Title of Invention

A DNA CONSTRUCT FOR GENE SILENCING IN RICE

Abstract A DNA construct comprising a silencing vector derived from a virus which induces the silencing of an endogenous gene when introduced into plants.
Full Text Title: A method of Virus-induced Gene Silencing in rice using a vector derived from a DNA virus.
FIELD OF INVENTION:
The present invention relates to the development of a Virus-induced gene silencing (VIGS) vector using Rice tungro bacilliform virus (RTBV) for inducing transient gene silencing rice. The present invention relates to the use of this vector as a means to "knock down" or silence the expression of rice genes. This method can be useful in determining the function of any gene in rice plant by studying the phenotype in the silenced plants.
BACKGROUND OF THE INVENTION:
VIGS is a technology that exploits RNA interference (RNAi)-based antiviral defence mechanism in plants as a tool for plant reverse genetics. In plants, the RNAi pathway is triggered as a natural defence response against invading viruses. Double-stranded RNA (dsRNA) produced as replication intermediates of RNA viruses or highly structured regions of viral transcripts produced during viral infections are efficient inducers for RNAi (Szittya et al.r 2002, Moissiard and Voinnet, 2006). In VIGS, recombinant viruses carrying plant cDNA fragments trigger the silencing of homologous host transcripts by RNAi, enabling this powerful tool to be applied for the analysis
of plant gene function (reviewed by Purkayastha and Dasgupta, 2009). In the last decade it has become an extremely popular tool to create transient "gene knockdowns" in plants, in which there is a transient silencing of the expression of a target gene. Such experiments are a great help in deciphering the functions of a gene and contribute towards functional genomics.
Several viral genomes have been modified for use as VIGS vectors in the last 10 years, most of them for use in dicot species (reviewed by Purkayastha and Dasgupta, 2009). Two VIGS vectors have been reported for use in monocot species, a Barley stripe mosaic virus (BSMV)-based vector for use in barley and wheat (Holzberg et al., 2002, Hein et al.r 2005; Scofield et al., 2005) and a Brome mosaic virus (BMV)-based vector for barley, rice and maize (Ding et al., 2006). Both the above vectors are based on the genomes of RNA viruses and their inoculation procedure involves in vitro transcription, which is a cumbersome and expensive procedure. RTBV is a rice virus containing DNA as its genomic material. Such a DNA-based VIGS vector for rice would be very useful for the study of the functions of rice genes and has great potential in discovering new genes which can contribute towards increasing rice production.
Rice is arguably the world's single most important crop being the staple food for over a third of the world's population. Furthermore, the entire genome of rice has been
sequenced and the crop is truly poised for research,on gene functions. The process for the analysis of genes functions in rice needs to be amenable to high-throughput processes, such that a large number of genes can be analyzed in a short period. With that background, the present invention was developed.
We have developed an RTBV-based VIGS vector for creating transient loss-of-function systems in rice. The vector has been used to silence an endogenous marker gene, encoding rice phytoene desaturase (pds). PDS is an enzyme involved in protecting chlorophyll from photo-bleaching. Silencing of pds leads to bleaching of the leaf lamina in dicots and the appearance of white streaks in leaves of monocots. It is widely used as a marker gene to test the efficacy of VIGS vectors in a wide range of plant systems.
The present invention provides a binary plasmid-based VIGS vector derived from RTBV DNA. The vector includes two of the four Open reading frames (ORFs) of the Indian isolate of RTBV. The vascular bundle-specific RTBV promoter (Mathur and Dasgupta, 2007) has been replaced with the constitutive and highly expressed constitutive maize ubiquitin promoter (MUP) in the VIGS vector. Because of the above change in the promoter sequences, a tRNA-binding site has been included in the primer designed to amplify the relevant viral DNA fragments, such that the site is placed immediately after the promoter sequences. This is because
replication of pararetroviruses such as RTBV pass through a reverse transcription phase, during which, reverse transcription primed by a tRNA on the RTBV promoter region is used as the starting step of the negative-strand DNA synthesis. A multiple cloning site has been included at the 3' end of the vector to allow easy cloning of target genes to be silenced.
OBJECTS OF INVENTION:
An object of the present invention is to develop a VIGS vector based on RTBV (pRTBV-MVIGS) for gene silencing in rice.
Another object of this invention is to provide the sequences of the primer used to construct the vector pRTBV-MVIGS (SEQ ID NO. 1 and SEQ ID NO. 2) .
Yet another object of this invention is to provide sequences of the primers used to amplify rice phytoene desaturase cDNA before cloning in pRTBV-MVIGS (SEQ ID NO.3 and SEQ ID NO. 4).
Yet another object of this invention is to provide sequences of primers used to amplify endogenous rice phytoene desaturase transcript by Real-Time PCR (SEQ ID NO.5 and SEQ ID NO. 6).
Further object of this invention is to provide gene sequences of the primer for rice ubiquitin 5 Real-Time PCR (SEQ ID NO.7 and SEQ ID NO. 8).
Still further object of this invention is to provide gene sequences of the rice phytoene desaturase cDNA (SEQ ID NO. 9).
Still another object of this invention is to provide a method for agroinoculation into rice plants for the purpose of studying gene silencing.
Yet another object of this invention is to provide a method of assay of the target gene transcripts whose silencing is being studied using Real-time PCR.
SUMMARY OF INVENTION:
According to this invention there is provided a DNA construct comprising a silencing vector derived from a virus which induces the silencing of an endogenous gene when introduced into plants.
The present invention relates to the development of a VIGS vector from RTBV to create transient gene silencing in rice.
The present invention provides information about binary vector-based VIGS using RTBV genome.
The present invention provides a method for agroinoculation of plants with the VIGS vector.
The present invention also provides information about the assay to detect accumulation of the VIGS vector in agroinoculated plants using slot-blot analysis.
The invention further provides a clone in which partial cDNA of phytoene desaturase (pds) is cloned in antisense orientation in the VIGS vector.
The invention further relates to the use of this vector for silencing pds gene in agronoculated rice plants and molecular analysis of the pds transcript levels by using Real-time PCR.
The invention further relates to the use of this vector for silencing any gene in agronoculated rice plants and molecular analysis of the corresponding transcript levels by using Real-time PCR.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS:
FIG 1: Dot-blot analysis of plants agroinoculated with pRTBV-MVIGS; Slot Al- C1 and D2-D10 plants agroinoculated with pRTBV-MVIGS, C2-C3, mock inoculated plants; C10- MQ negative control, Cll plants inoculated with pRTBV-MVIGS in E. coli, C12- mock-inoculated plant, Dl, pRTBV-MVIGS plasmid positive control; D11-D12, naturally infected RTBV plants. Total DNA for each sample ranged between 1.0-1.2 ug, pRTBV MVIGS (Dl) was as a positive control at 2.5 ng.
FIG 2: Silencing phytoene desaturase (pds) gene in rice inoculated with pRTBV-MVIGS- pds. a) The second leaf originating at 21 dpi in pRTBV-MVIGS-pds inoculated plants. White streaks indicated by the black arrows show photobleaching phenotype b) the second leaf originating at 21 dpi in plants inoculated with pRTBV-MVIGS. c) the second leaf of 5 week old uninoculated plants grown under same conditions.
FIG 3: Accumulation of pds transcripts in rice plants inoculated with pRTBV-MVIGS- pds. The levels of transcripts were determined by Real Time PCR and heights of bars indicate the values of the accumulated transcripts. Ui: plant inoculated using pRTBV-MVIGS; pds-plants inoculated with pRTBV-MVIGS-pds.
FIG 4: Sequence of primers used in the amplification of RTBV genes, showing the restriction enzyme recognition sites and tRNA binding sites. Numbers indicate the nucleotide residues of RTBV according to Nath et al. (2002). a)MVIGS-FP b)MVIGS-RP
FIG 5: Schematic representation of the pRTBV-MVIGS.
DETAILED DESCRIPTION OF THE INVENTION:
A preferred embodiment of the present invention relates to the development of a VIGS vector (pRTBV-MVIGS) and its use,
derived from the DNA virus RTBV for investigating gene silencing in rice.
Another embodiment of the present invention relates to
providing the nucleotide sequences of the primers used to
amplify selected genes of RTBV for the development of
pRTBV-MVIGS (SEQ ID NO. 1 and SEQ ID NO. 2) .
Another embodiment of the present invention relates to the process of cloning a gene of interest in pRTBV-MVIGS.
Another embodiment of the present invention relates to the transformation of the chimeric pRTBV-MVIGS containing the cloned gene of interest into Agrobacterium cells.
Another embodiment of the present invention relates to the method of agroinoculation of plants, where the plant can belong to the Family Poaceae.
Another embodiment of the present invention relates to the providing the nucleotide sequences of the primers used to amplify the cDNA for rice pds gene from rice plants (SEQ ID NO. 3 and SEQ ID NO. 4).
Another embodiment of the present invention relates to the providing a method of growing rice plants to study gene silencing.
Yet another embodiment of the present invention relates to the providing a method of assay for the accumulation of
transcripts specific for the target gene to be silenced using Real-time PCR after inoculating pRTBV-MVIGS containing the cloned gene of interest.
The construction of pRTBV-MVIGS was confirmed by plasmid isolation and restriction digestion with several enzymes. Before proceeding to use pRTBV-MVIGS as a VIGS vector, the capability of the vector to replicate and accumulate in plants was tested. Rice plants of the variety TN-1, which were 12-15 days old were agroinoculated with pRTBV-MVIGS by injecting 100 µl of a suspension of Agrobacterial cells containing the plasmid in the meristematic region of the plant. The process of agroinoculation is described in Example II. Total DNA was isolated from the plant leaves at 14 dpi and slot -blot analysis was performed. It was seen that more than 80% of all plants inoculated with pRTBV-MVIGS showed clear accumulation of the vector molecule. None of the uninoculated plants showed any " cross-hybridizing signal. The signal intensities shown by the above plants were similar to the infected plants containing RTBV, used as positive control (Fig. 1).
Having ascertained that pRTBV-MVIGS was capable of independently replicating and accumulating in agroinoculated rice plants, a partial cDNA fragment of rice pds was cloned into the Mlul and Pad site of the MCS of pRTBV-MVIGS in antisense orientation. Details of this process are described in Example 1. The pRTBV-MVIGS-pds was confirmed by restriction digestion with Mlul and Pad which led to the release of the pds insert.
Typical photobleaching phenotype associated with silencing of pds in monocot plants was observed in most plants agroinoculated with pRTBV-MVIGS-pds at 3-5 weeks post-inoculation (Fig. 2, Table I). The phenotype was observed in all emerging leaves for the subsequent 8-10 weeks, after which the emerging leaves remained green in color. The experiment was performed at 27°C and at 30°C to determine whether there was any effect of temperature. It was observed that pds silencing occurred only at 27°C. Repeated attempts to induce pds silencing at 30°C were not successful.
Table 1: Development of streaking phenotype in rice plants indicative of pds silencing following agroinoculation. The DNA constructs or medium used are indicated. Figures indicate number of plants showing phenotype /number of plants inoculated).

(Table Removed)
To determine whether there was a change in the levels of accumulation of the pds transcript, total RNA was isolated from plants showing photobleaching phenotype and Real-Time PCR analysis was performed, targeting the pds transcript. The analysis revealed that there was 60-90% decrease in the
levels of accumulation of pds transcript in agroinoculated plants injected with pRTBV-MVTGS-pds as compared to plants inoculated with pRTBV-MVIGS (Fig. 3) .
The DNA construct of Claim 4 wherein the viral genome comprises a nucleotide sequence of GenBank accession numbers AJ292232 and AJ314596).
The DNA construct of Claim 4 wherein the viral genome comprises DNA fragments derived from nucleotide sequence of GenBank accession numbers AJ292232 and AJ314596, as illustrated in Fig. 1.
The DNA construct of Claim 3 wherein the endogenous gene is as described in GenBank accession no. AF049356.
EXAMPLES:
The examples given are merely illustrative of the uses, processes and products claimed in this invention, and the practice of the invention itself is not restricted to or by the examples described.
Example 1: Construction of RTBV-derived VIGS vector (pRTBV-MVIGS).
The DNA of the Indian isolate of RTBV (Nath et al.t 2003) was used as the starting material for the construction of the VIGS vector. A cassette consisting of MUP, a multiple cloning site and Nos terminator was digested out of the
binary vector pB4NU (Ph.D. thesis, Saurabh Raghuvanshi, University of Delhi, 2001) by digestion with EcoRI and Hindlll and cloned into the promoterless binary vector pCAMBIA2300 to obtain the first intermediate vector pINTl. Only the MUP fragment was digested out of pB4NU by digestion with PstI and cloned into pBSK. The orientation of the MUP fragment in pBSK was determined by digestion with Sail and XbaI. MUP fragment was obtained by digestion with Kpnl and BamHI from the clone in the sense orientation and inserted into pINTl to obtain the second intermediary vector, pINT2, containing two MUP in a direct repeat orientation. Primers were designed to amplify RTBV ORF III and ORF IV from pRTBV204 (Nath et al., 2002) corresponding to nucleotide position 999 to 7183 of RTBV DNA.
The restriction sites for BamHI, SalI and tRNA binding site (to enable the essential tRNA-mediated initiation of reverse transcription to take place, Hay et al., 1991) and Kozak sequence (to ensure translation of ORF III amd ORF IV products, Kozak, 1987) were introduced into the 5' end of the primer in the order mentioned in MVIGS-FP as shown in SEQ ID NO. l(Fig. 4). To insert the Kozak sequence the nucleotides ACC were introduced after the tRNA binding site and a single A nucleotide at position 1003 in the RTBV genome was replaced with G. The concensus Kozak sequence ACCATGG was thus introduced. The revevrse primer fragment, MVIGS-RP containing the recognition sites of ApaI, PacI, AatII, MluI and BamHI shown as SEQ ID No. 2 was introduced into the 3' end of the primer for ease in cloning a test
gene. The primer pair MVIGS-FP/MVIGS-RP was used to PCR amplify approximately 6.1 kb fragment from a pRTBV204 (Nath et al., 2002) using Phusion polymerase (Finnzymes) and the product was cloned into TOPO cloning vector (Invitrogen, USA). The cloned fragment was subsequently released from the TOPO vector using BamHI. The 6.1 kb amplification product was inserted in pINT2, digested with BamHI, and ligated to obtain pRTBV-MVIGS, which was checked for the correctness and orientation of the insert by digestion with restriction enzymes. The vector was named pRTBV-MVIGS (Fig.5).
It has been reported that gene silencing by VIGS is best achieved if the gene to be silenced is inserted in the antisense orientation in the VIGS vector. To construct pRTBV-MVIGS with pds cDNA in antisense orientation, total RNA was isolated from rice using Trizol reagent
(Invitrogen) to amplify a cDNA corresponding to the pds. A 529 bp cDNA fragment of pds corresponding to nt 211-740 of the rice pds cDNA sequence as shown in SEQ ID NO. 9
(AF049356) was amplified from the cDNA pool of rice using oligonucleotide primer pds-FP and pds-RP shown in SEQ ID No. 3 and SEQ ID No. 4. The primers were designed to include a PacI and Mlul restriction site at the 5' and 3' end of the pds-FP and pds-RP respectively. The cDNA fragments thus obtained was cloned into T/A vector (InsTA cloning Kit, Fermentas), digested with MluI and PacI and cloned in a reverse orientation into the MluI-PacI sites of MCS of pTRBV-MVIGS to give rise to pRTBV-MVIGS-pds.
Example 2: Agroinoculation of plants.
For agroinoculation, all vectors were transformed into Agrobacterium tumefecians strain EHA105 by heat-shock method. Approximately lµg of plasmid DNA for each contract was added to competent cells of Agrobacterium and plunged in liquid nitrogen for two minutes. The tubes were then incubated at 37°C water bath for 5 minutes, followed by on ice for 10 minutes. One ml LB broth was added to each tube and was incubated at 28°C overnight and shaken constantly at 200 rpm. Out of the 1 ml, 200ul was spread on LB plate supplemented with appropriate antibiotics. The transformed colonies obtained were screened by colony PCR methods.
A primary culture was initiated from a single transformed Agrobacterium colony in LB medium supplemented with appropriate antibiotics and grown at 30°C in a shaker overnight. A secondary culture was grown to an OD600. of 0.6-0.8 using similar growing conditions, the cells were harvested and resuspended in l0mM 2-[N-morpholino] ethanesulfonic acid (MES), l0mM MgCl2, 200µM acetosyringone to an OD600 of 20. Approximately 15 day-old rice plants grown in Yoshida's medium (Yoshida et al., 1976) were used for agroinoculation. About 50ul of the bacterial suspension was injected into the meristematic region and the plants were then transferred onto sterile Whatman No. 1 filter paper immersed in Yoshida's medium placed on a solid support with its ends dipped into a reservoir containing the medium. The plants were covered with moist tissue paper
and transferred to tubes containing Yoshida's medium 24 hours post-inoculation and were maintained under conditions mentioned in Example 6.
Example 3: Assay for the accumulation of viral vectors in inoculated plants
DNA slot-blot analysis was performed to measure the accumulation of pRTBV-MVIGS in rice leaves. Total genomic. DNA was isolated from agroinoculated plants 15 days post-inoculation (dpi) using the Purgene method described below. About 15 cm of rice leaf was ground using liquid nitrogen in a mortar-pestle. About 1.5 ml of extraction buffer [10mM Tris-HCL (pH-8.0), lmM EDTA (pH-8.0), 1% SDS] was added to the finely ground tissue and transferred to a fresh microcentrifuge tube. The contents were mixed by vortexing the tube for several seconds. The tubes were incubated at 65°C for 20 minutes followed by cooling on ice for 5 minutes. About 500 µl of 6 M ammonium acetate was added to each of the tubes and the contents were mixed thoroughly. The samples were centrifuged for 10 minutes at 13, 000 rpm at 4°C and the supernatant was transferred to a fresh tube and centrifuged again under similar conditions to remove any residual debris. The clear supernatant was transferred to two fresh microcentrifuge tubes and to each of the tube 750 µl of isopropanol was added. The tubes were incubated at -20°C for 30 minutes to precipitate the DNA. The samples were centrifuged for 10 minutes at 13, 000 rpm at 4°C to pellet the DNA. The supernatant was discarded and the DNA
pellet thus obtained was washed with 75% ethanol for 10 minutes at 13, 000 rpm at 4°C. The pellet was air-dried and resuspended in 20 µl of buffer (10mM Tris-HCL, pH-8.0; 1mM EDTA, pH-8.0). For DNA slot-blot analysis 2 ug of DNA was spotted for each of the inoculated, mock inoculated and uninoculated sample. As a positive control 200 ng of total genomic DNA from RTBV infected plants and 10ng of pRTBV-MVIGS DNA were also spotted onto the membrane. The probe for hybridization was derived from a 900 bp MP fragment of RTBV corresponding to nucleotide 999 - 1898 on RTBV, WB genome. Megaprime DNA labeling System (Amersham) was used for hybridization of the blots.
Example 4: RNA extraction and cDNA synthesis
Total RNA was isolated from plants inoculated with pRTBV-MVIGS-pds or controls with pRTBV-MVIGS at 28 dpi, using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNasel (Fermentas, USA) prior to cDNA synthesis following the manufacturer's recommendations. The DNasel treated RNA was cleaned using RNeasy Kit (Quiagen). The first strand cDNA was generated by using high capacity cDNA archive kit (Applied Biosystems, USA).
Example 5: Real time (RT-) PCR analysis
For RT-PCR analysis, primers that anneal outside the region of the pds cDNA cloned into pRTBV-MVIGS were used to ensure that only the endogenous pds mRNA is reverse-transcribed.
Rice ubiquitin 5 (UBQ5) cDNA was used as an internal constitutively expressed control. Real-time RT-PCR analysis was carried out with a Real time PCR detection system (Applied Biosystems, USA). Primers were designed using Primer Express software. For SYBR Real-time RT-PCR experiments, primers RTpds-FP and RTpds-RP as shown in SEQ ID. NO. 5 and SEQ ID NO. 6 respectively were used for detection of pds transcripts while primers RTubq5-FP and RTubq5-RP as shown in SEQ ID. NO. 7 and 8 respectively were used for UBQ5 as the internal control.
Example 6: Plant Material and growth conditions
TN1 variety of indica rice was used in this study. Plants were grown either in tubes in Yoshida's medium at 27°C and a 16 hours light and 8 hours dark cycle using artificial lighting with 70% humidity at 30°C, the other parameters remaining the same.
ADVANTAGES OF THIS INVENTION
1. Silencing of rice genes can be detected within 14 days of agroinoculation of pRTBV-MVIGS containing the target gene sequences by looking for the development of silenced phenotype.
2. A multi-cloning site has been introduced into the vector for cloning any rice gene to be silenced-.







CLAIM:
1. A DNA construct comprising a silencing vector derived from a virus which induces the silencing of an endogenous gene when introduced into plants.
2. The DNA construct in Claim 1 wherein the endogenous gene is selected from any gene whose silencing results in an altered phenotype.
3. The DNA construct as claimed in claim 1 wherein the endogenous gene is phytoene desaturase.
4. The DNA construct as claimed in claim 1 wherein the viral genome is derived from a pararetrovirus.
5. The DNA construct as claimed in claim 4 wherein the viral genome is from Rice tungro bacilli form virus.
6. The DNA construct as claimed in claim 3 wherein the endogenous gene is derived from GenBank accession numbers of all rice genes whose silencing results in a visible phenotype and a measurable change in the plant.
7. The DNA construct as claimed in claim 1 containing the primer pairs selected from a group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8.
8. The DNA construct as claimed in claim 1 containing a DNA fragment amplified using primer pairs selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8.
9. The DNA construct as claimed in claim 1 where the plant is selected from a group consisting of Family Poaceae.

10. The DNA construct as claimed in claim 1 where the plant is selected from a group consisting of rice.
11. The DNA construct as claimed in claim 1 where the plant shows streaking in its leaves.
12. A method of inducing silencing of any gene in a plant, comprising the steps: introducing the DNA construct as claimed in Claim 1 into rice plant and growing the plant under conditions which result in gene silencing.
13. The method as claimed in claim 12, wherein the gene is any rice gene, silencing of which results in a visible or measurable phenotype.
14. The method as claimed in claim 13 wherein the gene is rice pds.
15. The method as claimed in claim 12 wherein the step of introducing the DNA into plants is agroinoculation.
16. The method as claimed in claim 12 wherein the plant is selected from Family Poaceae.
17. The method as claimed in claim 12 wherein the plant is rice.
18. The method as claimed in claim 12 wherein the endogenous gene regulates male fertility.
19. The method as claimed in claim 12, wherein the step of introducing the viral silencing vector is selected from the group consisting of particle bombardment, Agrobacterium-mediated transformation, Agrodrench, abrasion of plant surfaces and plasmid inoculation.


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Patent Number 278167
Indian Patent Application Number 2476/DEL/2009
PG Journal Number 53/2016
Publication Date 23-Dec-2016
Grant Date 15-Dec-2016
Date of Filing 01-Dec-2009
Name of Patentee UNIVERSITY OF DELHI
Applicant Address DELHI -110007, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 ARUNIMA PURKAYASTHA DEPARTMENT OF PLANT MOLECULAR BIOLOGY, UNIVERSITY OF DELHI SOUTH CAMPUS, BENITO JUAREZ ROAD, NEW DELHI-110021, INDIA
2 INDRANIL DASGUPTA DEPARTMENT OF PLANT MOLECULAR BIOLOGY, UNIVERSITY OF DELHI SOUTH CAMPUS, BENITO JUAREZ ROAD, NEW DELHI-110021, INDIA
PCT International Classification Number A23J
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA