What effects does this murderer leave to the victim?

What CMV provoques on the victim after having commited his job is a disease called Cucumber mosaic virus, which is present worldwide and affects a wide viriety of plants, aproximately 190 different ones' that include cucumber,  tomatoes, pumpkins, carrots, spinachs, beans, cellery,... and not only affect vegetables, but flower plants as begonias, tulips, dahlias, etc.

Image 3.1. Image showing
clearly the mottling leaves.

What means suffering the damage of Cucumber mosaic virus?

Having the disease implicates a change in the physical structure based on a modification between hosts. This can be simply external, what means that only change it appearance, for example, mottled leaves, and circular spots. 

Image 3.2. Image showing
dwarfism effects of CMV.

However, what farmers and scientists are really worried about, is the harmful modifyings that the plant can suffer from the CMV, and this changes goes from dwarfism and distortions of the fruit and flower, going through elongation and narrowing of the leaves, and finishing with the stunting of the fruits (has no comercial value). This effects has a big repercution on economy, thus can make a farmer being bankrupt.

But what si more worring is the fact that the effects of the murderer in the victim are completely different depending on the specimen affected, and also the season time is being infected, as if it happens in an early season, the effects are devastators and kills the plant, transforming it into a vegetable unmarketable, but if instead it happens in an older season, sympthoms doesn't expresses so agressively.

Image 3.5. Effects on
tulipas' flowers of CMV.

Image 3.3. Effects of
CMV in red pepper.

Image 3.4. Distortion effects
of CMV in a Curcubita pepo
crop.

Unforunately, there is no tratment to solve the infection of the murderer, neither resistant plants to this Bromoviridae family virus. Thus, we can only applicate preventive measures.

How the murderer achieves to penetrate a really complex security system?

I am gonna explain the mechanism of commiting murder used by this relentless virus. 

Image 4.1. Insects being 
vectors of the virus

First of all, this murderer has to be transmitted from somewhere, and this Bromoviridae family virus, arrives to the host in two possible ways. The first, by a mechanical inoculation in plant by an insect, and the second is even simple, staying in contact one plant with another. 

Image 4.2. Vegetable cells that
shows easily the tonoplast

After have penetrated on it, Cucumovirus has succeed one of his objectives necessaries to commit the crime: entering to a vegetable cell. When staying inside it starts to uncoat, what implicates to release the viral nucleic acids from the capsid, liberating the viral genomic RNA into the cytoplasm, contained inside the virus cell sorrounded by the capsid; causing the expression of the first and second protein, what produces the recplication proteins, responsible for synthetising the RNA strands, in a viral factory, that can be different types of organelles, in concrete, the accomplice of giving home to this criminal is the vacuoles membrane or also called tonolpast. 

Image 4.3. Electronic micrscope
photo of a bascular bundle cells

And the crime starts! 
It produces a transcribement of the complete RNA genome, providing new viral RNA genomes in order to multiply the CMV to the whole plant, but it can really go somewhere alone? No, there must be something controlling it and this is not other than the capsid, a organelle that mantains everything to it position, so can, now, moves. Finally the viral movement protein triggers the formation of tubular structures called plasmodesmatas what allows the murderer to move from one host to the other next to it.

Image 4.4. Picture displaying the transport plasmodesmata system

But "when more you have, more you want", hence the Cucumovirus not happy by infecting mature cells, it transports cell-to-cell, traspassing different cells as Sclerenchyma, Collechyma, Parenchyma and Xyle, aiming to arrive to the phloem so can be transported easily to the green shots, his ambitious objective.

Image 4.5. Explicative picture of bascular bundle cells

CMV virus inoculation, let's make it slay

Before doing this experiment, it has to be done in a crop plant, in a controlled atmosphere (temperature of 22 degrees, humidity, containment), of Cucumis melo during 7 to 10 days so you dispose of a large number of youth individuals. Then you choose between most healthy melon plants.

Image. 5.1. Montse Martin working on the
selection of healthy melon plants

Image 5.2. Mixing green
shots in the mortar

After doing the selection with everything prepared, you proceed to extract a huge amount of virus contained in a pumpkin plant which has been yet infected with CMV, as it grants more efectiveness in the inoculation. To extract the Cucumovirus, you have to cut with an scalpel the green shoots (that contain most of the virus) and letting it sit in a mortar, you mix this part of the plant with coal and a phosphate buffer, and finally we grind it all together.

Image 5.3. Rubbing the Cucumis melo crop

We rub the youth plants, which had been before treated with an abrasive, with our fingers in an slowly way, the finger is wet with the virus solution, hence it is absorbed into the Cucumis melo from the scars (made by the rub of the abrasive).

Image 5.4. Watering melon
plants after being rubbed

When this is finished you wait for more or less thirty seconds and water it so CMV that has not entered in the plant, and is remaining in the superficial part of the melon plants goes away.

DNA extraction, let's deep inside the murderer!

Image 6.1 Me and Fuensi 

working together and having 

fun.

What we pretend to get from this expirience (an extraction) is the substance contained in each cell like fear of the murder, hence we can use it with a wide variety of objectives, as analyzing simply the DNA of the studied individual or instead looking up if the transgenic gene has been inserted to the host, or ... But there is needed an essential machine called PCR, that can multiply the DNA chain, by making copies of a region of the original DNA (a unique chain) stablished by the scientist, who add primers, a molecule that can read the information and starts duplicating the strand by one concrete point, and finishes into another one fixed too. My experimentation had the objective to identify if the transgenic gene had been inserted to the Cucumis melo DNA.

Image 6.2 Liquid nitrogen.

First of all, of course, you need different possible individual you suspect has acquired this transgenic gene. Then, you maintain this alive substain in cold conditions, since you are prepared to use the liquid nitrogen in order to crush the leaf into little pieces. After having it all crushed in the eppendorfs, you start preparing the Doyle buffer solution.

Image 6.3 Vortexing the solutions
in the eppendorfs.

After having prepared it a part in a fume chamber, you added into the eppendorfs, thus you mantain the pH stable, keeping the leaf in good conditions. When this proces is finished, you proceed to gentle vortex the solutions and then putting them in a metal microtiter plate warming up.

Image 6.4 Me pipetting.

The process to break the cell walls and start isolating the DNA starts by additing isoamyl chloroform and then making a vortex, as well as centrifugating it. The result will be a solution separated in different phases, that the scientist will separate by using a pipette, taking apart all the aqueous solution that contain the DNA, and leaving apart all the cell "trash" (cytoplasm, wall, organelles,...). 

Image 6.5 Photo of the different
phases of the solution after
centrifuge.

After this, you combine the aqueous solution with cold isopropanol, and moving the solution slowly, you get very thin wires, the DNA. 

This deoxydoribonucleic acid will be isolated and will be let dry before resuspending the DNA into HPLC water.

Now it is time to let the PCR work and some time after, you get a lot of copies of the strand fragment. 

Image 6.6 Fuensi filling the
holes of the electrophoresi.

How do you know this fragment has acquired the transgenic gene?

Very simple, you only have to do an electrophoresis between the different copies of the different strands you have obtained from the PCR. You compare them with the transgenic gene you add at the first hole.

Image 6.7 Photo of a PCR in process