 7890-7894. 1989. 22. Lam E. and Chua N.H. Plant Cell 1, 147-1156-1989. 23. Medberry, et al. Plant Cell 4, 185-192. 1992. 24. Bao Y. and Hull R. Virology 197, 445-448, 1993.

. Guillley, et al. Cell 30, 763-773. 1983. 26. Coffin, et al. In Virus Taxonomy: Classification and nomenclature of viruses. Eds Murphy F.A., Fauquet C.M. Bishop D.H.L., Ghabrial S.A., Jarvis A.W., Martelli G.P., May M.A. and Summers M.D. Pp. 193-204. 1995 Springer Verlag. 27 Comstock J. C. and Lockhart B.E.L. Plant Disease 74, 53 0. 1990 EXAMPLE 2 - EXPRESSION FOR A HETEROLOGICAL SEQUENCE OPERATIONALLY LINKED TO A BSV PROMOTER All the conventional methods of molecular biology are from Ausubel et al. , Current Protocols in Molecular Biology, Wiley, 1996.

Subcloning of the BSV leader / promoter fragment In a portion of the BSV genome (fragment length of 1614 bp), it is amplified by the plasmid pSac21 by a polymerase chain reaction with the addition of suitable cloning sites for the creation of a chimeric gene.

The primer defining the 5 'end of the promoter / leader element is 5'-GCCAGATCTAAGCTTCCCGGGATAATCAGAACTGACAGTCA-3'.

The primer defining the 3 'end of the promoter / leader element is 3' -GCCCCATGGATTGTATGCAAGGTGAA-3 '.

This region of the BSV genome comprises the promoter and the leader (pBSV / BSV1) for the expression of the BSV genes. The BglII and Smal sequences are added upstream of the promoter element, and a Ncol sequence is added at the end of the BSV leader region. This fragment is cloned with its free ends into the EcoRV sequence of pBluescriptKS + (Stratagene, La Jolla, CA, USA), and various clones are sequenced to identify one with the correct sequence.

Structuring the pBH850 To prepare a LUCintron chimeric gene, a PCR overlap strategy is used, in which three different fragments are generated as follows: 1) Amplification of fragment (1) of pJJ3792 (source of LUC) with the primer set in NC389 ( 1) and NC392 (4) to give a 5 'portion of the imbricated CDS LUC fragment (2); 2) Amplification of fragment (2) of pAR4401 (source of intron st-ls ivs2, described in Vancanneyt et al., (Mol Gen Genet, 220, 245-50, 1990)) with primers NC390 (2) and NC393 (5) to give the intron st-lS ivs2 overlapping the fragment (i); 3) Amplification of fragment (3) of pJJ3792 with primers NC391 (3) and NC394 (6) to give a portion in the 3 'direction of the overlapping LUC fragment cds (2).

All reactions were carried out with the Pfu polymerase using 20 cycles. Subsequently, lμl of the fragment (1) and lμl of the fragment (2) are mixed and amplified using the Taq polymerase (15 cycles) with the primers NC389 and NC393.

In a separate reaction, lμl of the fragment (2) and lμl of the fragment (3) are amplified under the same conditions using primers NC390 and NC394 to produce fragments (1-2) and (2-3). In the final reaction, lμl of the fragment (1-2) and lμl of the fragment (2-3) are mixed and amplified using the Pfu polymerase (15 cycles) to generate the full-length product using the NC389 primers and NC394. This fragment is cloned into a pUC-based vector in the manner of a fragment of inactive ends, and the sequence of an isolated product is confirmed. Primers: 5'-NC389 AACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCCCT- 3 'NC390 5'-GTAAGTTTCTGCTTCTACCTT-3' NC391 5'-GTGGCCCCCGCTGAATTGGAA- 3 'NC392 5'-AGGTAGAGCAGAAACTTACCTGATACCCTTTGTATTTA --3i' NC393 5'-TTCCAATTCAGCGGGGGCCACCRGCACATCAACAAATTT-3 '5'-NC394 ACTCTAGATTACAATAGCTAA-3 The resulting LUC-int sequence including the complete luciferase CDS is used to construct pBH850, which has the LUCint sequence linked to the SpMas promoter (structure 6 of Ni et al., Plant J., 7, 661-76, 1995) and the 3 'end we (Bevan et al., Nuc Acids Res, 11, 369-85, 1983). The sequence LUC-intse isolates as a Ncol-Xbal fragment, after digestion and gel purification. A pUC plasmid having the LUC sequence (de Wet et al., Mol Cell Biol, 7, 725-37, 1987) bound to the SpMas promoter and the 3 'end is digested with Ncol and Xbal and the fragment containing the vector , the promoter and the 3 'end are gel purified. These two fragments are ligated together to produce pBH850, which comprises a chimeric SpMas-LUC-int-3 'gene.

Structuring a chimeric pBSV / BSV1-LUC gene . The pBSV / BSV1 fragment is excised from the plasmid described above using Xhol and Ncol, and the fragment is gel purified. A plasmid based on pUC (pBH850) containing a chimeric SpMas-LUCintron-3 'gene is digested with PstI and Ncol to release a fragment containing the LUC-INT CDS and nos • 3 'end, which are purified in gel. PBluescriptKS + is digested with PstI and Xhol, and the vector fragment is gel purified. The three fragments are ligated together to create a plasmid derived from pBluescript with the BSV / BSV / LUC-INT / 3 'chimeric gene.

Plasmid isolates with the expected pattern of digestion restriction are identified. One of these isolated products is named pMM7393 and is retained for a transient analysis for expression. Plasmid DNA is prepared using a conventional alkaline lysis method.

Reference Plasmid for Expression As a reference plasmid for the transient analysis of expression, a pUC-based plasmid (pJJ3792; available from DNA Plant Technologies, Oakland, CA, USA) contains a CaMVp35S-LUC-3 'gene we chimeric is used. Plasmid DNA is prepared using a conventional alkaline lysis method.

Biolistic control plasmid As an internal control for bullet-to-bullet variation for the biolistics of the DNA supply, a pUC-based plasmid (pP07119) comprising a 2xCaMVp35S-GUSintron-3 'or chimeric gene is used. This is one of a wide range of similar plasmids that can be used as a biolistic control for banana, including plasmids such as pZO1052 (which has a chimeric CaMV 35S-GUS gene, Ha et al., Plant Cell Rep, 11, 601-604, 1992) or pBI221 (which has a similar chimeric gene, Clontech, Palo Alto, CA). Plasmid DNA is prepared using a conventional CsCl gradient method.

Biolistics of DNA supply Cultures of banana suspensions that are derived from male flowers of cv. Grand Nain, and remain in liquid according to Grapin et al. (In Vitro Cell, Dev. Biol.-Plant, 32, 66-71, 1996). The cells in suspension are removed from fresh cultures in suspension, the cells in suspension are seeded successively through a nylon mesh to produce a fraction between 250 μm and 600 μm, and the embryonic fraction of the suspension is then extended in thin form on the filter paper discs. Approximately 20-30mg of cells in suspension are used for the biolistic bullet.

The plasmid 7DNA is precipitated in tungsten particles using a conventional step of precipitating CaC12 / spermidine (Vain et al., Plant Cell Rep., 12, 84-88, 1993). 20 μg of each LUC gene plasmid is mixed with 10 μg of control GUS plasmid and coated in 2 mg of tungsten particles. 3 μl of suspended tungsten particles (taken from 15 μl in a single preparation) are supplied per shot, when using a particle inflow gun (Finer et al., Plant Cell Rep., 11, 323-28, 1992 ).

Enzyme titrations The cells in suspension are incubated for 19 hours and then the luciferase activity (result of the expression of the chimeric LUC gene) is monitored using a program for the counting of isolated photons in a Beckman scintillation counter. The beta-glucuronidase activity (resulting from the expression of the chimeric GUS gene) is quantified by determining the fluorescence of the methyl-umbelliferone that is released from the methylumbelliferyl-glucuronide substrate. 12 DNA introductions are carried out in each preparation; 8 of the samples are taken for their luciferase assessment, and 4 of the samples are taken for glucuronidase titration.

Results The results demonstrate a good expression that is driven by the BSV promoter in plant shown in Table 4 below.

Example 3 - Detection of the BSV Virus isolation Musa plants with typical chlorotic veins are collected from Onne Research Station, UTA, Nigeria. Viral minipreparations are prepared from 5g of leaf tissue samples following the protocol of Ahlawat et al. (nineteen ninety six) . The identity of the BSV is confirmed by ISEM.

DNA extraction and sample preparation The DNA is isolated from the Musa leaf tissue by the method of Li et al. (1994). Crude extracts are prepared from growing leaves for a direct PCr and an IC-PCR by grinding it in a ratio of 1:10 (w / v) either in two different protocols. The first protocol uses 1 x phosphate buffered saline (PBS) / 10 mM mercaptoethanol as a milling medium, followed by centrifugation at 13,000 g for 1 minute. The supernatant is received and used in a subsequent evaluation. The alternative protocol uses PBS / 2% polyvinyl pyrrolidine (PVP) / 1% sodium sulfite as a milling medium followed by filtration through glass wool. The resulting filtrate is used.

For DB-PCR, the plant extracts are prepared by the second method using a carbonate buffer (1.59 g Na2CO3, 2.93 g NaHCO3, per liter, pH 9.6) as a milling medium.

PCR and Primers The 50μl PCR reaction mixture contains 1x buffer supplied by (Gibco, BRL), 200μM each of dATP, dCTP, dGTP, d-TTP, 2.5mM MgCl2, 10 pmoles of a reverse primer and a progressive primer , 2 units of Tag polymerase (Gibco-BRL). PCR conditions are 94 ° C for 1 minute, (94 ° C x 1 min, 50 ° C x 1 min, 72 ° C x 2 min) x 33 cycles and a final extension at 72 ° C x 5 min. The primers used to detect the BSV are the pair v3012-vl573 of which the sequences are given above. They are selected from aligned sequences of amino acids corresponding to the AP and RT regions derived from the isolated Onne BSV sequence. The primers have been analyzed for amplification of a precedent product of 644 bp and for its specificity to BSV which has been confirmed by Southern hybridization and a sequence analysis of the product (Harper et al., 1996). Musa nuclear DNA is detected by PCR using the pair of primers 5'ACTAAAACGCCTATAACTCC and 5'GCTCCAATACCCATAAGAA to amplify a 260 bp fragment of a repetitive intermediate sequence of Musa (Baurens et al., 1996).

A region of the ACC oxidase gene encoded in the nucleus is amplified using the degraded primers for two regions that are conserved ASFYNPGS (progressive 5'GCGTCGTTCTAYAACCCGGTAGC) and EPRFEAM (inverse 5 'SCYWNCGAAKCTT GGMWCC). EMBL entry x91076, accol gene from Musa acumina ta.

Mitochondrial DNA is detected using a pair of primers n lB-nadlC to amplify an intron sequence of the mitochondrial nadl gene (Demesure et al., 1995). Chloroplast DNA is detected by PCR using the pair of primers tbcLPl-a pBPl to amplify the intergenic region between rbcL and tpB (Al-Janabi et al., 1994).

IC PCR and DB PCR For IC-PCR, the rabbit polyclonal antiserum for BSV or a mouse polyclonal antiserum for BSV are used for coating. Sterile 0.5 ml polypropylene microcentrifuge tubes are loaded with 25 μl of purified IgG (1 μg / μl diluted to 1/500) in a carbonate buffer, and incubated at 37 ° C for 2 hours or at 4 ° C for the night. After three washes with PBS / 0.05% Tween-20, 25 μl of a plant extract is loaded and incubated at 37 ° C for 2 hours or 4 ° C overnight. For DB-PCR the plant extract in the coating buffer is added directly to the tubes without a previous coating with anti-BSV IgG and incubated at 4 ° C overnight. For both techniques the tubes are washed similarly with PBS three times and dried briefly. The PCR is carried out directly on the tubes, as described for PCR, without a specific interruption treatment for the virion particles.

Analysis and RE analysis of PCR products After amplification, 10-15μl of the PCR reaction products are separated by electrophoresis through 1.2% agarose in either lx TBE or lx TAE. After the agglutination of ethidium bromide, the DNA is visualized in an ultraviolet transilluminator. DNA markers are supplied by Gibco-BRL. The PCR products are directly restricted in the PCR reaction mixture by the addition of 0.5μl of the appropriate restriction enzyme to 10μl of each PCR reaction and incubated at 37 ° C for 1 hour before electrophoresis of the full sample.

Southern Hybridization The products of the reaction are separated by electrophoresis through a 1.5% agarose gel and detected by the fluorescence of the ethidium bromide under ultraviolet light. After photography, the products of the reaction are transferred to a nylon membrane (Hybond-N, Amersham). A probe labeled with 32p is synthesized by random priming when using the full-length product of the BSV PCR as a template (Feinberg and Vogelstein 1983). Hybridization and stringent washing are carried out at 65 ° C using the solutions and protocols described by the membrane supplier. The autoradiography is carried out as described by Sambrook et al. (1989).

Cloning and Sequencing The PCR products are cloned into a TA vector (Invitrogen) followed by a purification using a set of implements for the preparation of the PCR according to the manufacturers protocol (Promega). All DNA manipulations were carried out according to Sambrook et al. (1989). The plasmids containing the cloned grafts of the PCR product are sequenced by the chain termination method of dideoxynucleorides (Sanger et al., 1977) by using the Secuenase version 2.0. (United States Biochemicals) and the forward and reverse M13 primers.

Results Conventional PCR and amplifi cation conditions The expected 664bp BSV DNA fragment is amplified from leaf tissue samples that are harvested from plants grown in the fields with or without the characteristic symptoms of the chlorotic or necrotic stria of BSV. The same amplification product is also obtained from samples of leaves collected from plants grown in a greenhouse or in a cold room (22 ° C) again with or without the symptoms of BSV. More interestingly, the expected amplified product is obtained from tissues that are harvested from seedlings in vi tro. The amplified product is hybridized to the cloned BSV probe and the hybridization signal is detected after highly stringent washing conditions. The sequence of the PCR product is identical to that of the episomal BSV sequence. No product is amplified from extracts of nucleic acids from the traveler's palm. { Ravenala madagascari ensi s) or Heliconi um sp. nor of samples without a DNA template. Hybridizations using a cloned fragment mated with digoxigenin, from a BSV isolation indicate a seemingly low homology between the probes and the amplification products. Even under mild washing conditions, PCR products that are amplified from some clones that appear intense in gels stained with ethidium bromide produce only mild hybridization signals.

The optimal conditions for amplification are determined. At a lower set temperature (37 ° C) that is used in the initial cycles of the PCR program, they lead to non-specific amplification products which are visible in the gels. However, only a single PCR product is detected after Southern hybridization. Running all 40 cycles with a fixing temperature of 55 ° C leads to a slight decrease in the intensity of the bands observed in agarose gels. A fixation temperature of 60 ° C greatly reduces the intensity of the amplified products of the banana samples that are analyzed. Altering the concentration of MgCl2 between 4 mM and 2.5 mM has little effect on the observed intensity of the amplification products. A concentration of 1 mM MgCl2 results in an inconsistent amplification of the 664-bp DNA fragment. Under optimal conditions, a product is easily obtained from 0.1 picograms of full length BSV DNA.

Boiling the DNA template with the primers, followed by ice cooling, increases the sensitivity for the detection of ScBV in most samples of sugarcane (Brait waite et al., 1995). This procedure is not necessarily for the fixation of solutions containing either the cloned genome of the BSV virus or the purified virions of the BSV.

Comparison of techniques for indexing the BSV Twenty plants are examined for the typical BSV symptoms and samples are taken from leaves that show different appearances. The typical chlorotic or necrotic symptoms of BSV are shown in nine plants (13 symptomatic samples), the remaining 11 are asymptomatic (23 asymptomatic samples) (Table 3). ISEM detected BSV in 10 of the 36 samples, whereas BSV was detected using a spot blot hybridization assay in only 6 of the 36 virus miniprep preparations. However, PCR generates amplification products of the expected size (644 bp) in 32 of the 34 viral samples of minipreparations and produce strong hybridization signals with Southern analysis when using a BSV-derived probe. The two PCR negative samples (TMP3x 15108-1 and FHIA-1) are also negative to ISEM (Table 3). These results indicate that PCR is a much more sensitive technique for indexing BSV than the use of symptoms, ISEM or blot hybridization.

IC PCR and DB PCR Both the PCR of IC and the PCR of DB consistently amplify the product of 664 bp specific for BSV from raw leaf extracts or from viral minipreparations that are prepared from Musa spp. infected with BSV. No similar product is amplified from raw leaf extracts prepared from other plant species (traveler's palm, tobacco, Heliconi um sp., Sugar cane) or PCR solutions that do not contain a DNA template.

Pre-treatment of the tubes for one hour at 37 ° C with either 1% BSA or 5% evaporated skim milk can reduce the apparent agglutination of the virus both in the IC- and in the DB-PCR. In contrast, Img / l of herring sperm DNA as a blocking agent had no effect on viral agglutination.

The intermediate nuclear sequence of repetitive DNA of Musa, the mitochondrial nadl gene and the DNA of the intergenic region of the chloroplast can be detected in the genomic DNA of Musa. However, they can not be detected in IC-PCR tubes where the PCR sequences can be detected.

The comparative results of 10-fold serial dilutions of crude leaf extract indicate that IC-PCR and DB-PCR are at least as sensitive, and for some samples, more sensitive than conventional PCR probably due to the removal of inhibitory substances by the washing steps in the IC- and DB-PCR.

The sensitivity of IC-PCR and DB-PCR to detect BSV is compared with that of conventional PCR, ELISA and ISEM using viral minipreparations from plants previously infected by ISEM and ELISA: the sensitivity of PCR-based methods are comparable with others and they are much better than for the other detection methods. The IC-PCR distinguishes the integrated BSV and the episomal sequences of the BSV and is therefore useful in the context of the present invention.

Discussion Despite the concentrated efforts of many researchers (Lockhart and Olszeswki, 1993; Harper et al., 1996; Braithwaite et al., 1995) and international centers (INIPAB, 1995), reliable detection of BSV from infected plants it has become a serious impediment to the safe mobilization of improved Musa germplasm. At present, there is no report or development of protocols for reliable detection of BSV from in vitro shoots that are suitable for the movement of germplasm. Our results presented in this study demonstrate a PCR technique that detects BSV from these suckers in vi tro and is suitable for the initial large-scale detection of Musa in vi tro germplasm, therefore reducing the number of 'BSV-free' plant materials for greater confirmatory indexing.

The methodology used here is to use a "compound" antiserum that is generated against several different BSV isolates to trap the viral particles and a subsequent PCR with the primers based on the sequence of a Nigerian BSV isolation. The degraded alternative primers are available (Lockhart and Olszes ki, 1993) for the amplification of isolates with a widely varied sequence. The washing steps after immunocapture effectively remove the inhibitory substances from the PCR which improves the reproducibility of the results. A detailed comparative analysis of all degraded primers that are reported in combination with the restriction analysis can be useful information regarding the variability of BSV sequences and the possibility of differentiating and detecting all BSV isolates.

The PCR primers and the conditions described herein provide a specific, sensitive method for diagnosing bananas / plantains infected with BSV I from both in vitro propagated or field grown plants. This method is currently applied to detect and probe plantain germplasm from quarantine plant materials, and in the field to ensure the full extent and distribution of BSV infection.

Integration The health conditions of infected plant material for BSV only through PCR can be questioned due to the possibility of integration of BSV DNA into Musa spp. (LaFleur et al., 1996). By using the degraded primers, the integrated BSV sequences have been detected from Musa germplasm collected from around the world (LaFleur et al., 1996). Other results with Musa DNA PCR using specific BSV primers confirm that essentially all Musa cultures appear to have integrated BSV sequences. Additional evidence is shown by a Southern genomic analysis when carefully isolated, the very high molecular weight Musa DNA hybridizes to the BSV sequences. The apparently high degree of infection of Musa sp. For the BSV shown in this study, it is mimicked by the widely distributed infection of sugarcane by the related ScBV (Comstock and Lockhart, 1990, Braithwaite et al., 1995).

Our results show that the episomal virus can be detected in a specific way with a high sensitivity and specificity. Nuclear, mitochondrial or Musa chloroplast DNA that may contain possible integrated sequences of BSV are not captured by the antiserum or by the tubes in which the titration is carried out.

Development of Valuation The ease of sample preparation for this technique is adequate for handling a large number of samples. Under limited conditions in a laboratory facility, either the empty Eppendorf tubes and those that are coated with antibodies can be carried by hand or sent by collaborating scientists, and the tubes can be returned after loading the samples and washing the complete PCR assessment, A similar system for virus detection by mail-order ELISA has been reported to be successful (Gaikwad and G. Thottappilly, 1988).

Spot blot hybridization can be a more efficient detection system, and economically and technically less demanding to handle a large number of samples. However, our results show that the ISEM, ELISA and PCR evaluations are much more reliable methods than this assessment and confirm the results of Lockhart and Olszeswki, (1993).

The ELISA test is not sensitive or reliable enough to detect BSV, (a low titre virus) in infected plants without the characteristic symptoms, and its broader application is limited. However, the ELISA test has proved useful in confirming the BSV infection of symptomatic plants. The ISEM has proven to be sensitive enough to detect the BSV of plants with a low BSV titre. Our results that are presented in this study also show that PCR linked to serological techniques provide a rapid and sensitive assessment system.

The possible improvements for the current PCR protocols can allow a wider application of Musa virus diagnosis. For example, CMV infection of Musa is generally observed under field conditions as a single or mixed infection with the BSV. Symptoms caused by CMV are usually confused with those caused by BSV. An IC-RT-PCR is being developed for the detection of CMV with the ultimate purpose of developing a simultaneous detection of both BSV and CMV in a single test tube from individual samples.

PCR Due to its high sensitivity, PCR titers can produce false negative or false positive results. ' The small-scale DNA extraction procedure allows the detection of a very small portion of tissue. The distribution of the virus may not be uniform in all portions of the tissue of a plant, for example in some portions of the leaf tissue the viral particles may be absent. Therefore, if the valuation of an entire plant is based on a single small sample, it is possible that it results in a false negative. This problem can be reduced, however, by taking multiple samples of different leaves and combining them into composite samples. Our experiments so far have not shown that viruses are absent from certain parts of the leaf, and the leaves are an appropriate tissue for their assessment.

Experimental errors (eg contamination) or non-specific amplification are possible sources for positive "false" results. Hybridization of the DNA of the amplification product with the homologous probe rules out any possibility of a false positive. These problems can also be minimized by multiple analyzes of individual plants and the use of independent confirmatory assessments (eg, ISEM) for reliable diagnosis.

Our results show a rapid, sensitive and accurate assessment for episomal BSV. So far, the ISEM is the confirmatory diagnostic analysis especially for the international movement of Musa germplasm (Diekmann and Putter, 1996). However, since a larger application for the ISEM to index large numbers of samples is limited by the work, equipment requirements and time required to carry out the assessment, the IC-PCR should be considered as its replacement rating. .

References to Example 2 and detection of banana striator virus. 1. Ahlawat, et al. Plant Disease 80, 590-592. 1996 2. Al-Janabi, et al. Theor. Appl. Genet 88, 933-944 1994 3. Baurens, et al. Mol. Gen. Genet. 253, 57-64. 1996 4. Bouhida, et al. Journal of General Virology 74, 15-22. 19.93. 5, Braithwaite, et al. Plant Disease 79, 792-796. 1995. 6. Comstock, J, C, Lockhart, B. E. L. Plant Disease 74 1990. 7. Demesure, et al. Molecular Ecology 4, 129-131. 1995. 8. Diekmann, M., Putter, C.A. J. PAO / IPGRI Technical Guidelines for the Safe Movement of Germplasm. No. 15. Musa. 2nd Edition. Food and Agriculture Organization of the United Nations, Rome / International Plant Genetic Resources Institute, Rome, 1996. 9. Feinberg, A. P., Vogelstein, B. Analytical Biochemistry 132, 6. 1983 10. Gaikwad D.G. and Thottappilly, G. J. Phytopathology 121: 366-369. 1988. 11. Hagen, et al. Virology, 196, 619-628. 1993 12. Harper, et al. Detection of banana streak virus. In (Ed) Marshall, G. Diagnostics for Crop Protection. BCCP Proceedings 65, BCCP, Surrey, UK. pp 47-51. 1996 13. Hay, et al. Nucleic Acids Research 19, 2615-2621. 1991. 14. LaFleur, et al. Phytopathology 86, S1OO-S101. nineteen ninety six . Lassoudierre, A. Fruits 29, 349-347. 1974 16, Li, et al. Plant Mol. Biol. Rep. 12, 215-220. 1994 17. Lockhart, B. E. L. Phytopathology 76, 995-999. 1986 18. Lockhart, B. E. L. Phytopathology 80, 127-131. 1990. 19. Khart, B. E. L., Olszewski, N. E. Serclogical and genomic heterogeneity of banana streak badnavirus: implications for virus detection in Musa germplasm. In Ganry, J. (Ed.) Breeding banana and plantain for resistance to diseases and pests. CIRAD / INIBAP, Montpellier, France, pp. 105-113. 1993. 20. Medberry, et al. Nucleic Acid Research 18, 5505-5513. 1990 21. Qu, et al. Virology 185: 354-364. 1991 22. Sambrook, et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY. 1989. 23. Sanger, et al. Proc. Natl Acad. Sci. USA 74, 5463-5467. 1977. 24. Takahashi, et al. Phytopathology. 83, 655-659. 1993. 25 Table 1 . Comparison of the nucleotide homology of the Badnavirus.

Table 2, Table 3. Comparative detection of BSV.

Table 4 It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Having described the invention as above, the content of the following claims is claimed as property: ID NO: I TGGTATCAGAGCAAGGTTCGTTTTTATGGCTTTCATGGGGTAATTCCTTT AGATAGGAGCCGAAGGGCTCTGCTTTTCTCTAATTGAGTTACAAGTTTAT GATTTAAATTGTTTAAATTGGAGCTGTATTCAGTCTTTCTTAGAAAAATG AGCATGATTTCGATTATAGCTGGTCAAGGCTGTAGGGAAAAATGATTATG TTTTATGCTAGTTGGTCCAAGAGAGCATGCCTACCCAAGAAAAAGTACCC GAAGAGAATGGGGGAAAAATTGGTTCTTCGCATGTATGAGGATAATATCC TAGAAACAGAACATAACCTGCGTGAAGTACTTACAAAGCCAAGATAATAT CCTATATAGAAACATGGGCATCGTGAAGTATAAGACTGACTGAACTACAA GCTCCTTTATAAACAAAAGGATCATAGACCTCTGTACGTCAATACGGGTT AAGCATCCTGGAGGAGTACTCCTATCTAGTTTACGAAAAGAAAGTCATTC ACCTTGCATACAATTTATGGTCGAGAAAACTTGGGATCAAAAATTCCAGG AATTTCTAAACTCATCTGAACTCACTCAAGCTCAACTTGAGTATCTTGAC TTGGCAACGGAAGCCAAAGTCTCAAACAAAGATCTTGCTCATAATTTGCA CATCAACACTTATCGACTAAGCCTTACAGGGAAGGTCCTTTGGACTTCTG GAAGGAAAAATCGGGATTTACTTGTGCAGATAATCGGTGAGCAGGAGGCT CAAAAGAAGGACTTATTGGAGTTGCAGAACTTAAGTAAGATTGTGCGCAG CCAGCGTAACGATCTTAAGAAGGCTCACGAAAAGCTGGACGTCTTGTCTG ATGAGCTTAAAGCTCTCAGAAAGGACTATCTGAAGAGGCGCCCCTTGAGC AAAGAAGACGTTGAGGAATTAGTTTTGCGTATCTCTGAACAACCTAAGTT CATAGAGAAGCAAACTGAGGCCTTAACTGAGGAGCTGACTAAAGAAGTTC AAAATCTGAAAAAAATTATTCATGACTTTGAGCGAAAGCTCATGGGATGA GTAATTCTATCACAAGCTCTGCAGTATACCAACAAGCAATTGCTGGAACA ACTGGTGACTGGGAATCCCCTGGAGTAGGGATATCTGACCGTGGATCGGT GAACAACACCCAGCTCACAAGACAACTCAACACCATTATATTCCTGTGTA CAAAGACACAACAGGAAGTCTTAGCCTTAAAAGACACAGTAGCGGAC TC CAAAACCGCTTGAGAATACTTGAAAGGACCGGTGCAACATCAGCCGGTAC CCCACAACTTAAGGGTGAAATTGACGCCATCAACGAGAAGTTATCAAGAA TTCAACAAATTCAAGGGAGTCAACCAAGGAAAGACGGTGGTACTGCGGCC ACTAGCAAAGTATTTCAAGACCCCTACAAACTTCTCAGAAATTTGAAGTA ATGGCTCAAAGACCCAGAATTACCGGATCAAGAACCACCACTGCTGAAGA AGGGACACCCTTAATTGACGACCAAATAAGGGAATATAGAAGCTCCAGAA GAGCAGCATACGAAGCCCAACGAATTGCCAGGCAGACCGGCAATATTATT GGAAGGGTTGTAGGACGACAACCCCGGGAGCATACACTATCTATGGTGGT AGACCCGAATTCGGAGCTCAGCAGAAGTCTTGCCCACAGAGCAAGAACAG TACCAGGAGAGGTACTATATATGACTCAACGAGACAGTCCTGTAAATAGA ATCTACAGAAACAGGACAGAAGAAAGAATGCTCGTAACCAACGGGCAACA AGATAGATCTTTCATATATCAAGAATCATTTGAGGAGCTAGCCTCAGCAG GATTTGAGTACATCCATCTGGGTGTATTACAAGTCAGGATCCAGATCATG CATAGGACATATGCAGGCACGATGGCCTTAATAGTCTTTCGTGATACACG ATGGACTCAGGAAGGAGAAGAAGGCAGATCCATTATTGGAGCTATGGAGG CTGATCTGTCACAAGGCCACCAACTAATCTACGTCATCCCGGATATAATG ATGACCATAAGGGATTTCTACCAACATGTACAAATCAGTATCCTAACTAA GGGATACCAGGGATTCCAAGGTGAAGCAAATCTGCTGATTACCAGGAGCT GTAGATGCAGACTCACCAATGTTCCCAACGTCGGATTCGCCTATAATATT CAAAGAGTAGTGGAGTACTTAAACTCCAAAGGCGTTAAAGCTATACAAGC TCAGAAGCTGAGTACAACAAAGTATCAGGGCACTGAATGGAACATCAAGC CCTCCAATGTGGTTGTACCAATGCAACCAACGAATTTGATCACCAGAGTC AACTACGACAACTCACGGAGTATACGATTTGGTAATTATCAAGCAAGCAC ATCCTCAGCGCCACCAAAATACAACGAAGATGGAGATTCAGATGATGACA TCCAAGCCACAATAGAGCATGTTAACATGCTTTATATTGAAGATACATCT GATACTGACTATCCAGTCATGGCAGCTGAAGAGGAAATCTTTCCTCTTGA AAACATGGTGGGAGAAGATGACATTATCTCCCAATTTTTGGAAAATTTGG ATATCACGGATGATGAGGAAGATTCCAGATCACAATACGTGATGAATCTG GAAGACAATGAAGAATTCCCACAACTCAGGGAAATTGAAAAAGTCTTATC CTCAGTAGCTGAAACAGCAATCAGTTCATATAGACCACCAGATGCTGAAA TGGGTGAAGAAGCACCCGCATATGCACCAGCTACAAGTGCAACAGGATGG GCTGGATCAAGGCCTTTCCCCTTTATGCCCAAAGGAGGACCAAGGAGGTG GGATTCCAACAATGAATTTTATTCATTACCTCCAGCACAAAGTCGGCAAG GAGCCATGTTCGTCATGCCAATGGACTTTGACATCAAAGTATTCGAAAGG TGGGAGAGCATTACCCTCCTACACATGACGGAAAAGATTTTTGATAATGC TGACGACAAAATGAGATACATGGAGAACCTTCTCGGAGAAGACGAGAAGA AGCACTTCATTGAATGGAGGATGAAGTATACAGCAGAATATGAGACAATG AAAGCTCAAGCACTCGGAGACCAAGGTACACAAAATATCATCAATCAAAT TCGATTGATATTCTTTTTGGAAAATCCGCAAGTAGGAACTACTACTACTACAC AAGATGCGGCCTATAAAACCTTGAAGAGCCTGGTCTGCACAGAGATCACA GACACAGCAATCTACAGATACATGAATGATTATTTCCATCTGTCAGCCAA AACAGGAAGAGCATGGGCCTCAGAAGAATTATCCAAGGAATTCTTTACCA AACTACCAAGAGGTCTAGGAGATGAGGTTGAAAAGGCATTCATGGAAAAA CACCCAAGTAACACAGTAGGGATCACCGCAAGGATCACCTTTACCAAAAG ATACCTAAAGGAACTGTGTGAAAAGGTAGCACTACAAAAAAGTATTGGCA AAATGGATTTCTGCAGAAGCACGCCAGTACATGGTTTATACAGAGACAAG TCATACAGAAAGTATGGAGCTAGAAAAAGTACATCCTACAAGGGAAAGCC CCATAAATCCCATGTTAGGATTGGTAAGAAGAAATATTTATCCTTGAGAA AGAAAAATTGCAGGTGTTATGCCTGTGGAGAAGAGGGACATTTCGCCTCT GAATGCAAGAATCCAAGAAAGATCATGGATAGAGTCAAGGTTCTGGACTC TCTAGACTTGGAAGATGGATTAGACGTTATCTCAGTCGGCTTTGATGAAG ATGACGTATCAGACATCTATTCAATAGATGAAGAAGCTGATAACTACAGG TTCACAAATGAAGAAATGGAAGGCTTCAAGAACTACGAGGTCTATATGTT AAGAATGGAAGAGATGGATGAGCCAAGGGAATATCTCGTAGGAGAACCAT CTGAATGGAGATCTAAGATGAAAGTCTCCAGAAGACAGTATTTCTGCAAG CATGAGTGGAAATTTGAAGAGACTCATGTGACTATCTGCAAGGCATGCGG ATCTGAAGCAGCTCCTAAGCATAGGATTGACTGCTTGAAATGTGAGATGA CTGTTTGTCTCATGTGCCAACCCTGGTTCTATTTTTTCGTCAACACTGAA GAAGTTAAGTTTTCCAGAGTTCGGATAGAAAGGGTAATTGATTGGAAGGA TATTGCACTAAAACAACTCGAAGTTCTCAAGACCAGCATTGCAAATGAGA AACAACTCTCAGAGGAAGTAGAAATCTTGAGGAAACAAAGCAAAGAGCTG AAGGAAAAAGAACCAATCATCTTTGAAGAAGACACGGAGGAAACAGCTCA ACTGATACAGAAGCTAGAAGACGTGGAAAGAGAAAATGAGCTTCTAAATA TCCTTATCAAGCAGAAGGAAAAGGATGAAATCCAATACCTCAATGAGATT ATAGAGCTCAAGGAAAGAATAAAAGATTTAGAGCAGCAACAGAAGGACAA GGAAGAACAAGTAAATGTCCTTGAAGAAGTCTCGATTAACGCTCTGAGGC CAAGGAACAACCATCTCAATATCAAATGTGAGATAGAAGTCAAAAACAAG AAGGTAGTCCTGAACGCAATTCTTGACACTGGAGCTACAGTCTGTGTAGC AGATGAGAGGATGATACCTTCAGGAATGAAAGAGCAGGCCAAAAACAAAA TCATTATTCGAGGAGTCAACGGAGTCACTGAAGTAAACGAGGTGACATCA GCGGGAAAGCTATGGGTTGGTAAGCAATGGTTCTACCTCCCTCAAACTTT TATTATGCCTTCATTAGCTGATGGAGTTCATATGATCATAGGCATGAATT TTATTAGAACTGTTGGCCTAAGGATAGAAAATGGTGAGGTCACAATTTAT AAGATCATGACAACAGTACAAGCCCCACCAATAGTTCATGAGCTGAATTA TATTGATGAACTAGAACTGGAACTTCATGAATACTATAACATATGTGCAG CTGAGAGTTCTAGAGGGGAAATTTCTGAAGAATTTATATCTCCTGACATT ATTGGAAAAATGAAAAAATTGGGATATATTGGAGAAGAACCTCTCAAACA TTGGGAGAAAAATCAGGTGAAATGTAGGATTGAAGTAAAAAACCCTGATA TGATTATTGAAGACAGGCCATTAAAACATGT TACCCCTACAATGAAAGAA ACCATGGCTAAGCATGTCCAGAAGCTTTTAGAACTTAAAGTGATCAGGCC TTCAAGCTCAAAACATAGAACAACGGCAATGATAGTAGAATCTGGGACAG AGGTTGACCCAATGACTGGAAAGGAAAGAAGAGGAAAGGAGAGATTGGTG TTTAACTATAAGAGGCTGAATGATAACACTGAAAAAGACCAGTACAGTTT GCCTGGAATTAACACCATTATTAAGAGAATTGGAA? TGCCAAGATCTATA GCAAGTTTGATTTAAAGAGTGGGTTTCATCAAGTAGCAATGGACCCAGAG TCTATTCCCTGGACGGCATTTTGGGCTATAGATGGGCTGTATGAGTGGCT AGTTATGCCCTTTGGACTTAAAAATGCTCCTGCCATATTTCAACGAAAAA TGGACAATTGCTTTAGAGGGACAGAGGATTTTATTGCGGTATACATTGAT GACATACTAGTATTCTCTGAAACAATACACCAACATAAAGAGCATTTGAA GAAATTTATGACAATCTGCGAGAAAAATGGTTTAGTCTTAAGTCCGACAA AAATGAAAATTGGAACAAGACAGATTGACTTCCTAGGTGCAACTATTGGA AACTCAAAAATTAAGTTGCAGCCTCATATTATTAAAAAGATCATCGAAAT GAAAGATGAAGAACTAAAGGAAGTGAAAGGATTAAGGAAATGGCTGGGAA TCCTTAATTATGCTAGGAGCTACATTCCGAAACTGGGAAAAATCCTTGGA CCACTGTATGCCAAAACGAGTCCTAATGGAGAAAGAAGAATGAATACTCA GGACTGGAAAATTGTCAAAGAGGTCAAGGAAGTTGTAGCTAATCTGCCAG AACTTGAGTTACCCCCCGAAAAAGCTATCATGATAATTGAAACGGATGGC TGTATGGAAGGCTGGGGAGGGGTATGTAAGTGGAAAACTGATAGTCTGCA GCCAAGATGGTCAGkAAAGATCTGTGCTTATGCGAGCGGGAAATTCACTC CCATCAAAAGCACAATTGACGCAGAGATACAGGCTGTAATAAACAGTTTA GACAAGTTCAAGATATATTATCTTGATAAGAAAGAGCTCATAATCAGAAC TGACAGTCAAGCAATAGTGAGCTTTTACAAGAAAAGCAGTGATCATAAAC CATCAAGAGTCAGATGGCTTGCCTTCACAGATTACATTACCGGGACAGGT CTTGAGATTAAGTTTGAACACATTGACGGAAAGGACAACGTTTTAGCAGA CACTCTGTCAAGACTGGTAAAGATTATTCTCCATCCGGAAAAGCATCAAT CTGAAGGTGTGTTGATCAATGCAGTGGAGGAGGTATTTCACAAGGGAAAC ACCGATGCAAAACAGAGAGTTAATGATGTTGTAAAAAGATATGAAGACTG GTTGACCAAAGGCTACAGGTTGCATCAAATCAATGTGCTAACACTAAGTG AAGAGCCGGTTTTCAAATGTGGATGCAATAAACCAGCAAAACTGAAGATC TCCAGAACATCCAGAAATCCTGACAGGGAGTTTTACTCCTGTGAAACTAA CACTTGTTTTACTTGGGTCTGGAAAGACAAATTGACTCGTTTMTGCAGGA AAAGATCAGATGGGAGAAGAAACTTGAAGAAATATCAGAAGACTCACTGT GGGAGGAATTACTGAGGGAGCAAGAAAATCTGCGTGCGAAACAAGAATAT CTTATTGAAGATGCTCTAGATCTGCTGGATATCAGTAATGATGACTGAAG CGGAAGTGGCGGACCCCTACCACGTGTTGATACCAACCGGTGTGAAGACT GATAAGATGCGGAGTGAGCTGGATACCACTCACTTTATGTAAAGAGGAGA CAAAGTATAATGTCTCTTTATTTTAAGTTTGTCGGTGTCGTTGTCTAGTC ACGCACGATGACCTTTAGTGACTTTGCAGGATTCTTACGCAAAGTTGTTA GGCCAGAGACATGTGATGATGCTTATCTGCATTATTGGTGGATGCCACCT AACGATGCCAGAAAGCTCCACAACTCTCTATATAAGGAGCCTTGTATTCA GGTTGCAAACACGCACCACAACGCGAGTTTACTCCTGATTTGAGAAATAA AAACTTCTGTGCTTGAAACACACTTTGTGCGAGTTCACTTTGTGCGAGTAGAGCGCAAGATCCTAGTTCCGCGAGCGTAGACCCGTC SEC:; 2

Claims (20)

1. CLAIMS 1 . An isolated polynucleotide characterized erqué s nsis e in the promoter sequence of the Banana Screener Virus that is selected from: (a) the promoter sequence of a Nigerian isolation of the Banana Streamer Virus shown in SEQ ID NO:. 2; (b) a promoter sequence for an isolation of the Banana Screener Virus whose promoter sequence is an allelic variant of the promoter sequence of the SEQ ID NO:. 2; (c) a fragment of (a) or (b) which, when operably linked to a transcribable sequence, promotes transcription of the transcribable sequence in a cell of the Musaceae plant.
2. 2. An isolated polynucleotide, according to claim 1, characterized in that said promoter sequence is shown in SEQ ID NO:. 23. An isolated polynucleotide consisting of a promoter sequence, characterized in that the promoter sequence: (i) hybridizes, selectively, under stringent conditions with a polynucleotide complementary to a polynucleotide which has the nucleotide sequence shown in SEQ ID NO:. 2; Y (ii) when operably linked to a transcribable sequence the transcription of the transcribable sequence is promoted in a Musaceae plant cell. Four . An isolated polynucleotide according to claim 2, characterized in that it has a nucleotide sequence that is found in a strain of the Banana Screener Virus. 5. An isolated polynucleotide characterized in that it consists of a promoter sequence, whose promoter sequence is at least 75% identical to the promoter sequence shown in SEQ ID NO: 2 and, when it is operably linked to a sequence transcribible, promotes the transcription of the transcribable sequence in a Musaceae plant cell. 6. A nucleic acid structure comprising the promoter sequence of a polynucleotide according to any one of claims 1 to 5 and a sequence that is not from the Banana Streak Virus. 7. A nucleic acid structure according to claim 6, characterized in that the promoter sequence is operably linked to a transcribable sequence. 8. A nucleic acid vector suitable for the transformation of a plant cell and including the promoter sequence of a polynucleotide according to any of claims 1 to 5. 9. A nucleic acid vector according to claim 8, characterized in that the promoter sequence is operably linked to a transcribable sequence. 10. A plant cell containing a heterologous polynucleotide, a nucleic acid structure, or a nucleic acid vector according to any one of claims 1 to 9. 11. A plant cell according to claim 10, characterized in that it has a heterologous structure of polynucleotides or nucleic acids within its chromosome. 12. A plant cell according to claim 10 or claim 11, characterized in that it is comprised in a plant, in a plant part or in a plant propagule, or in an extract derived from a plant. 13. A method for producing a cell according to claim 10 or claim 11, characterized in that the method includes incorporating this polynucleotide, the nucleic acid structure or the nucleic acid vector, into the cell by means of transformation. 14. A method according to claim 13, characterized in that it includes recombining this polynucleotide or this nucleic acid structure with the nucleic acids of the cellular genome in such a way that it is stably incorporated therein. 15. A method according to claim 13 or claim 14, characterized in that it includes regenerating a plant from one or more transformed cells. 16. A plant comprising a plant cell according to claim 10 or claim 11. 17. A part or propagule of a plant characterized in that it comprises a plant cell according to claim 10 or claim 11. 18. A method for producing a plant, characterized in that the method includes incorporating a polynucleotide, a nucleic acid structure or a nucleic acid vector, according to any of claims 1 to 9, into a plant cell and regenerating a plant from from another plant cell. 19. A method according to claim 18, characterized in that it includes propagating, sexually or asexually, or cultivating the shoots or a descendant of the plant that is generated from this plant cell. 20. The use of a nucleic acid vector according to claim 8 or claim 9, characterized in that it is used for the production of a transgenic plant.