Difference between revisions of "Team:Bordeaux/Description"

Our Solution: Curdlan

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 as an Extracellular PolySaccharides (EPS) in it's capsule [1]. 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 for the bacteria are due to the fact that Curdlan forms a capsule that completely surrounds the outer cell surface of bacteria.

Figure 4: Representation of the Curdlan molecule showing the beta 1,3 links between the glucose units

More precisely, applied to grapevine plants, sulfated ß-glucans induce the accumulation of phytoalexins (organic antimicrobial substances) and the expression of a set of Pathogenesis-Related proteins . In plants, the fact that oligosaccharides must carry crucial sulfates for their biological function suggests that chemical sulfation of oligosaccharides can improve their biological properties. In recents studies, compared to Laminarin (ß-glucan), its sulfated derivative triggered an enhanced immunity against P. viticola in V. vinifera and a stronger immunity against TMV in Nicotiana tabacum. The results indicate that the chemical modification of an elicitor, such as sulfated derivative of ß-glucans, could improve its resistance-inducer efficiency. Moreover, if a ß-glucan is a substrate for plant ß-1,3 glucanase, its sulfation clearly protects the molecule from its enzymatic degradation. Thus, a basal activity of plant glucanases can degrade ß-glucans and consequently releases short inactive ß-glucans; whereas sulfated derivatives still remain active molecules during a longer period. This might explain the higher resistance induced by ß-glucan sulfates compared to ß-glucans.

Furthermore, non-sulfated Curdlan doesn't trigger the hypersensitive response [3] 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? »

« That is a good question, let me tell you how we approached the subject! »

Figure 5: Schematic representation of the β 1,3 glucan synthetic pathway [2]

Before starting the project, we took a few weeks to decide which host organism we would use and how they could be useful. To begin with we looked at three different organisms: Escherichia coli , Bacillus subtilis and Saccharomyces cerevisiae and compared their β glucan metabolic pathways. We rapidly eliminated Bacillus subtilis from our possible hosts due to it's lack of enzymes involved in the metabolic pathway of β 1,3 glucans (Figure 5). However, we found that Saccharomyces cerevisiae naturally produces Curdlan in it's cell wall, like Agrobacterium. Furthermore, Escherichia coli is only missing one enzyme (the β Glucan synthase) to synthethize Curdlan. We therefore concluded that we could keep these two organisms: one where we would overexpress the β 1,3 glucan synthase using a constititive promoter and one where we would insert the ability to create curdlan by adding the enzyme that is needed throught the crdASC putative operon.

Literature Cited:

[1] M. McIntosh (2005) Curdlan and other bacterial (1→3)-β-D-glucans mini review. Appl Microbiol Biotechnol 68: 163–173

[2] M. McIntosh (2012) Recent advances in curdlan biosynthesis, biotechnological production, and applications. Appl Microbiol Biotechnol 93:525–531

[3] Adrien Gauthier (2014)The Sulfated Laminarin Triggers a Stress Transcriptome before Priming the SA- and ROS-Dependent Defenses during Grapevine’s Induced Resistance against Plasmopara viticola. Plos one 93:525–531

To sum it up, we would like to produce Curdlan in Escherichia coli or Saccharomyces cerevisiae and then sulfate it before spraying it on grapevine leaves as a preventive treatment against Downy mildew infections. That way vineyards can continue to produce good wine and make everyone happy.