For a fruit, tomatoes are strangely ubiquitous, appearing in everything from ketchup to BLT sandwiches. In fact, the average American eats about 23 pounds of tomatoes each year, with half of the weight located in tomato sauce.
When Sarah Refi Hind, a research associate at Boyce Thompson Institute, began work as an undergraduate, she became intrigued by the fruit and began research involving tomato defense against insects.
Why did Hind choose to study the tomato? Part of the intrigue of tomatoes is that, unlike most plants used in research, they are not weeds.
“It’s a plant that’s important for people because they eat it,” Hind said.
When she began as a researcher at Boyce Thompson Institute, Hind knew that she wanted to learn new skills, so she switched from plant-insect interactions to studying microbial pathogens, which are viral or bacterial microorganisms, and plant-microbe interactions while keeping tomato as her “common denominator.”
Hind began conducting her graduate research in the lab of Prof. Gregory Martin, plant pathology and plant-microbe biology. In his lab, she explained, they have been taking advantage of natural variation in tomato as a tremendous genetic resource, with 12 closely related wild species largely underutilized in research.
To obtain a big picture view of the genetic diversity in tomato, the lab screens specific varieties of tomatoes to figure out their resistivity to pathogens. After screening a specific heirloom tomato called yellow pear, they found it was completely resistant to the region of a flagellin protein — a protein that builds the flagellated tail of bacteria — called flgll-28. Intrigued by this quality unique to the yellow pear, Hind performed a crossbreed between the yellow pear and a wild species of tomato in order to map a genetic screen.
A crossbreed involves breeding two different organisms with the intended goal of creating offspring with both of the parent traits. In the cross, she found that a new receptor called Flagellin-Sensing 3 binds to a region of the flagellin protein called flgll-28. This new receptor, FLS3, is unique in the sense that it has been found in very few plants and is exclusive to nightshade plant species including tomato, potato and pepper.
The discovery of FLS3, according to Hind, is particularly useful since “some bacteria have been able to evade FLS2 mediated recognition.”
“These bacteria,” she said, “had an altered flg-22 region of flagellin that the FLS2 receptor [a well-conserved receptor found in higher plants] does not recognize anymore.”
However, since FLS3 is found only in a small subset of plants, it is likely that bacteria have not been under as much evolutionary pressure to evade this flgll-28 region. Theoretically, FLS3 could be moved from tomato, potato and pepper to other plant species to resist bacterial attacks.
Martin, the lab director, emphasized his optimistic vision for the future of this research.
“One of our next goals in this work is to look for natural variants of FLS3 that might allow tomato to detect and resist diverse bacterial pathogens,” Martin said.
One of the main challenges Hind experienced was proving a direct interaction between bacterial pathogens and tomatoes. Before Hind and her team found FLS3, there had been very few true receptors shown in plants. Most had been found using radioactive isotopes such as iodine that labeled the peptide, hormone or signaling molecule. Essentially, if the protein targeted became radioactive, the team had demonstrated direct interaction.
There are, however, major drawbacks involved with using radioactivity.The radioactive properties of iodine, which if ingested can affect the thyroid, are dangerous to work with.
“That was my line in the sand that I didn’t want to cross,” Hind said. “The risk was a problem in general in the field, and we anticipate that the method we developed and used for this project can be used by other scientists who study receptor-ligand interactions.”
Such receptor-ligand interactions involve a ligand, a molecule, that in this case bonds to a receptor, which is also a molecule that performs a function based on commands from hormones or neurotransmitters.
To overcome this problem, Hind and her team turned to a non-traditional technology in the plant world called click-chemistry, which is a method of tagging specific biomolecules and to chemically interact with target substrates. Through collaboration with the Schroeder Lab, a synthetic chemistry lab also located at the Boyce Thompson Institute, Hind and her team developed a click-chemistry system that proved this direct interaction with flg 228, leaving behind the issue of radioactivity. In this sense, Hind and her team created an alternate method to prove direct interaction between proteins and receptor ligands.
This new method did not happen overnight, however. Because the method involved utilizing techniques from all different fields, it took more than two years to piece everything together and get it working correctly. Of course, there are still ways to improve the tactic – in fact, there are students who worked on the Nature paper who are now looking to refine the method. In total, it took Hind and her team three and a half years to make the discovery, three generations of chemistry students, several postdoctoral fellows, and two additional years of tough experimental demands from reviewers to ultimately publish in Nature.