Erwinions

Description

ABSTRACT

Erwinia amylovora causes fire blight disease in some of the most important crops such as apple, pear and most of the members of the Rosaceae. Here the team Tec-Chihuahua presents its proposal to address this environmental issue by using synthetic biology techniques to synthesize three different enzymes that might inhibit most, if not all, the virulence factors of E. amylovora. The use of N-Acyl homoserine lactonase, encoded by the aiiA gene, would directly affect the AHLs by hydrolyzing and inhibiting the main quorum sensing molecule. Then, the Cyclic-di-GMP phosphodiesterase from the yhjH gene would linearize the c-di-GMP avoiding the formation of biofilm and encouraging the motility. For this last virulence factor, the team proposes to arrest flagellar rotation with a Putative Glycosyltransferase encoded by epsE. Also, we had already designed a biocontrol product that goes by the name of "Erwnions" which main value proposition it's our modified Erwinia. We had considered all the entrepreneurship, social and legal aspects of how this project will affect locally, nationally and worldwide.

INTRODUCTION

The Rosaceae, commonly called the rose family, is a family of flowering plants; it consists of about one hundred genera and three thousand species. It's distributed throughout the world, being especially common in North America, Europe, and Asia.

One of the most conspicuous characteristics of Rosaceae is the variety of fruits produced by its species, which much of them are eaten by people in temperate regions; for example apples, pears, strawberries, and cherries. Also, many members of the family are important as ornamentals because of their foliage and flowers plus with the vast variety of species they are large enough to be sources of wood (Folta, 2009). Also, it is essential to highlight the economic impact of the Rosaceae. For example, the total world production of edible rosaceous fruits in 2005 was about 113 million tonnes. If this amount is converted to money considering a very conservative farm gate value of US$400 per tonne, this represents an economic value of $45 billion (Hummer, 2009).

This family commonly suffers from different diseases, the majority caused by pathogenic bacteria. One of the most harmful diseases is known as Fire Blight, which can be developed in the flowers, fruit, branches, trunks, scaffold limbs, root systems and practically the whole plant (UCANR, 2011). Fire Blight was the first disease proven to be caused by bacteria, it was originally found in North America, but through time it has vastly expanded all around the globe, including many countries from Europe, Asia and other American countries like Canada and Mexico. Although it's been many years since its discovery, nowadays there's still not a highly effective way to counterattack this problem (Johnson, 2000).

Plague control and loss costs are estimated in approximately $100 million a year in the USA. Specifically, in Michigan in the year 2000, $42 million in losses were estimated because of the removal of about 400,000 apple trees, while approximately $68 million is estimated in losses in Washington and northern Oregon. The decline of apple and pear trees from their landscape can be expensive to replace and could have a negative effect in many areas. Although there are no recent investigations in Mexico about the epidemy, it was estimated that in 1994, Chihuahua, one of the most important producing regions of apples in Latin America, had 10% of its crops presenting the usual symptoms of the disease. In the long-run, the disease of the fire blight is a critical factor for economy and society (Johnson, 2016; Ramirez, 1994).

It's important to highlight that Fire blight is caused by a phytopathogenic bacteria; Erwinia amylovora. It is a Gram-negative, facultative anaerobic, rod-shaped bacteria widely distributed in forty-six countries in every continent except Antarctica. The main characteristics that define this plant pathogen as a significant threat are:

Fast plant propagation that could induce the disappearance of the affected crop in a single vegetative period.
Great capacity to disseminate by different means.
Its ability to survive in the tissues of host plants.
Devastating effects with high economic impact.
No effective control methods.

Also, it is relevant to mention that E. amylovora counts with four virulence factors, which play an important role in its pathogenicity. The first two are the injectisome and the secretion of exopolysaccharides (EPS), both produced via quorum sensing. The other two factors, biofilm formation, and motility are regulated by the concentration of C-di-GMP. As a solution to this problem, we propose the use of synthetic biology to attack these virulence factors, this proposal will be thoroughly explained in the detailed project description.

Detailed Project Description

The four virulence factors of Erwinia amylovora mentioned before play an important role in its phytopathogenicity, giving it the necessary qualities to become an important threat to the agricultural sector. Our project objective is to dismiss the Fire Blight problem by inhibiting the quorum sensing, biofilm formation and motility of E. amylovora, consequently stopping its virulence. With the help of genetic engineering, synthetic biology, and the iGEM registry, our proposal involves the use of three genes: aiiA, yhjH, and epsE. These genes encode for different enzymes that will inhibit the virulence factors.

During the quorum sensing process of some Gram-negative bacteria (as is the case for E. amylovora), communicating molecules called AHLs (acyl-homoserine lactones) are used. These molecules regulate the expression and synthesis of two key EPS, amylovoran and levan (Molina et al., 2005). The aiiA gene encodes for the autoinducer inactivation enzyme A, better known as N-Acyl homoserine lactonase, an enzyme that hydrolyzes and inactivates the AHLs, thus provoking the degradation of the communication signals. When the quorum sensing is interrupted, inhibition of the injectisome and EPS expression is expected as a consequence. At the same time, the AHLs inhibition is expected to impact directly in the T3SS. This supposition is based on the recurrent reports that had established a relation between this virulent system and the quorum sensing in Vibrio fischeri. Nevertheless, it is still unknown how it will affect on E. amylovora but this can be considered as an opportunity for better characterization of the aiiA gene

Concerning C-di-GMP there we found that it is a key component of the regulatory networks that govern the expression of virulence traits in E. amylovora. This secondary intracellular messenger molecule is involved in the regulation of many cellular processes in numerous bacterial species, including the transition from a motile to sessile lifestyle, virulence, biosynthesis of exopolysaccharides and adhesion structures. Thus it is demonstrated that at high concentration of c-di-GMP induces a positive regulation of biofilm formation and represses motility. The gene yhjH encodes for Cyclic-di-GMP phosphodiesterase, which makes di-GMP linear. As consequence of this molecule disruption, the biofilm formation would be inhibited, but it will promote an increase in motility due to a low or nonexisting c-di-GMP concentration (Jonas,2009).

At this point it is necessary to inhibit the fourth and last virulence factor for this pathogen, motility. Here we include the third gene epsE which encodes to a Putative Glycosyltransferase, arresting flagellar rotation, by disengaging motor force-generating elements in cells embedded in the biofilm matrix. According to the bibliography, it is believed that the espE enzyme interacts with FliG interrupting the MotA-FliG interaction, stopping the flagellar spinning resembling a clutch. The specific reaction and region that interacts with FliG are still unknown (Jonas, 2009)

Applications and Implications

As described in the past section, the project will involve the synthesis and expression of four biobricks. Three of them will express each of the genes described above with the objective of making an individual characterization against the pathogen. On the final stage of the project, the fourth biobrick would be created as a combination of them to establish the efficiency these enzymes would have over E. amylovora at the same time and medium. The team expects a complete or partial inhibition of the virulence from the phytopathogen, if this happens to be true, then the project could be extrapolated to several applications. In this section, the process of selection of a specific application.

For example, the team could create a GMO that serves as a biocontrol in the Rosaceae crops competing and inhibiting the most important pathogen. Nevertheless, this would implicate a more complex genetic circuit which takes into consideration things like the environment, inducible promoters, and secretion of the enzymes through proteic capsules. Also, it would be important to analyze which bacteria would be the ideal considering that it would be in direct contact with crops for human consumption.

A more simple application could be the creation of a spray product that contains the enzymes in the solution. In this case, it would be necessary to consider facts like the concentration of the proteins in the product, storage, proteic capsules, the protein's lifetime, as well as economic viability.

At last, the team’s vision of the project was the creation of a transgenic plant able to defend itself against E. amylovora. This vision could mean that there wouldn't be a need to use antibiotics anymore having a positive impact on the economy of the agriculture sector. However, creating a transgenic plant for the human consumption would imply several issues to consider like the genetic circuit, briefly described above, as well as the possible effects that the enzymes could have inside a human body (toxicity, bioavailability, etc.).

To be honest, at the beginning of the project, the whole team was really lost on the final application of the project due to several ideas that emerged along the project development. It wasn't until we started our Integrated Human Practices that we were able to establish this edition project as a biocontrol which the main component is modified/sterile Erwinia. We took the Sterile Insect Technique as an idea where the sterilization means that Erwinia has lost all of his virulence factors. It is important to highlight the fact that all of the genetic requirements to be a biosecure product and the implications were reviewed with gorvernamental entities (also as part of our Integrated HP and implemented directly into our design).

REFERENCES

Hummer. (2009). Rosaceae: Taxonomy, Economic Importance, Genomics. may 31, 2017, de US Department of Agriculture Sitio web: https://hort.purdue.edu/newcrop/janick-papers/rosaceae.pdf

Johnson, Kenneth B. "Fire Blight of Apple and Pear." The American Phytopathological Society. The Plant Health Instructor, 2000. Web. 15 Nov. 2016.

Jonas, K. (2009). Regulation of c-di-GMP metabolism in Biofilms. 2017, de Future Medicine Sitio web: http://www.futuremedicine.com/doi/abs/10.2217/fmb.09.7

K.M. Folta, S.E. Gardiner (2009), Genetics and Genomics of Rosaceae, Plant Genetics and Genomics: Crops and Models 6, DOI 10.1007/978-0-387-77491-6 1

Molina, L et al.. (2005). Autoinduction in Erwinia amylovora: Evidence of an Acyl-Homoserine Lactone Signal in the Fire Blight Pathogen. JOURNAL OF BACTERIOLOGY, 187, p. 3206–3213. 25/05/201, De NCBI Base de Datos.

Ramirez. (1994). Manejo del tizón de Fuego del Manzano en la Sierra de Chihuahua. 31 may, 20017, de INIFAP Sitio web: http://biblioteca.inifap.gob.mx:8080/jspui/bitstream/handle/123456789/2521/Manejo%20del%20tizon%20d e%20fuego%20del%20manzano%20en%20la%20sierra%20de%20chihuahua.pdf?sequence=1

UCANR. (2011). Fire Blight. 23 may, 2017, de UCANR Sitio web: http://ipm.ucanr.edu/PMG/PESTNOTES/pn7414.html

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