Difference between revisions of "Team:Bordeaux/Description"

The Problem

This year, iGEM Bordeaux’s project is focused on Downy Mildew

This disease, caused by an oomycete (fungus-like eukaryotic microorganism) called Plasmopara viticola , is unfortunately famous in the Aquitaine region because it affects tens of hectares of Bordeaux vineyards every year and threatens wine production (How much?) . It was originally observed in the United States of America in 1834 and has been most abundantly found in the northern and midwestern areas of the United States. Shortly after, the pathogen was introduced in European countries where it played a devastating role in the yield and production of their wine. In 1878, the first cases of Downy mildew were observed in France (in the region of Lyon) and also in Swizerland and Italy. Common symptoms include necrosis of the stem or shoot, discoloration, brown spotting and yellowish-green tips of the leaves and mycelium invasion of the grapes. While some North American species have become resistant to this parasite, European species such as Vitis vinifera (the grapevine used for wine) are extremely sensitive. Depending on the year, production of grapes in France has been estimated to be at a loss of 50% or more ref and the Aquitaine region is particularly affected due to the favorable climate and the economic importance of the wine industry. Thus, Downy mildew has been considered the most devastating disease caused by a filamentous pathogen to affect European vineyards and this has lead vineyards to search for effective measures to protect their vines. Unfortunately, most of these mesures have a bad environmental impact and pollute the surrounding soils.

In 2015, vineyards are still threatened by the disease

Our iGEM team has been following this year's effect of mildew closely reading the official vineyard mildew bulletins available on the vinopole website. It is clearly shown that there is a significant increase of mildew infection on parcels that haven't been treated with copper sulfate compared to those that have been treated. Furthermore, the infection of mildew on treated parcels appears to be much more easily controled on parcels treated with copper sulfate. Evidently, without any alternative treatment, wine production in the region would be affected and this shows just how important our project is!

Different models (Caffi model, Potential systems model) take into account pluviometry, temperature, relative humidity and plant morphology to decide when are the best moments to apply the fungicides. However, even if these models have allowed vinyards to drastically reduce the quantities of fungicides used, they still cause environmental and sanitary problems in the surrounding regions.

Since repairing damaged tissues infected by downy mildew is impossible, the main solutions available to vinyards are preventive solutions, mainly through preventing primary infections. This is mainly done by spraying fungicides on the organs that are most infected: leaves and stems. The most efficient preventive treatment was discovered at the end of the 19th century: a solution made of copper sulfate also known as "Bouillie Bordelaise", the only treatment used until the end of the 20th century. Recently, synthetic fungicides have replaced this chemical treatment and more and more research is being done on alternative eco-friendly preventive treatments.

Natural defenses of plants

Immunization of plants is based on the same principle than humans: activate the natural defenses before contamination by the infectious agent. The concept is simple; it is to put the plant in contact with a molecule able to activate plant's natural defenses: an elicitor . In nature there are many elicitors produced by micro-organisms (exogenous elicitors) or by the plant itself when it is attacked (endogenous elicitors). The presence of an elicitor in the plant triggers a series of cellular reactions including the production of molecules to strengthen the resistance of walls , but also the production of plant antibiotics such as phytoalexins or defense proteins. These compounds have antifungal and antibacterial properties. The external application of a natural elicitor or a similar synthetic molecule thus results in the production of phytoalexins or defense proteins in the absence of any pathogen. The plant "immunized" is ready to fight back if attacked. First, the cell wall forms a physical barrier which prevents the penetration of most microbes. Then, if some pathogens are able to cross this wall, the infection depends on the ability of the plant to perceive and to trigger defense reactions that would prevent the development of the disease. This recognition is done with certain compounds, known as elicitors from the pathogen or plant. The fixing of an elicitor to a plant cell receptor initiates a cascade of events that leads to the synthesis of defense compounds . The best known are antimicrobial compounds such as phytoalexins and Pathogenesis Related proteins .

There are two types of defenses: "passive" and "active". Natural defense reactions of plants are passive when these components were preformed prior to infection. They remain active until the pathogen penetration. Active defenses are induced upon recognition of the pathogen and remain active during infection.

Consequences of the infection

Delta-viniferin is a grapevine phytoalexin produced during infection by Plasmopara viticola . Phytoalexins are antimicrobial and often antioxidative substances synthesized by plants that accumulate rapidly at areas of pathogen infection. Phytoalexins produced in plants act as toxins to the organism. They damage the cell wall, delay maturation, disrupt metabolism or prevent reproduction of the pathogen.

When phytoalexin biosynthesis is inhibited, the susceptibility of infection in plant tissue increases showing its importance in defense mechanism . While, when a plant cell recognizes particles from damaged cells or from the pathogen, the plant launches a two-pronged resistance: a general short-term response and a delayed long-term specific response.

During the short-term response , the plant deploys reactive oxygen species such as superoxide and hydrogen peroxide to neutralize invading cells. The short-term response corresponds to the hypersensitive response, in which cells surrounding the site of infection are signaled to undergo apoptosis (programmed cell death), in order to prevent the spread of the pathogen to the rest of the plant.

The long-term response , also called systemic acquired resistance (SAR), permits a communication of damaged tissues with the rest of the plant using plant hormones such as jasmonic acid, ethylene, abscisic acid or salicylic acid. The reception of the signal leads to changes within the plant inducing genes that protect from more pathogen intrusion like genes coding enzymes involved in the production of phytoalexins. Also, if jasmonates or ethylene is released from the wounded tissue, neighboring plants also synthesize phytoalexins in response.

Our Solution

What is Curdlan?

To start with, let's talk about glucans. Glucan molecules are polysaccharides of D-glucose monomers linked by glycosidic bonds. One of them is called Curdlan, a (1→3)-β-D-glucan. This molecule is a linear homopolymer which may have as many as 12,000 glucose units. It is naturally produced by Agrobacterium sp. ATCC31749 which uses it like an Extracellular PolySaccharides (EPS) in it's capsule. The capsule formation is correlated with cell aggregation (floc formation) and it is suggested that the capsule and floc formation together function as protective structures in cases of Nitrogen-starvation of the post-stationary phase. The protective effects are due to the fact that Curdlan forms a capsule that completely surrounds the outer cell surface of bacteria.

Curdlan belongs to the class of biological response modifiers that enhance or restore normal immune defenses including antitumor, anti-infective, anti-inflammatory, and anticoagulant activities. As a matter of fact, this β1,3 glucan can stimulate the plant's immune system.

More precisely, applied to grapevine plants, sulfated Curdlan induces the accumulation of phytoalexins (organic antimicrobial substances) and the expression of a set of Pathogenesis-Related proteins .

However, non-sulfated Curdlan doesn't trigger the hypersensitive response characterized by the rapid death of cells in the local region surrounding an infection, avoiding a complete contamination of the plant. This response has been studied in Arabidopsis thaliana through a mutant gene: pmr4 . This mutant is resistant to mildew infections but is unable to induce Pathogenesis-Related proteins expression .

Also, activation of a Pathogenesis-Related protein called PR1 in grapevine is regulated by the salicylic acid signaling pathway . The lack of PR1 expression in non-sulfated Curdlan-treated grapevine could be explained by a negative feedback of glucan. This is demonstrated by the study of a double mutant of pmr4 which restore the susceptibility to mildew. It suggests that linear β-1,3 glucan negatively regulates the salicylic acid pathway. So, sulfation of the glucan would counteract the negative feedback effect.

To conclude, activation of the innate immune system before the invasion of pathogens is a way to improve the resistance of plant against infection and to reduce the use of chemicals products.

« Ok i understand the value of producing Curdlan. How will you proceed? »

« This is a good question i'll answer. »

In Agrobacterium , three genes (crdA, crdS and crdC) are required for Curdlan production. The putative operon crdASC contains crdS, encoding β-(1,3)-glucan synthase catalytic subunit, flanked by two additional genes : crdA and crdC. The first assists translocation of the nascent polymer across the cytoplasmic membrane and the second assists the passage of the nascent polymer across the periplasm. However, all Curdlan biosynthesis is dependent of nitrogen starvation and various parameters. We want to simplify all of this.

Using Bacteria: Escherichia coli

Firstly, we decided to produce curdlan with Escherichia coli , because Agrobacterium and it are Gram negative bacteria and have a lot of membrane similarity. Moreover Escherichia coli is a little pretty good bacteria, it can be grown and cultured easily and inexpensively in a laboratory setting unlike Agrobacterium .

Secondly, we are going to put these three genes into Escherichia coli under an easier control as N-starvation.

Thirdly, when we produce curdlan with Escherichia coli , we will sulfate Curdlan by chemical method. In fact sulfated Curdlan, it is the better form of Curdlan to activate immune plant system.

To sum up, we would like to produce Curdlan in Escherichia coli and then sulfate it to use it as a preventive treatment for the vine against the mildew infection and continue to produce good wine and make everyone happy.

Using Yeast: Saccharomyces cerevisiae

Yeast cell walls are made up of various layers which are represented in the following diagram. First there is a layer of chitin, then a layer of beta glucans and finally a mixed layer of proteins and mannan. Commonly, the yeast cell wall is made of 5-10% of beta 1,6 glucans and 50-55% of beta 1,3 glucans and beta 1,6 glucans.

Since the layer of mannan and proteins as well as chitin is insoluble in alkali solutions, beta glucans are easily separated from the rest of the yeast cell wall. Therefore, the only alkali soluble components are a mix of beta 1,6 and beta 1,3 glucans. (aimanianda et al 2009) In order to separate the two we plan on using beta 1,6 glucanases in order to obtain a solution of beta 1,3 glucans and therefore our curdlan molecule.

We therefore decided to over-express the curdlan metabolic pathway by inserting into yeast an inducible promoter (gal1) for the glucan synthase gene (Fks1) hoping that this would allow the cell to produce curdlan in greater quantities. This would allow us to compare our curdlan production in E. coli to the natural production in an organism and the enhanced production through the addition of a promoter.
To do this , we will extract the FKS1 gene yeast DNA and amplified it by PCR. We will then insert FKS1 in one hand, into plasmid pYES2 to integrate the modified plasmid in Saccharomyces cerevisiae and boost production of curdlan . On the other hand , we will integrate the plasmid FKS1 iGEM to get our famous BioBrick that we'll send to Boston. However, site-directed mutagenesis may be necessary when integrating the gene into the plasmid because there are restriction sites ( EX and SP) unwanted within the gene.

« It's very clear now. It looks like cool. But, i have a last question: why did you choose this subject? »

« Because, as explained above, downy mildew is a real problem for the Aquitaine region. That is why we wanted to bring an ecological solution to this problem. And also, we are SWAG (Secretly We Adore Glucan) »

Other useful properties of Curdlan

Curdlan belongs to the class of biological response modifiers that enhance or restore normal immune defenses, including antitumor, anti-infective, anti-inflammatory, and anticoagulant activities. CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a Nout–Cin disposition

Sulfation of Curdlan. Acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) is a severe disease that can destroy the body’s immune system, so the discovery of methods to prevent AIDS infection is of great importance. All the other curdlan clinical applications in cancer, diabetes, hypertension, hypertriglyceridemia etc. are listed here.

Curdlan is also neutral and insoluble in water. If it is heated in aqueous suspension , it adopts simple helical conformations ( 55-80 ° C) or triple helical connected ( 80-130 ° C). It then acts as a gelling and form two types of gels (low-set gel or high-set gel) . This property is widely used in the food industry since Indeed, curdlan is a food additive ( E424 ).

Results

How much curdlan we produced and how did we determin how much we produced.