# Patent

## Title
Isolated nucleic acid molecules from the genome of citrus sudden death virus and uses thereof

## Abstract

The present disclosure relates to nucleic acid molecules comprising the genome of the Citrus Sudden Death Virus (CSDV), the causative agent of Citrus Sudden Death (CSD) disease. The cloned CSDV nucleic acid molecules can be used as probes or can be used to design oligonucleotide primers useful in assays, such as a polymerase chain reaction, for detecting the presence of CSDV in biological samples, particularly leaves, roots and other tissues or organs of plants, such as plants from the genera Citrus and Poncirus. The disclosure also comprises the nucleic CSDV nucleic acid molecules, in whole or part, as well as transgenic plants, such as monocots and dicots, containing the CSDV nucleic acid molecules, in any kind of combination, so that expression increases resistance to CSD disease.

## Background
RELATED APPLICATIONS
This application claims priority of PCT International Application PCT/BR2004/000179, filed Sep. 20, 2004, which designated the United States, and is a continuation in part of provisional application Ser. No. 60/506,520, filed Sep. 26, 2003, provisional application Ser. No. 60/508,979, filed Oct. 6, 2003, provisional application Ser. No. 60/529,246, filed Dec. 12, 2003, and provisional application Ser. No. 60/560,466, filed Apr. 7, 2004, all of which are incorporated by reference in their entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 4, 2011, is named 05999401.txt and is 93,350 bytes in size.
FIELD OF THE INVENTION
The invention relates generally to the fields of molecular biology, biochemistry, plant pathology, and agriculture. More particularly, the invention relates to polynucleotides and proteins from a phytopathogenic virus, suitable for disease diagnosis and generation of transgenic plants with resistance to infectious viruses strains.
BACKGROUND OF THE INVENTION
Several Citrus diseases have been shown to be caused by infection with pathogenic viruses (Derrick, K. S. and Timmer, L. W., Annu. Rev. Phytopathol., 38. 181-205 (2000)). One of the most important of these viruses is Citrus Tristeza Virus (CTV), a member of the Closterovirus group which induces serious disease syndromes in citrus. For example, CTV induces quick decline that causes the death of trees grafted on sour orange rootstock, and stem pitting of scion cultivars regardless of the rootstock used (Bar-Joseph et al., Annu. Rev. Phytopathol., 27. 291-316 (1989)). These diseases cause significant losses in the citrus industry worldwide.
In Brazil, citrus tristeza, first detected in 1937, destroyed millions of trees of sweet orange grafted on sour orange rootstocks. The problem was solved by exchanging sour orange rootstock with Rangpur lime rootstock. Today, more than 85% of the 200 million citrus trees in Brazil are grafted on Rangpur lime rootstock (Gimenes-Fernandes, N. and Bassanezi, R. B., Summa Phytopathologica., 27. 93 (2001)).
In 1999, a new citrus disease, named Citrus Sudden Death (CSD) was discovered in Brazil (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologica., 27. 93 (2001)). This disease affects sweet orange (Citrus sinensis) grafted on Rangpur lime rootstock (Citrus limonia), and causes the death of trees within a few months after the symptoms manifest (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologca., 27. 93 (2001)). Although the disease was first observed in the sweet orange/Rangpur lime scion/rootstock combination, it has also been observed in orange trees cvs. Hamlin, Natal, Valencia, Pera, and Rubi, all grafted onto Rangpur lime (Bassanezi et al., Phytopathol., 93. 4. 502-512 (2003)).
Plants with CSD symptoms present generalized leaf discoloration, partial defoliation, decreased number of young shoots and absence of internal shoots (Bassanezi et al., Phytopathol., 93. 4. 502-512 (2003)). As the symptoms become more pronounced, the disease progresses rapidly and leads ultimately, to the death of the plant. The physiological status of the plant is important for the disease progression, since the severity of the symptoms increase at high water demand (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologica., 27. 93 (2001)). The root system of the symptomatic plants is severely damaged and dies quickly as the disease progresses. CSD is also characterized by the development of a strong yellow stain in the phloem of the Rangpur lime rootstock (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologica., 27. 93 (2001)). The time between the appearance of the first visible symptoms in the canopy and the death of the plant ranges from 1 to more than 12 months depending on season and citrus variety (Bassanezi et al., Phytopathol., 93. 4. 502-512 (2003)).
The number of symptomatic trees in one affected area (north of São Paulo State and south of Triângulo Mineiro region, west of Minas Gerais State, Brazil), where the disease was originally found, increased from 500 in 1999 to more than 300,000 in February, 2002, and more than 1 million in June 2003 (Bassanezi, et al., Phytopathol., 93. 4. 502-512 (2003)); (Román et al., Plant Disease, 88. 5. 453-467 (2004)). The pattern of CSD dissemination is similar to that of quick-decline, a disease caused by certain CTV isolates that elicit a graft union incompatibility when infected sweet orange scions are grafted onto sour orange rootstocks (Bassanezi et al., supra) however, CSD affects several sweet oranges grafted on Rangpur lime, a rootstock/scion combination that is not affected by the CTV strains that causes quick-decline (Bassanezi et al., supra).
Based on the spatial and temporal patterns of CSD dissemination, it has been hypothesized that CSD may be caused by an insect-vectored pathogen, potentially a new, undescribed strain of CTV (Bassanezi et al., supra.) Alternatively, a new virus could be the causative agent of the CSD disease.
To test if CSD is caused by a variant strain of CTV or is caused by a new virus, a genomic approach using shotgun sequencing of genomic viral RNA that had been randomly reverse transcribed and cloned in a plasmid vector, was used to study the disease described herein. Bioinformatic tools were developed for the identification and assembly of viral sequences. Using this approach, it was possible to obtain a saturated database of viral sequences from individual trees.
Genomic viral RNA isolated from citrus trees symptomatic or asymptomatic for CSD were reverse transcribed and the first strand cDNA was used to construct random-primed cDNA libraries. Around 2,000 cDNA clones from each tree were sequenced and the sequences were analyzed using BLASTX, BLASTN, and TBLASTX searches against public databases. A viral genome assembled consensus sequence of 6820 nucleotides (SEQ ID NO: 1) encoding a viral polyprotein (SEQ ID NO: 2) sufficient to assemble a viral particle, was identified.
The 6820 nucleotides viral genome sequence, when translated in all possible frames, give rise to, at least, the following polypeptides: a Major Capsid Protein (Coat Protein 1) encoded by the Nucleotide Sequence Domain (NSD1) starting at nucleotide position 6028 and ending at nucleotide position 6675 of SEQ ID NO: 1, whose translation product give rise to the Amino Acid Sequence Domain (AASD) of the polypeptide of SEQ ID NO: 2, starting, at amino acid position 1974 and ending at amino acid position 2188; a Minor Capsid Protein (Coat Protein 2) encoded by NSD2 of SEQ ID NO: 1 starting at nucleotide position 6082 and ending at nucleotide position 6675, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 1992 and ending at amino acid position 2188; a Putative Movement Protein encoded by NSD3 of SEQ ID NO: 1 starting at nucleotide position 6260 and ending at nucleotide position 6724, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 3, starting at amino acid position 1 and ending at amino acid position 154; a Methyltransferase Domain encoded by NSD4 of SEQ ID NO: 1 starting at nucleotide position 487 and ending at nucleotide position 1119, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 127 and ending at amino acid position 337; a Protease Domain encoded by NSD5 of SEQ ID NO: 1 starting at nucleotide position 2797 and ending at nucleotide position 3114, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 897 and ending at amino acid position 1002; a Helicase Domain encoded by NSD6 of SEQ ID NO: 1 starting at nucleotide position 3358 and ending at nucleotide position 4053, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 1084 and ending at amino acid position 1315; a RNA-dependent RNA polymerase encoded by NSD7 of SEQ ID NO: 1 starting at nucleotide position 4528 and ending at nucleotide position 5778, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 1474 and ending at amino acid position 1890.
The viral genome sequence showed strong similarity to several viruses from the Tymoviridae family of plant viruses, especially the oat blue dwarf virus (FIGS. 1 and 2). Analysis of CSD-symptomatic or asymptomatic trees for the presence of these viral sequences revealed that only trees presenting the CSD symptoms contain the viral sequences. It was therefore assumed that these sequences belong to an undescribed virus of the Tymoviridae family which is the causative agent of the CSD disease.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid molecules of and from the genome of a virus that is the causative agent of CSD disease. Sequence comparison with a number of viral genome sequences revealed that this new virus belongs to the Tymoviridae family. This new virus is herein named “Citrus Sudden Death Virus,” or “CSDV”. It is hereafter an object of the invention to provide nucleic acid molecules which encode infectious CSDV. Such nucleic acid molecule is referred to throughout the application as “CSDV nucleic acid molecules”.
For the purposes of this application, nucleic acid molecules refers to RNA, DNA, cDNA or any variant thereof with functions equivalent to RNA, gDNA, and cDNA, such as the synthesis of CSDV polypeptide domains. Also, the polypeptide domains encoded by CSDV nucleic acid molecules are referred to throughout the application as “CSDV polypeptides” or “CSDV proteins”.
The invention relates to the use of the CSDV nucleic acid molecules to produce polypeptides. “Nucleic acid molecules of the invention” refers to, e.g., CSDV nucleic acid molecules, mutations of CSDV nucleic acid molecules, chimeric nucleic acid molecules and so forth.
In one embodiment, polypeptides are produced by cells transfected with nucleic acid molecules of the invention. In another embodiment, the polypeptide or polypeptides are produced recombinantly from a fragment or portion of the nucleic acid molecules of the invention. In yet another embodiment, the polypeptides are chemically synthesized.
Since, in nature, the CSDV proteins are ultimately synthesized from the information contained in the genome sequence of CSDV, the invention also relates to the use of the CSDV particles isolated and purified from infected plants tissues and/or organs. In one embodiment the particles can be used to produce antibodies against the CSDV proteins. In another embodiment the purified CSDV particles can be used to isolate and purify CSDV protein domains that can further be used to produce antibodies against CSDV.
The polypeptides of the invention can serve, e.g., as immunogens in the development of diagnostic assays for detecting the presence of CSDV in biological samples, as they provoke antibody production, and the antibodies can then be used in assays.
The invention also relates to the use of the CSDV nucleic acid molecules for diagnosis purposes, in which oligonucleotide primers containing from 5 to 100 nucleotides presenting from 90 to 100% identity with the CSDV nucleotide sequences can be used in, e.g., RT-PCR reactions, so that parts of the CSDV nucleic acid molecules can be amplified and detected in ordinary agarose gels serving as a diagnostic for the presence of the virus.
The invention also relates to methods of transforming plants, such as monocots or dicots, with constructs containing the CSDV nucleic acid molecules, to produce plants that are resistant to CSDV. Such methods include the introduction of constructs containing at least one CSDV nucleic acid molecule into plant parts, such as scions, rootstock cultivars, and so forth, as well as into citrus germplasm and breeding lines. Transformed CSDV-resistant germplasm and breeding lines can be used in conventional breeding programs, to create new cultivars that carry and express the resistance genes.
Accordingly, the invention features (i) isolated and/or purified CSDV nucleic acid molecules that have at least 65% sequence identity with the nucleotide sequence of SEQ ID NO.: 1; (ii) sequences complementary thereto or nucleotides whose complement hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO.: 1; as well as with the coding regions of the domains encoded therein (iii) polypeptides or portions of polypeptides that have at least 70%, more preferably 80% sequence identity with the amino acid sequence of SEQ ID NO.: 2, or with any of the domains within SEQ ID NO:2 as defined herein.
“Stringent conditions” as used herein, refers to parameters with which the art is familiar, i.e., hybridization in 3.5×SSC, 1×Denhardt's solution, 25 mM sodium phosphate buffer (pH 7.0), 0.5% SDS, and 2 mM EDTA for 18 hours at 65° C., followed by 4 washes of the filter at 65° C. for 20 minutes, in 2×SSC, 0.1% SDS, and a final wash for up to 20 minutes in 0.5×SSC, 0.1% SDS, or 0.3×SSC and 0.1% SDS for greater stringency, and 0.1×SSC, 0.1% SDS for even greater stringency. Other conditions may be substituted, as long as the degree of stringency in equal to that provided herein, using a 0.5×SSC final wash.
The invention also features expression vectors or constructs in which the CSDV nucleic acid molecules are operably linked to one or more expression control sequences or promoters.
The invention further features a transgenic plant, such as one of the genera Citrus and Poncirus, into which a CSDV nucleic acid molecule has been introduced. Exemplary are citrus scions and rootstock cultivars (e.g., from sour orange [Citrus aurantium], Rangpur lime [Citrus limonia], rough lemon [Citrus limonia and Citrus jambhin], mandarin “Cleopatra” [Citrus reshni], Sunki [Citrus sunki], Volkamerian lemon [Citrus volkameriana], “Caipira” orange [Citrus sinensis]) and intrageneric hybrids (e.g., tangelos [Citrus paradisi×Citrus reticulate], tangors [Citrus reticulate×Citrus sinensis], citrumelo [Poncirus trifoliata×Citrus maxima], and citrange [Citrus sinensis×Poncirus trifoliate]). The plant can be a breeding line. It can also be one from Fortunella and Citrofortunella species, including calamondin and kumquat. The nucleic acid molecule in the plant preferably includes a selectable marker such as an herbicide resistance gene.
The invention relates to nucleic acid molecules, and the polypeptides they encode, which were identified using shotgun sequencing of viruses genomes from citrus plants with or without symptoms of CSD. These sequences come from a newly identified virus of the Tymoviridae family which, when it infects a plant, such as a citrus plant, causes CSD disease, leading to the death of the plant. Methods of genetic transformation to produce plants that are resistant to CSDV strains are within the invention. Such methods include the introduction of constructs containing the CSDV nucleic acid molecules into scions and/or rootstocks, so that commercial varieties or any other germplasm useful for breeding programs can be produced and used to create new cultivars resistant to CSDV.
Accordingly, the invention involves purified CSDV nucleic acid molecules, which may be isolated from citrus plants manifesting symptoms of CSD, that when used in appropriate constructs have the ability to confer resistance to pathologies caused by CSDV infection in plants infected with CSDV. The nucleic acid molecule can be a purified portion or a gene present in the complete genome of CSDV, e.g., a nucleic acid molecule whose nucleotide sequence encodes a polypeptide or a portion of a polypeptide that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO.: 2. The nucleotide sequences can be that of SEQ ID NO.: 1 or one that define polynucleotides whose complement hybridizes under high stringency conditions to the nucleotide sequences of SEQ ID NO.: 1.
The invention also features expression vectors, isolated recombinant cells, and plants and plant parts containing the nucleic acid molecule of the invention, e.g., one of the nucleic acid molecules described above. The nucleic acid molecule in the vector, cell, seed or plant can be operably linked to one or more expression control sequences or promoters.
In addition, the invention features the purified proteins encoded by the nucleic acid sequence domains present in the genome sequence of CSDV. The proteins include those with amino acid sequences that (a) share at least 80% sequence identity with SEQ ID NO.: 2 or (b) includes the amino acid sequences of SEQ ID NO.: 2. Proteins of the invention can be expressed in bacteria either as “neat” proteins, or as heterologous or fusion polypeptides. The invention also features antibodies raised against the proteins of the invention, e.g., those described above. The antibodies can further include a detectable label.
The invention also features a plant transfected with one of the nucleic acid molecules of the invention, e.g., any purified CSDV sequence. The plant may be of the genera Citrus and Poncirus (e.g., sweet orange [Citris sinensis], mandarin [Citrus reticulata], sour orange [Citrus aurantium], Rangpur lime [Citrus limonia], rough lemon [Citrus limonia and Citrus jambhin], mandarin “Cleopatra” [Citrus reshni], Sunki [Citrus sunki], Volkamerian lemon [Citrus volkameriana], “Caipira” orange [Citrus sinensis]) and intrageneric hybrids (e.g., tangelos [Citrus paradisi×Citrus reticulata], tangors [Citrus reticulate×Citrus sinensis], citrumelo [Poncirus trifoliata×Citrus maxima], and citrange [Citrus sinensis×Poncirus trifoliate]). The plant can be a breeding line. It can also be one from Fortunella and Citrofortunella species, including calamondin and kumquat.
The invention features a method of producing a disease resistant plant by introducing constructs containing purified nucleic molecules under the control of a suitable plant promoter, into the plant, transforming the plant with Agrobacterium strains or microprojectile bombardment.
All technical terms used herein are terms commonly used in biochemistry, molecular biology, phytopathology and agriculture, and will be understood by one of ordinary skill in the art to which this invention belongs. Those technical terms can be found in, e.g., Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing Associates and Wiley-Interscience, New York, 1988 (with periodic updates); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 5th ed., vol. 1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002; Genome Analysis: A Laboratory Manual, vol. 1-2, ed. Green et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997. Methods involving plant biology techniques are described herein and are described in detail in methodology treatises such as Methods in Plant Molecular Biology: A Laboratory Course Manual, ed. Maliga et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995. Various techniques using PCR are described, e.g., in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, 1990 and in Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003. PCR-primer pairs can be derived from known sequences by known techniques such as using computer programs intended for that purpose (e.g., Primer, Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Caruthers (1981) Tetra. Letts., 22:1859-1862 and Matteucci and Caruthers (1981) J. Am. Chem. Soc., 103:3185.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention can be more readily understood by reference to the accompanying drawings, wherein:
FIG. 1 shows the alignment of the nucleotide sequence of the CSDV genome (SEQ ID NO: 1) with that of the Tymoviridae Oat Blue Dwarf Virus/OBDV-GI 9629255 (SEQ ID NO: 13).
FIG. 2 shows the alignments of the amino acid sequence of the polyprotein encoded by the CSDV genome (SEQ ID NO: 2) with those encoded by other Tymoviridae genomes [Oat Blue Dwarf Virus/OBDV—GI 9629255 (SEQ ID NO: 14), Grapevine Fleck Virus/GFkV—GI 18138525 (SEQ ID NO: 19), Grapevine Asteroid Mosaic-Associated Virus/GAMaV—GI 29335718 (SEQ ID NO: 15)]. FIG. 2 (b) discloses SEQ ID NOs: 15-16, respectively, in order of appearance. FIG. 2 (c) discloses SEQ ID NOs: 17-18, respectively, in order of appearance. FIG. 2 (d) discloses SEQ ID NOs: 19 and 3, respectively, in order of appearance.
FIG. 3 shows the PCR amplification of a DNA fragment amplified with primers based on the CSDV nucleotide sequence of SEQ ID NO.: 1. CDNA samples were prepared from symptomatic and asymptomatic citrus trees. Only trees presenting the CSD symptoms contain an amplifiable CSDV sequence of SEQ ID NO.: 1.
FIG. 4 shows the results of a Northern Blot experiment in which RNA samples from symptomatic and asymptomatic plants were subjected to an RNA gel blot analysis to verify the presence of RNA species able to hybridize with a DNA probe derived from the CSDV sequences. Only lanes containing RNA samples from symptomatic trees possess a band sizing 6.5 kbp representing the complete CSDV viral RNA genome.
FIG. 5 depicts the results of a RT-PCR experiment performed on total RNA taken from insect bodies, to determine the vectors that transmit CSD.
FIG. 6 shows the results of a western blot experiment in which total protein extract from symptomatic and asymptomatic plants where subject to a protein gel blot analysis developed with antibody against the CSDV Coat Proteins 1 and 2, respectively.
FIG. 7 shows an electron micrography of virus particles purified from citrus plants presenting CSD symptoms.
FIG. 8 shows a Northern blot of RNA extract from purified virus particle.
FIG. 9 shows a drawing of the vector pTYMO-AS that contain part of the RNA-dependent RNA polymerase sequence domain (SD7, as defined in SEQ NO.: 1) operably linked to the CaMV 35S promoter. This vector was used to transform citrus plants by Agrobacterium mediated transformation.



DETAILED DESCRIPTION OF THE INVENTION
Nucleic acid molecules from the genome of an undescribed virus that causes Citrus Sudden Death (CSD) have been cloned and sequenced. Such nucleic acid molecules are referred to throughout the application as “CSDV nucleic acid molecules”. Polypeptides predicted from CSDV nucleic acid molecules have been analyzed using software programs including BLAST, and have been shown to encode, inter alia, an RNA polymerase, a methyltransferase, a protease and a helicase that are involved in the replication of the CSDV, a movement protein involved in the translocation of the virus throughout the plant, and the capsid proteins responsible for the encapsulation of the virus genome.
The molecular cloning of CSDV nucleic acid molecules provides the means to develop diagnostic methods to detect the presence of CSDV in biological samples, including tissues, cells and organs of plants, such as plants of the genus Citrus. The molecular cloning of CSDV nucleic acid molecules also provides the means to create CSD-resistant plants of the genus Citrus through genetic transformation. Genetic transformation of plants of the genus Citrus, can be obtained using Agrobacterium mediated transformation methods. Such methods include cloning constructs containing CSDV nucleic acid molecules operably ligated to promoter and enhancer regions, initiation and termination sequences. These constructs can also contain genes for selectable markers, such as herbicide resistance. These constructs may be cloned in the Ti plasmid of Agrobacterium. Plasmid vector-containing constructs are used to transform commonly used Agrobacterium strains, which are subsequently used to transform plants, such as those of the genus Citrus. Plasmid vector-containing constructs may be also introduced into plants by microprojectile bombardment. The constructs containing the CSDV nucleic acid molecules are useful for creating CSD resistant plants such as all common types of citrus fruits, including but not limited to sweet oranges, grapefruit, mandarins, tangerines, pummelos, lemons, limes, citrons, bergamots, limequats, meyer lemons, silver limes, key limes, kaffir limes, lavender gems, blood oranges, satsumas, oroblancos, melogolds, bergamots, intrageneric hybrids such as tangelos and tangors, and citrus-type fruit such as calamondins and kumquats (Fortunella spp.). For example, CSDV nucleic acid molecules can be introduced into commercially utilized rootstock cultivars, including but not been limited to, Rangpur lime, sour orange, rough lemon, various mandarins, and citrus intrageneric and intergeneric hybrids. CSD resistant citrus plants, composed of genetic modified scions and rootstocks, can then be used by citrus growers to counter CSD disease, and to avoid decreased productivity and/or tree death and replanting costs.
The invention provides purified nucleic acid molecules (polynucleotides) that encode polypeptides having an amino acid sequence such as that of SEQ ID NO.: 2.
The CSDV nucleic acid molecules of the present invention can be obtained from CSDV infected plants. The molecules of the present invention may be in the form of RNA or in the form of cDNA. The cDNA may be double- or single-stranded, and, if single-stranded may be the coding (sense) strand or noncoding (anti-sense) strand. The sequence may be identical to a nucleotide sequence of SEQ ID NO.: 1. It may also be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as polynucleotides of SEQ ID NO.: 1. Other nucleic acid molecules within the invention are variants of CSDV nucleic acid molecules such as those that encode fragments, analogs and derivatives of native CSDV nucleic acid molecules. Such variants may be, e.g., naturally occurring polymorphic variants of native CSDV nucleic acid molecules, or a non-naturally occurring variant of native CSDV nucleic acid molecules. For example, the nucleotide sequence of such variants can feature a deletion, addition, or substitution of one or more nucleotides of native CSDV nucleic acid molecules. Naturally occurring variants of native CSDV nucleic acid molecules within the invention are nucleic acids isolated from CSDV infected plants that have at least 65% sequence identity with native CSDV nucleic acid molecules, and encode polypeptides having at least one functional activity in common with native CSDV nucleic acid molecules encoded proteins.
Shorter oligonucleotides (e.g., those from 6-200, preferably 12-200, more preferably 20, 30, or 50 to 200 (100, 125, 150 or 200) base pairs in length), e.g., that match perfectly to the CSDV nucleic acid molecules or hybridize with CSDV nucleic acid molecules at stringent conditions as defined herein can be used as probes, primers, or antisense molecules.
Longer polynucleotides (e.g., those of 300 to 800 base pairs) that encode or hybridize with CSDV nucleic acid molecules, can be used in place of a native CSDV nucleic acid molecule in applications where it is desired to modulate the functional activity of a native CSDV nucleic acid molecule.
Nucleic acids molecules that hybridize under stringent conditions as defined herein to CSDV nucleic acid molecules of SEQ ID NO.: 1 or the complement of the SEQ ID NO.: 1 are also within the invention. For example, such nucleic acids can be those that hybridize with the CSDV nucleic acid molecules of SEQ ID NO.: 1 or the complement of the SEQ ID NO.: 1, under low stringency conditions, moderate stringency conditions, or high stringency conditions, and are within the scope of the invention. Preferred nucleic acids molecules are those having a nucleotide sequence that is the complement of all or a portion of the CSDV nucleic acid molecules of SEQ ID NO.: 1. Other variants of nucleic acid molecules within the invention are polynucleotides that share at least 65% sequence identity to the CSDV nucleic acid molecules of SEQ ID NO.: 1 or the complement of SEQ ID NO.: 1.
Other CSDV nucleic acid molecules encoding polypeptides are also within the invention. Such polypeptides can be made by preparing a construct (e.g., an expression vector) that expresses CSDV nucleic acid molecules encoding polypeptides, when introduced into a suitable host. Variant CSDV nucleic acid molecule-encoding polypeptides can be produced by those skilled in molecular biology procedures using standard nucleic acid mutagenesis techniques or chemical synthesis, or the polypeptides can be isolated and purified from CSDV particles isolated and purified from infected plants, as the CSDV particles encoded by nucleic acid molecules of SEQ ID NO.: 1 and polypeptide domains of SEQ ID NO.: 2. Antibodies can be produced against the isolated and purified CSDV particles and can be used for serological diagnosis of the virus.
Another aspect of the invention relates to the use of purified antisense nucleic acids to inhibit expression of CSDV nucleic acid molecules. Antisense nucleic acid molecules within the invention are those that specifically hybridize under cellular conditions to cellular mRNA and/or genomic RNA of CSDV in a manner that inhibits expression of the nucleic acid domains encoded by the CSDV nucleic acid molecules.
The antisense nucleic acid molecules can be delivered into cells that express CSDV genes. For instance, constructs expressing antisense molecules under the control of a strong promoter can be introduced into citrus plants by genetic transformation using Agrobacterium or microprojectile bombardment (Ghorbel et al. Tree Physiology., 20. 1183-1189 (2000); Bespalhok et al., Crop Breed. Appl. Biotech., 1. 27-34 (2001); Bespalhok et al., Braz. Arch. Biol. Technol., 46. 1. 1-6 (2003); Molinari et al., Scientia Horticulturae., 99. 34. 379-385 (2004); Jia-Long et al., Plant Science, 113. 2. 175-183 (1996)).
The expression of CSDV nucleic acid molecules can be modulated by RNA interference (RNAi) (Lee et al. Nature Biotech., 19. 500-505 (2002); Voinnet, O. Trends Genet., 17. 449-459 (2001)) by which a construct driving the synthesis of sequence-specific double-stranded RNA (dsRNA) is introduced into an organism or cell in order to silence the targeted gene (Hannon, Nature, 418. 244-251 (2002)). Selected sequences corresponding to CSDV nucleic acid molecules can be used to create, after expression, a sequence-specific dsRNA that can interfere with accumulation of endogenous RNA encoded by the CSDV nucleic acid molecules.
The CSDV nucleic acid molecule can be altered by using molecular biology techniques to produce a mutant recombinant virus that work as a vaccine. This is well known in the art as cross-protection system, in which a mild, non-infective virus is introduced in a plant and after replication induces in the host plant a defense response against the severe, infective strain (Costa, A. S. and Muller, G. W. 1980, Plant Dis., 64:538-541). Plants inoculated with recombinant CSDV mutant become resistant to the wild-type CSDV infective strains.
The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and are not to be construed as limiting the scope or content of the invention in any way.
EXAMPLES
Example 1
This example describes the identification and cloning of nucleic acid molecules corresponding to the complete genome sequence of CDSV. cDNA libraries were constructed from double strand RNA isolated from citrus plants presenting symptoms of CSD by shotgun cloning of cDNAs generated by RT-PCR. Clones were randomly sequenced using an ABI 3700 sequencer. Sequences were trimmed for vector bases and low quality bases and BLASTX-analyzed against the non-redundant (NR) GenBank database. Two cDNA clones were identified as having nucleotide sequences similar to viral nucleotide sequences of the Tymoviridae family. By primer walking PCR using a cDNA library or total RNA from CSD plants as templates, cDNA clones were identified that, after sequencing on both ends, gave rise to a consensus CSDV nucleic acid sequence of 6820 nucleotides. This consensus sequence contains nucleotide sequence domains that encode the complete set of the viral proteins comprised of: a Major Capsid Protein (Coat Protein 1), a Minor Capsid Protein (Coat Protein 2), a Putative Movement Protein, a Methyltransferase Domain, a Protease Domain, a Helicase Domain and a RNA-dependent RNA polymerase. The gene and protein domains organization of CSDV is similar to that found in several virus strains of the Tymoviridae family, especially to the Oat blue dwarf virus (FIGS. 1 and 2).
Example 2
This example describes RT-PCR analysis of citrus plants to determine if the CSDV nucleic acid molecules of the invention could be used to design oligonucleotide primers that amplify CSDV sequences and could be use in diagnostic assays. Oligonucleotide primers designed on the basis of CSDV nucleic acid molecules of SEQ ID NO.: 1 were used to amplify the CSDV nucleic acid sequences using RT-PCR from RNA isolated from CSD symptomatic or asymptomatic citrus plants. Bark from young citrus branches was peeled and ground to a powder, in liquid nitrogen. Total RNA was purified from 100 mg of bark tissue by using Trizol reagent according to the manufacturer's instructions. The resulting total RNA was suspended in 50 ul of DEPC (diethylpyrocarbonate)-treated sterile water and used for cDNA synthesis. First strand cDNA was synthesized using 8 ul of total RNA (approx. 8 ug) as template. Two microliters of random primers (1 ug) were added to the total RNA that had been denatured at 97° C. for 5 min. The solution was then incubated on ice while adding 1 ul of 10 mM dNTP mix and 3 ul of First Strand buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2) to the tube. The mixture was incubated at room temperature for 2 min, 1 ul (200 U) of the enzyme reverse transcriptase SuperScript II was added and the solution was further incubated at room temperature for 10 min. followed by 60 min. at 42° C. For PCR, 1 ul of the synthesized cDNA was added to a 20 ul reaction containing 0.5 mM of each of primers “TYMOF2” 5′-GTCAGCTGTCCAACCAGTTCC-3′ (SEQ ID NO: 4) and “TYMORR”: 5′-GTGAAGATCAATGAGAGCCTG-3′(SEQ ID NO: 5), 0.125 mM each dNTP, 2.5 mM MgCl2, 1× reaction buffer (20 Mm Tris-HCl, pH 8.4, 50 mM KCl), and 1 U of Taq polymerase. The reaction was heated for 2 min. at 94° C. and subjected to 40 amplification cycles (30 s at 94° C., 30 s at 55° C., 1 min at 72° C.). Ten microliters of the RT-PCR reaction were combined with 10 ul of 2× digestion buffer (100 mM potassium acetate, 40 mM Tris-acetate, 20 mM magnesium acetate, 2 mM DTT and 4 ug BSA, pH 7.9) and 0.5 ul of the restriction enzyme Apal (5 U). Digestion was carried out for 2 hours at 25° C. DNA fragments were separated in a 1.5% agarose gel, stained with 100 ng/ml ethidium bromide and compared under UV light.
The results are depicted in FIG. 3. Lanes a-f and m-t are from asymptomatic plants, while g to l, and u to z are from symptomatic plants. There is no banding at all in lanes a-f and m-t, while clear banding is evident in the samples from symptomatic plants in lanes g to l and u to z, which generate fragments of 500 and 250 base pairs long when digested with Apal enzyme. Data was further expanded to 512 plants (351 symptomatic and 161 asymptomatic) and 99.7% of yellow bark plants shows the CSDV nucleic acid fragment amplification.
Example 3
In these experiments, Northern blotting was carried out on samples of RNA taken from asymptomatic and symptomatic plants. A DNA probe taken from CSDV nucleic acid molecules was used. FIG. 4 shows that a band of 6.5 kbp was identified in symptomatic plants, while no banding was identified in any of the asymptomatic material.
Total RNA from bark tissue of symptomatic and asymptomatic plants was extracted using the Trizol Reagent (Invitrogen) according to the manufacturer's protocol. Fifteen micrograms of total RNA from each sample were separated by denaturing electrophoresis on a 1% agarose gel containing 1×MOPS and 0.6M formaldehyde, according to Sambrook and Russell (Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). The gel was subsequently transferred to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) by capillary transfer in 10×SSC (1×SSC is 0.15M NaCl; 0.015M sodium citrate) for 16 h. The membrane was baked at 80° C. for 2 h, prehybridized in ExpressHyb hybridization buffer (Clontech) for 2 h at 65° C., followed by hybridization in a fresh aliquot of ExpressHyb solution containing 20 ng/ml of probe (2×107 cpm/ml) for 16 h at 65° C. The probe consisted of a 772-bp CSDV nucleic acid fragment amplified by PCR from a plasmid containing part of SEQ ID NO.: 1 using SEQ ID NOS: 4 and 5. The probe was radioactively labeled by random-priming with [α-32P]dCTP (6000 Ci/mmol) using the Random Primers DNA Labeling System (Invitrogen). After hybridization, the membrane was washed at room temperature in 2×SSC, 0.05% SDS for 4×10 min, and then twice at 55° C. for 20 min each in 0.1×SSC; 0.1% SDS. The blot was exposed for three days and analyzed by phosphoimaging on a FLA-3000 Fluorescent Image Analyzer (Fujifilm).
Example 4
This example describes RT-PCR analysis of citrus insect vectors to determine if the CSDV nucleic acid molecules of the invention could be found in insect vectors and therefore determine the kind and species of insects that could act as a vector for transmission and dissemination of CSD in citrus plantations.
A number of insect species including aphids such as Toxoptera citricida, Aphis spiraecola and Aphis gossypii, and leafhoppers/planthoppers representing, but not limited to, Deois flavopicta, Xerophloa viridis, Ferrariana trivittata, Hortensia similis, Erytrogonia sexguttata, Macugonalia leucomelas, Dechacona missionum, and Copididonus hialipennis were sampled at citrus plantation areas affected by CSD and at areas without symptoms of CSD. In the areas affected by CSD the insects were collected from individual trees presenting symptoms of CSD. Leafhoppers/planthoppers were separated into 6 groups according to morphological characteristics, while aphids were separated according the respective species. Around fifty individuals of each leafhoppers/planthoppers group or aphids species were used to extract total RNA.
Total RNA from whole bodies of insects was extracted using the Trizol Reagent (Invitrogen) according to the manufacturer's protocol. Eight micrograms of total RNA from each sample were used for RT-PCR analysis using CSDV specific primers designed on the basis of the CSDV nucleic acid molecules of SEQ ID NO: 1.
RT-PCR was carried out on samples of total RNA using SEQ ID NOS: 4 and 5. FIG. 5 shows that a band of 772 Kbp corresponding to part of the CSDV nucleic acid domain encoding the RNA-dependent RNA polymerase was amplified from samples taken from the aphids Toxoptera citricida, Aphis spiraecola and Aphis gossypii collected from symptomatic trees in the affected areas, while no band was amplified in any of the RNA samples isolated from leafhoppers or planthoppers of CSD affected areas, or from aphids collected in trees from the non-affected areas.
Table 1 presents a summary of the results of the assays for the presence of CSDV in samples of insects collected in CSD affected and non-affected areas. The virus was found only in the aphid samples from citrus trees of CSD affected areas. This suggests that these aphids could act as vectors for CSDV transmission and dissemination.
Example 5
This example illustrate the use of polyclonal antibodies raised against the CSDV Coat Proteins 1 and 2 to determine the presence of CSDV in plant extracts. The polyclonal antibodies was produced using two different sources of immunogens: 1) Peptides were designed based on the deduced amino acid sequences of Coat Proteins 1 and 2 of CSDV, taking into consideration b-turn structure prediction (Chou, P Y and Fasman, Biophys J., 1979 June; 26(3):367-73) and hydrophobicity level (Kyte, J. and Doolittle, R F, J Mol Biol., 1982 May 5; 157(1):105-32). Peptides corresponding to amino acids 7 to 20 (AGPAPSRDDRVDRQ (SEQ ID NO: 8)), 11 to 24 (PSRDDRVDRQPRLP (SEQ ID NO: 9)), 51 to 64 (DGSEAKNLSDDLSG (SEQ ID NO: 10)), 111 to 124 (PASASETSYYGGRL (SEQ ID NO: 11)), and 161 to 175 (RFSYSVYSNGGTKGT (SEQ ID NO: 12)) of the predicted Coat Protein 2 (amino acid positions 1974-2188 of SEQ ID NO: 2) were synthesized, KLH-conjugated (Genscript Corp, USA) and used to immunize rabbits. Polyclonal antibodies were delivered as antisera and used without further treatment for Western blot analyses. 2) Polyclonal antibodies were also obtained against the recombinant Coat Proteins 1 and 2. The CSDV nucleic acid sequences between nucleotide positions 6,028 to 6,672 (Coat Protein 1) and 6,082 to 6,672 (Coat Protein 2) were cloned into the pET-28a(+) expression vector (ClonTech) and expressed in E. coli BL21(DE3) strain. Coat Protein 1 was expressed as a fusion with a His-tag and thrombin site in the N-terminal (His-p22.5) and Coat Protein 2 was expressed fused to a His-tag in the C-terminal (p21-His) and also in a non-fused form (NFp21). Large scale expression was conduced under IPTG induction, bacterial cells were disrupted by French press, and the soluble fraction of the fused recombinant proteins were purified using the His-Trap purification Kit (Amersham). Purified p21-His and Hisp22.5 were used to raise polyclonal antibodies in rabbits.
Recombinant CSDV Coat Proteins and crude protein extracts from citrus trees were resolved in a 12% (w/v) acrylamide gel and proteins were transferred to nitrocellulose membrane by semi-dry electroblotting (BioRad apparatus). The membrane was blocked with a Blotto solution (PBS, pH 7.2 containing 0.1% Tween-20 and 5% (w/v) non-fat-milk powder), incubated with the polyclonal antibodies (1:1000 in PBS) against the peptides, washed with PBS-Tween 20 and incubated with anti-rabbit IgG alkaline phosphatase conjugate (BioRad, 1:2000 in PBS). Color development was carried out in freshly prepared substrate solution (1.5 mg NBT, 3 mg BCIP in 20 ml carbonate buffer, pH 9.2). Antisera obtained for the peptides were able to recognize both recombinant Coat Proteins as well as Coat Proteins present in the CSDV-infected plant extracts (FIG. 6). Two protein bands of expected size were evident only in infected plant extracts and may correspond to Coat Proteins 1 and 2, respectively. Similar results were obtained using antibody raised against the recombinant CSDV Coat Proteins.
Example 6
In this example, CSDV particles were purified from CSD-affected citrus plants according to the methodology described by Bar-Joseph et al. (Bar-Joseph, M., D. J. Gumpf, J. A. Dodds A. Rosner, and I. Ginsberg. 1985, Phytopathology, 75:195-198.). The purity of virus preparation was determined by electron microscopy using a negative staining methodology with 2% uranyl acetate (Gaméz, R., T. Fukuoka, and Y. Kozuka. 1977, Rev. Biol. Trop., 25:151-157). The electron micrography shows purified virions as expected isometric, non-enveloped,  ̃30 nm in diameter particles, with a rounded contour, and prominent surface structure. As typical Tymovirus, CSDV present under uranyl acetate staining two types of particles (Boulila, M., D. Boscia, B. Di Terlizzi, M. A. Castellano, A. Minafra, V. Savino, and G. P. Martelli. 1990, J. Phytopathol., 129:151-158), one with a negative stain (T form, represents non-infectious empty shells) and another with positive stain (B form, represents intact particles).
In order to confirm that the purified particles correspond to CSDV, total nucleic acids were purified from a preparation of virus particles by phenol extraction, resolved in a 1% agarose gel, transferred to Hybond-N+ nylon membranes (Amersham) and hybridized with a P32-labelled DNA probe from part of the CSDV nucleic acid molecules of SEQ ID NO.: 1. The results of FIG. 8, confirm the presence of the CSDV genomic RNA in the purified sample.
Example 7
This is an example of a construct produced using a CSDV nuclei acid molecule in order to generate transgenic plant resistant to CSDV. A 761pb nucleic acid fragment of the CSDV RNA-dependent RNA Polymerase of SEQ ID NO.: 1, were PCR amplified using the primers TY-Fw (5′-TGGAGCTCCCTGCCCACGACCCAAC-3′) (SEQ ID NO: 6), containing a Sac I restriction site and TY-Rv (5′-TCTAGAGCCTGGGGGATGGAGAGC-3′) (SEQ ID NO: 7), containing a Xba I restriction site. This fragment was cloned in PGEM-T easy vector (Promega corp, Madison, Wis., USA) and confirmed by sequencing. The RNA-dependent RNA Polymerase fragment was removed from pGEM-T by restriction digestion with Xba I and Sac I and cloned in the antisense direction in pA35S(2×) (Kay, R., Chan, A., Daly, M. & McPherson, J., 1987, Science, 236: 1299-1302), restricted with the same enzymes yielding the plasmid pATYMO-AS. The pA35S(2×) plasmid includes a herbicide resistance gene (Christensen A H, Quail P H., 1996, Transgenic Res. 15: 213-218.) under the control of the CaMV35S promoter. FIG. 9 represents a drawing of the TDNA insertion fragment of pATYMO-AS.
In order to produce the transgenic citrus containing the plasmid pATYMO-AS, explants from ‘in vitro’ germinated Rangpur lime were used for transformation experiments with Agrobacterium tumefaciens strain EHA105 carrying the plasmid. The explants were inoculated with an overnight bacterial suspension for 20 min, blotted dry and platted onto MS medium containing 2,4D and acetoseringone. The co-cultivation was made in darkness for 3 days and then the explants were platted in MS medium containing BAP, PPT, cefotaxime and vancomycin. The developed shoots were transferred to MS medium containing IBA, PPT, cefotaxime and vancomycin. Transgenic regenerated plants were tested by PCR and Southern blot.
For the southern blot experiment genomic DNA from transgenic and non-transgenic plants were isolated according to Dellaporta et al. (Dellaporta S. L., Wood J., Hicks J. B, 1983, Plant Mol. Biol. Rep., 1:19-21,). Purified DNA was digested with EcoRI, separated by agarose gel electrophoresis (10 ug DNA/lane, 1% agarose) then transferred onto charged nylon membranes according to standard procedures (Sambrook et al., 2001 Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Probes were labeled using the Genes ImagesTM AlkPhos DirectTM labelling and detection system from Amersham Biosciences that is based on a dioxetane chemiluminescence system. Blots were hybridized to CSDV specific probes and the citrus chalcone synthase CitCHS2 gene (GenBank 5106368) as a positive control. Membranes were washed at 55° C. according to the instruction manual (Genes Images AlkPhos Direct labeling and detection system—Amersham Biosciences). Chemiluminescent signals were detected using a CDP-StarTM chemiluminescent detection reagent (Amersham Biosciences) according to the protocol recommended.







TABLE 1







RESULTS OF TEST FOR THE PRESENCE OF CSDV


IN INSECTS COLLECTED FROM CITRUS THREES


IN CSD AFFECTED AND NON-AFFECTED AREAS.









INSECT




Leafhoppers/planthoppers
Samples Analyzeda
Diagnosis for CSDV





Group I
1 (affected area)
Negative



2 (affected area)
Negative


Group II
1 (affected area)
Negative



2 (affected area)
Negative


Group III
1 (affected area)
Negative



2 (affected area)
Negative


Group IV
1 (affected area)
Negative


Group V
1 (affected area)
Negative


Group VI
1 (affected area)
Negative


T. citricida
1 (non-affected area)
Negative



2 (non-affected area)
Negative



3 (non-affected area)
Negative



4 (non-affected area)
Negative



5 (affected area)
Positive



6 (affected area)
Positive



7 (affected area)
Positive



8 (affected area)
Positive



9 (affected area)
Positive



10 (affected area)
Positive



11 (affected area)
Positive



12 (affected area)
Negative



13 (affected area)
Positive



14 (affected area)
Positive



15 (affected area)
Positive


A. spiraecola
6 (non-affected area)
Negative



7 (affected area)
Positive



8 (affected area)
Positive



9 (affected area)
Negative



10 (affected area)
Negative


A. gossypii
5 (non-affected area)
Negative



6 (non-affected area)
Negative



7 (affected area)
Positive



8 (affected area)
Negative



9 (affected area)
Negative



10 (affected area)
Negative





aeach sample was composed of >50 aphids or around 10 leafhoppers/planthoppers.

## Claims

1. A recombinant expression vector comprising a cDNA molecule comprising a nucleotide sequence encoding an amino acid sequence having amino acids 1974-2188 of SEQ ID NO: 2, operably linked to a heterologous promoter.

2. An isolated nucleic acid molecule of claim 1, wherein said nucleotide sequence further encodes amino acids 1992-2188 of SEQ ID NO: 2.

3. A recombinant expression vector of claim 1, wherein said nucleotide sequence further encodes amino acids 127-337 of SEQ ID NO: 2.

4. A recombinant expression vector of claim 1, wherein said nucleotide sequence further encodes amino acids 897-1002 of SEQ ID NO: 2.

5. A recombinant expression vector of claim 1, wherein said nucleotide sequence further encodes amino acids 1084-1315 of SEQ ID NO: 2.

6. A recombinant expression vector of claim 1, wherein said nucleotide sequence further encodes amino acids 1-154 of SEQ ID NO: 3.

7. A recombinant expression vector of claim 1, wherein said nucleotide sequence further encodes amino acids 1474-1890 of SEQ ID NO: 2.

8. A cDNA molecule comprising a nucleotide sequence consisting of nucleotides 6028-6675 of SEQ ID NO: 1, operably linked to a heterologous promoter.