Description

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. If the biobrick result to be functional, several applications are briefly reviewed in this proposal as well as some of the implications that would have to consider making the next step possible.

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 important 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 would translate to $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 still not a highly effective way to counterattack this problem (Johnson, 2000).

Control and loss costs are estimated 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 presented the usual symptoms of the disease. In the long-run, fire blight is a very important factor of 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.

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 is 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