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• RNAi and Crop Improvement

   

RNAi is thought to have many different applications in all sorts of organisms. We will look mainly at the applications in plants in this section. In plants, RNAi is thought to be a good technique to study gene function. This would increase our understanding, of the various genes within a plant.

There are several projects currently studying this. These include the CATMA group (Complete Arabidopsis Transcriptome MicroArray), which is generating gene sequence tags (GSTs) representing each Arabidopsis gene. These have been designed so that they will hybridise on Arabidopsis cDNA microarrays in a gene-specific manner. The AGRIKOLA consortium (Arabidopsis genomic RNAi knock-out line analysis) uses these PCR products to generate gene-specific RNAi constructs for each Arabidopsis gene for use in large scale gene silencing studies (Matthew,L. 2004). It is thought that RNAi also has applications in Plant Science, in terms of crop improvement.

RNAi and Functional Genomics

The major application that RNAi has in Plant Science, is in functional genomics. RNAi has only been used in plant functional genomics very recently. RNAi is a technique that can be put to good use in plant functional genomics. Identification of gene function is very important as gene knockouts in many plants do not result in phenotypes under normal conditions. Screens are therefore being set up with different growth conditions, so that we can see which mutant phenotypes are linked to which growth conditions. This is considered the first step in assigning gene function (Thakur, A. 2003).

The principle behind the use of RNAi in plant functional genomics is quite simple. The dsRNA is introduced into the cell. This activates the DICER gene and RISC complex which eventually leads to loss of gene expression. In this way, after studying the plant and which traits have not appeared, the function of the gene can be inferred (Thakur, A. 2003). As mentioned earlier, there are several projects underway that are working on plant functional genomics using RNAi.

Kit used to make siRNAs for use in RNA interference

(http://www.nature.com/cgi-taf/DynaPage.taf?file=/nbt/journal/v20/n4/full/nbt0402-407.html, Last Accessed: 1st May, 2005)

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Advantages and drawbacks of using RNAi in functional genomics

Of course, RNAi has advantages and limitations when used in plant functional genomics. RNAi has many advantages over the functional genomics strategies based on insertional mutagenesis. The first and foremost advantage is that RNAi gives us the ability to specifically target a gene. If the target sequence is carefully chosen, a specific gene or genes can be silenced. RNAi can also be used to achieve varying levels of gene silencing, using the same ihpRNA construst in different lines. This allows for selection of lines with varying degrees of gene silencing. In addition to this, the timing and extent of the gene silencing can be controlled, so that genes that are essential will only be silenced at chosen stages of growth or in chosen plant tissues (Matthew, L. 2004). So, RNAi provides us with a great degree of flexibility in the field of functional genomics.

There are also limitations however to RNAi. Unlike in insertional mutagenesis, for the use of RNAi the exact sequence of the target gene is required. Once this sequence information is available, the rest of the process is however relatively fast. Secondly, delivery methods for the dsRNA is a limiting step for the number of species which RNAi based approaches can be used easily. Due to this, improvement and further research into the kinds of vectors that can be used safely and reliably is needed. There have also been some reports that it has been difficult to detect mutants in which there has been subtle changes in gene expression. In plants, marker genes are being developed that will indicate if there has been a change in gene expression (Matthew, L. 2004).

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Vectors used in RNAi based functional genomics

There are currently many different vectors in use for performing RNAi for the use of functional genomics. These include such vectors as binary vectors used for expression of GUS and GFP proteins, the pHELLSGATE high-throughput gene silencing vector and a high throughput tobacco rattle virus (TRV) based Virus-induced gene silencing (VIGS) vector. One feature that is commom to all of these vectors is the inclusion of the Gateway recombination-based technology for cloning that was developed by Invitrogen. The Gateway system is used to replace conventional cloning steps that took up valuable time. This is being exploited by several projects including the AGRIKOLA project (Matthew, L. 2004).

Gateway is a cloning system developed by Invitrogen that is universal, and has sped up the process of plant functional genomics. It is based on the phage lambda system of recombination. It enables segments of DNA to be transferred between different vectors while orientation and reading frame are maintained. It can also be used for transfer of PCR products. It saves valuable time, because once the DNA has been cloned into a Gateway vector, it can be used as many genome function analysis systems as is required. In this way, the use of vectors in the process of plant functional genomics has been made much easier, while the process has also been made faster. This allows for higher throughput analysis to occur (Invitrogen, 2004).

Example of a Gateway compatible destination vector

(http://www.invitrogen.com/content.cfm?pageid=3376, Last Accessed: 1st May, 2005)

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Delivery of the vectors

There are many different ways in which vectors can be delivered. Firstly, there is microparticle bombardment with vectors that express intron-containing hairpin RNA (ihpRNA) or dsRNA. The second method of delivery is through the use of Agrobacterium carrying a T-DNA that expresses an ihpRNA transgene. Then there is virus induced gene silencing (VIGS), where the target sequence is integrated into the virus' sequence which is then used to infect the plant. These can also be expressed from transgenes introduced by Agrobacterium, or by stable transformation by ihpRNAs that express transgenes (Matthew, L. 2004).

Each of these methods of delivery have their advantages and disadvantages. Microparticle bombardment is a transient method of vector delivery. Its advantages are that it is rapid, has a wide range of species on which it can work, and is a valuable tool for work on single cells. The disadvantages are that this limits gene silencing to the cells on the surface of the leaf, and silencing is only temporary. The Agrobacterium method is also a transient system for vector delivery. The advantages of this method of vector delivery are that it is rapid and provides a high throughput, it is relatively easy to use and it has a low cost. The disadvantage is that it has not really been tested on most species, so we do not know the scope for use on different species (Waterhouse, P.M. & Helliwell, C.A., 2003).

Virus induced gene silencing (VIGS)is another method of vector delivery that is transient. It has many advantages. It is rapid and provides a high throughput, and it is easy to use. It can be applied to plants that are mature, and is considered to be good for use on species that difficult to transform. With these many advantages come many disadvantages. It has limitations on its host range. It might have restricted regions of silencing, and there may be size restrictions on the inserts. It is dependant on the availability of infectious clones. Viral symptoms could be superimposed onto the silenced phenotype. The ihpRNA method of delivery also has many advantages. It has no restrictions on host range, and provides heritable gene silencing. It has a high throughput, and one can control the degree of gene silencing that occurs. In addition to this, one can control the tissue specificity of the gene silencing. This method of vector delivery has one disadvantage: an efficient technique for transformation is needed (Waterhouse, P.M. & Helliwell, C.A., (2003).

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Example protocols

For the purpose of this section of the website, we will look at the protocol used by the AGRIKOLA project to conduct their work.

The first stage is amplification by PCR. The following components are required for this, and the procedure for the PCR is as follows (to be carried out over 35 cycles):

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The next stage is cloning a GST as a Gateway entry clone using BP cloning. The following components are required:

It is very important to add mix to PCR product and not the other way round.

• Incubate the reactions overnight at 25?C
• Add 0.5 ?l of proteinase K at 2?g/?l
• Incubate reactions for 15 mins at 37?C

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After this, one must carry out bacterial transformation in the 96-well plates. The procedure for this is as follows:

• Add 10 ?l of "ultra-competent" DH5 cells to 1 ?l of the LR reaction in 96-well PCR plates
• Incubate for 20 min on ice-water
• Heat shock it for 15 sec at 42?C in a water bath
• Incubate it for 5 min on ice-water
• Add 90 ?l SOC media to this
• Shake it for 1 h at 37?C
• Add 100 ?l of transformed bacteria to 1 ml 2LB media 15 mg/l kanamycin in 2ml 96-well plates
• Shake at 37?C until OD=1.4. The bacterial density is critical for miniprep yields. It is recommended to shake for 20 h at 800 rpm on a Heidolph Titramax 1000 shaker.

For the second PCR, the following components are required, and the following PCR conditions (carried out over 35 cycles):

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The next stage is to clone to GSTs into the hpRNA vector.

The following components are required for this:

It is very important to add the mix to the miniprep plasmid and not to do this the other way round.

• Incubate the reactions overnight at 25?C
• Add 0.5 ?l of proteinase K at 2?g/?l
• Incubate reactions for 15 mins at 37?C

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After this, one must carry out bacterial transformation in the 96-well plates. The procedure for this is as follows:

• Add 10 ?l of "ultra-competent" DH5 cells to 1 ?l of the LR reaction in 96-well PCR plates
• Incubate for 20 min on ice-water
• Heat shock it for 15 sec at 42?C in a water bath
• Incubate it for 5 min on ice-water
• Add 90 ?l SOC media to this
• Shake it for 1 h at 37?C
• Add 100 ?l of transformed bacteria to 1 ml 2LB media 50 mg/l kanamycin in 2ml 96-well plates
• Shake at 37?C until OD=1.4. The bacterial density is critical for miniprep yields. It is recommended to shake for 20 h at 800 rpm on a Heidolph Titramax 1000 shaker.

For the third PCR, the following components are required, and the following PCR conditions (carried out over 35 cycles):

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(Agrikola Cloning Protocols, Last Accessed: 10th April, 2005)

Now the cloning should have been carried out, and we will move on to the transformation aspect. The first step will be to create the competent Agrobacterium GV3101:pMP90:pSOUP cells. The following steps are needed for this:

• Grow 8 ml culture of Agrobacterium overnight in LB with 5 ?g/ml tetracycline + 50 ?g/ml rifampicin + 25 ?g/ml gentamycin (LB used at all stages for Agrobacterium has 5 g/l NaCl)
• Inoculate the 8 ml overnight culture in 192 ml LB without antibiotics
• Shake at 28?C until an OD of about 0.5 (generally 3-6 hrs but depends on strain used)
• Centrifuge this at 4000 rpm for 15' at 4?C
• Resuspend pellets in 10 ml of ice-cold 10 mM Tris-HCl (pH of 7.5)
• Resuspend pellets in 10 ml of ice-cold 10 mM Tris-HCl (pH of 7.5)
• Centrifuge this at 4000 rpm for 15' at 4?C
• Resuspend pellets in 20 ml cold LB and freeze in liquid N₂; these cells will stay competent for 6 months if kept at -80?C

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Next comes the actual transformation of the Agrobacterium in 96-well plates:

• 200 ?l competent cells + 1 ?g plasmid mixed in pre-chilled tubes or plates
• Put this on ice for 5 min
• Put this for 5 min in liquid N₂
• Put this for 5 min at 37?C
• Add 800 ?l LB to it
• Shake gently for 2 h at 28?C
• Liquid selection of 100 ?l in 1.3ml LB + 5 ?g/ml tetracycline + 50 ?g/ml rifampicin + 50 ?g/ml kanamycin + 25 ?g/ml gentamycin
• Shake for 2 days at 28?C on a Titramax at 600 rpm
• Make a 25% glycerol stock and conserve this plate at -80?C

For the fourth PCR, the following components are required, and the following PCR conditions (carried out over 35 cycles):

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After this we have selection and validation of the individual Agrobacterium clones:

• Make a 4.10⁵ dilution of glycerol stock
• Plate 150 ?l of this dilution on Q-tray with 200 ml of LB + 5 ?g/ml tetracycline + 50 ?g/ml rifampicin + 50 ?g/ml kanamycin + 25 ?g/ml gentamycin
• Grow for 2 days at 29?C
• Inoculate four individual colonies each into 1 ml selection medium (LB + Kan, Tet, Rif, Genta)
• Shake for 2 days at 29?C
• Conduct PCR 5 on culture with primers 51/56/64/69 (same as PCR 4 above)
• Make up 25% glycerol stock (add 75 ?l of 50% glycerol to 75 ?l of the culture)
• Select correct clones for plant transformation, based on PCR 5 results

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Now we have the actual plant transformation:

• Day 1: sowing; cultivation at 16 h 145 ?E, 20?C, 75% relative humidity / 8 h 6?C, 75% relative humidity
• Day 7: transfer to cultivation at 8 h 145 ?E, 20?C, 75% relative humidity / 16 h 16?C,75% relative humidity
• Day 14: transfer into pots: 9 seedlings per pot; continue cultivation at 8 h 145 ?E, 20?C, 75% relative humidity /16 h 16?C, 75% relative humidity
• Day 28: transfer to glass house, cultivation at 16 h 250 ?E, 20?C, 80% relative humidity / 8 h 18?C, 50% relative humidity
• Day 47: clip primary inflorescence
• Day 54: dip coflorescences into Agrobacterium suspension
• Day 75: cover inflorescences with bags
• Day 89: stop watering
• Day 103: collect bags, store at 15?C 15% relative humidity for ca. 2 weeks
• Day 119: harvest seeds into glass tubes, store at 15?C 15% relative humidity (for thrips control: for 48 h -20?C)

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Finally we have the selection of transformants

• Day 1: sowing (GA-treatment); cultivation in glass house at 16 h 250 ?E, 20?C, 80% rel. hum. / 8 h 18?C, 50% relative humidity
• Day 7: BASTA treatment (40 mg/l)
• Day 9: BASTA treatment
• Day 12: BASTA treatment
• Day 14: BASTA treatment
• Day 16: BASTA treatment
• Day 21: transfer resistant seedlings into pots; cultivation in glass house at 16 h 250 ?E, 20?C, 80% relative humidity / 8 h 18?C, 50% relative humidity
• Day 75: cover inflorescences with bags
• Day 89: stop watering
• Day 103: collect bags, store at 15?C 15% relative humidity for about 2 weeks
• Day 119: harvest seeds into glass tubes, store at 15?C 15% relative humidity (for thrips control: 48 h -20?C)

For a more detailed protocol, please look at the AGRIKOLA Project website.

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(Agrikola Transformation Protocols, Last Accessed: 11th April, 2005)

RNAi and Crop Improvement

RNAi also has applications in the field of crop improvement. This is however very much related to functional genomics. Functional genomics can be used to find out the function of genes, and therefore improve traits that various crops display. RNAi is considered to be especially good for this purpose as it is highly specific, and can down-regulate instead of knock-out a gene if required. A tool produced for this by Bayer CropScience, called SVISS, is currently used in research for this purpose (Metzlaff, M. 2005).

It has been shown by researchers that crops that were transformed using RNAi constructs, resulted in a stable modification of the biochemical pathways. This can result in much improved productivity and yield, as well as improved crop quality. Thus, RNAi has an economic application in the field of Plant Science (Metzlaff, M. 2005).

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