First expirience in the CRAG

The Center for Research in Agricultural Genomics (CRAG) has been very kind with me,and has decided to help me in my research work. Montse Martín, a renouned scientist, leader in the investigation group plant virus, they recieved me with the open hands and hostesd me inside the investigation group, so I could document myself with first hand information so I can develop my Batxillerat final work. She will be my orientator in this agrifood work.

Image 1.1. Logo CRAG

CRAG is an independent investigation center associated with the Consell Superior d'Investigacions Científiques (CSIC), Institut d'Investigació i Tecnología agroalimentaria (IRTA), Universitat Autònoma de Barcelona (UAB), and the Universitat de Barcelona (UB).

Image 1.2. CRAG building situated on the UAB

What is a gene?

A gene is a lineal sequence of nucleotides of DNA or RNA, located in cell nucleus, essential to carry out a manifestation of an inheritable characteristic of an alive individual. In terms of structure, a gen is a fragment of a large molecule of DNA that storage information to fabricate a determined protein. This protein is the one who determine the corresponding character, as the eyes color, skin color, presence of seeds, resistance to a disease,...

Image 2.1. Image of a chromatide and the material contained on it, showing in an augmentated way, a gene (yellow)

Image 2.2. An image that shows
in yellow and red two different 
locus.

Usually, genes develop its function through transcription and translation. In some cases, although, can happen that a gene can make its function doesn't necessarily requiring this genetic processes. 

Each gene occupies a certain position in the chromosome called locus. And if you take into account the whole chromosomes of an individual we talk about the genome, as it is the set of genes of species.

To understand it easily, maybe is better to think of the DNA or RNA that form an individual (genome) as an ecnyclopedia, in which the nucleotides are the letters, the genes are the amount of chapters that compose the volumes created by the agrupation of letters in a determined way, and the chromosomes are the different volumes that form the encyclopedia.

Image 2.3. Gene sequence showing the nucleotides and each aminoacid formed

What is the genetic engineering?

Image 3.1. Picture showing
how we can play with the
DNA.

Is an amount of techniques that allow you modify the characteristics of an alive organism through directed and controlled modification of its genome, adding, eliminating or modifiying some of its genes, that it is not other than a modification in the DNA of the organism all time oversight by humans who carry it out. 

Image 3.2. Picture showing how we can play
with the DNA.

Thus, among other applications, genetic engineering allows you to eliminate an undesirable characteristic as producing a toxine, annulling the corresponding gene. 

Also it allows you to introduce a new characteristic as an insect resistance, copying the corresponding gene resistant to this individual from a resistant specie and introducing it in the genome of the susceptible specie. 

As it exist an universality of the genetic code, genetic engineering can use the "information" in all organisms.

What is a transgenic?

Image 4.1. gold rice 

transgenic plant.

Is an individual whose genome has been modified through genetic engineering, to introduce new genes or to modify the function of an own gene. Due to this modification the transgenic plant shows a new characteristic. Once carried out the insertion or modification of the gene, this expresses, and transmites itself to the offspring as one more gene of the individual. In concrete, in transgenic plants this genetic modification is performed in a coontrolled way that only affects a reduced number of well known genes. As a result of this fact, the transgenic variety don't differ much from none transgenic ones.

Image 4.2. Photo taken by me of a crop of transgenic plants resistant to a virus which has been tested by putting the virus in the atmosphere, hence, showing wich plants has acquired the gene (red) and which not, and are dying (yellow).

Who is the guilty of this vegetable crime?

There is no other culpable than Cucumovirus, which can be identified if looking to it  you are able to diferenciate a non-enveloped, spherical virion of about 29 nanometers in diameter, which has a T=3 icosahedral symmetry composed of 180 coat proteins: 12 pentamers and 20 hexamers.

Image 2.1. Picture about the real 
morfology of CMV

How this murderer is formed of?

It is not an easy question, and there must have being other researchs before answering this issue. His structure is formed of a segmented tripartite linear RNA composed of RNA1, RNA2 and RNA3. Each genomic segment is 3' tRNA-like structure and a 5' cap.

Image 2.2. Position of dierent RNA that compose the 
segmented tripartite linear virus RNA

This Cucumovirus expresses himself by using the RNA as a messager RNAs. Then, RNA1 and RNA2 encode proteins 1a and 2a, involved in the genome replication and internal transcription of sgRNA4 from the negative copy of RNA3. HRNA3 is translated into a movement protein, while sgRNA4 it translates to a capsid one. Apart from this, ORF2b, present at RNA2, encodes a viral supressor of RNA silencing.

How does it commit the crimes?

When the expression is done, virus expresses the protein 1st and 2nd to produce replication proteins, as I have mentioned before, on the viral factory and after it transcribes/replicates providing new ssRNA genomic (new viruses).

Everithing continues translating RNA4 that produces the capsid that envolves the ssRNA genome, and assemblying this virus particles.

Image 2.3. Picture of diferent viral factories for viruses

Finally, it is the turn of the viral movement protein, that triggers the information of tubular structures that mediate viron cell-to-cell transfer via a tubule-guided mechanism.

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

Outstanding expirience at the Festival of Cience

Last 19th of June I had the pleasure to visit the Festival of Cience celebrated in the Parc de la Ciutadella, a really big park in Barcelona. This event apart from being really amazing, conmemorated its tenth birthdy.

Image 7.1 Addvertisement of the Festival of the Cience

I went to the Festival not only to deep a little bit more inside the agrifood and how people have selected differents kinds of vegetables to satisfy their own needs. But also to see in first person the continious evolution of cience applicated in a wide variety of research branches, as sport, medicine, physics, chemistry, biology,... As you can chech the photos below.

But what really caught my attention was CRAGs' exposition about agrifood and human selection of vegetables among ages, hence selecting some plants that benefit ourselves, and reporting really losts in vegetables biodiversity, as we selected some plants to make it grew up and others we leave it at their fate and leave apart making them disappear.

Image 7.2 Montse Martin 
exposing "the evolution 
of plants with virus 
and humans

I really got impressed about Montse Martin exposition as made me open the eyes in sense of importance of human acts, and virus in a profitable way. 

We see virus as something that conferes a bad effect on the host, and hurts it. However, we are completely wrong, as there are some viruses that allow the host survival in different ambients and exposures. 

The presentation also made me realize about the power of ignorance, that is blinding the transgenic world, as it is seen for poeple who don't deep inside the subject as something innatural against the nature, that also damage it, but this view is completely wrong as it has been proved that transgenic plants cannot damage humans. This transgenic plants are only resistance, and tolerant to an abiotic or a biotic agent. This demands of the individuals are making it advance really slowly the way law allows the sell of a concrete agrifood product. For example, gold rice has never been comercialized because of the amount of laws stablished in terms of transgenics, there are so many impediments that it is really difficult to commercialize gold rice, or whatever transgenic plant, although it has a lots of benefits in terms of proteins (vitamin A), and would safe a lot of lives around asiatic world. But some people don't understand that above all should be the people.

She ended the presentation explaining his own life, telling us why did she passed from studing SIDA (VIH) to study virus in plants. I really get shocked about his answer she gave at that moment, as I was not conscious of this since she said it:

"MOST DEATH IN ANIMALS OR PEOPLE IS NOT AIDS BUT HUNGER"