SUMMER 2025
Zander Ketwitz
During my summer at Nagoya University in Japan, I worked in Dr. Takemoto’s lab under my mentor, Dr. Maurizio Camagna, alongside Julia Yurkiv, another student from NC State. We worked together on this project, splitting the work when possible, and helping each other if needed. Our research focused on an antimicrobial pathway found within Solanaceae plants, also known as Nightshades.
When plants are infected by a pathogen, they begin to implement defense mechanisms to combat the infection. In many plants, this includes the creation of antimicrobial compounds known as phytoalexins. Within the Solenacea group of plants, the individual compounds within the biochemical production of the phytoalexin Rishtin is known. However, the genes that code for the transformation of one product into the next within the pathway are unknown.
A compound used for phytoalexin production within many species of plants, Farnesyl Pyrophosphate (FPP), is the starting point for the Rishtin pathway. However, only the first two genes within this pathway are known. From FPP, the gene PVS is known to create Premnaspirodiene and from that, the gene POX is known to create Solivetivone. The pathway continues with the creation of Lubimin, then Oxylubimin, and finally Rishtin, all of which the genes responsible for their conversion are unknown. The goal for our two months within the lab was to test candidate genes to figure out which converted Solivetivone into Lubimin. The plant species Nicotiana benthamiana (N. benth.) was selected to be our host for this experiment since it naturally produced FPP, but does not follow the Rishtin pathway nor contain any of the intermediate products of that pathway naturally.
Before we could test any candidate genes, we first needed to elicit Solivetivone production within the plants. This was done by infiltrating Agrobacterium containing the PVS and POX genes into the leaves of the plant which, in theory, would convert any FPP produced into Premnaspirodiene and then into Solivetivone. To ensure that FPP was being produced and that the plant would “want” to create these antimicrobial compounds, elicitins called INF1, which originate from plant pathogens, were also infiltrating into the same leaves to create a pathogen defence response within those cells.
When measuring chemicals and the amount of each chemical present within the leaves using GCMS, results continued showing no Soliveticone production. These issues persisted throughout the entire two months, where our goal changed from testing candidate genes to trying to figure out what was incorrect about our methods. By the end of the two months, we had replaced the old glycerol stock of POX and PVS used to ensure the genes we were adding were functional, tested optimal infiltration timing of both the agrobacteria and INF1, as well as sample collection timing and methods.
Due to the issue of us being able to produce Solivetivone in the plants, towards the latter half of our research, we began investigating the possibility of putting this pathway into yeast instead. This would ensure that there are no unknown pathways conflicting with our inserted genes and remove some of the variability that naturally exists due to working with more complex organisms like plants. Unfortunately, our initial tests on how to select for yeast that had taken up our desired genes while excluding those that had not taken up those genes saw no success and we were unable to pursue this matter further due to time constraints.
Our very last plant sample collection did yield some Solivetivone, but it was such a small amount that it would have been unusable for testing candidate genes. Despite all of the issues that arose within our research efforts, I feel that I gained a valuable understanding of what the reality of working within a research environment is like. Even with the frustrations of continued failures for seemingly no reason, we pushed through and still provided useful information for the field. When speaking with Dr. Maurizio Camagna about our efforts, he made sure to mention that all of our work has led to the greater development of the methods he will continue to use in his future research.
My time in Japan was extremely beneficial for me, not just in learning new lab techniques and experiencing research, but also in my own personal growth. All of my experiences in and outside of the lab in Japan helped me to gain more confidence in my own skills, be more willing to ask for help, and have a more open mind to new experiences and cultural differences. I am extremely appreciative of all of the members of Dr. Takemoto’s lab for their willingness to discuss their culture and lives with me to help me understand how their own experiences have shaped them and allowing me to gain a new perspective on life.
