Sribas Chowdhury, Adamas University, Kolkata
With scientific developments in genetics booming over a couple of decades, the identification and assay of genes in different organisms have become a topic of great interest. A lot of new methods have been developed to understand the genetic traits of various physiological phenomena. One such phenomenon is the resistance of plants to pathogen-induced diseases. Over the years, researchers have been trying to decode the traits that lead a plant to have resistance to certain diseases. However, the biggest challenge is most of the existing methods use indirect methods of quantification, which is both expensive and time-consuming.
Bioluminescent markers:
With a vision to solve this problem, Dr. Dinesh Pujara and his team developed a bioluminescent marker to identify the mutants that lead to the expression of resistance traits against certain pathogens. Conventionally, the markers are made using a protein from an organism which is then tagged (tagging in genetics refers to identification or introduction of new DNA to the DNA scientists are working with) with the strain of a pathogen or virus. The most common pathogen used is Pseudomonas syringae and its strain is referred to as Pst. One such organism whose protein is tagged with Pst is called Photorhabdus luminescens. The bioluminescent marker that is developed from it is called Pst_Lux (or LuxCDABE).
Development of marker:
The research team used a Pseudomonas syringe strain (Pst) and then tagged it with a protein named NanoLuc (NL) Luciferase. The protein was derived from Ophlophorus gracilirostris, a species of shrimp living in the deep sea. It resulted in a bioluminescent marker which was 100 times brighter than Pst_Lux. It was then used for imaging Arabidopsis thaliana with the aid of an automated table and a camera that moved in synchronization with the assay. The imaging was further enhanced by adding Silwet L-77, a co-polymer that acts as a surfactant and increases spreading.
Observations:
After imaging, it was observed that the bioluminescent marker was 100 times brighter than the conventional Lux operon Pst_Lux. The scientists were able to identify and analyze over 30 mutants in Arabidopsis thaliana that expressed resistant traits. They also established the involvement of epigenetic players in ETI and basal resistance in plants. Further, it was also revealed that the age of leaves further contributes to bacterial resistance when the species is treated with a spray.
Significance of the study:
Through this imaging, the researchers gained deep insights into complex mechanisms of plant resistance to pathogens. The new bioluminescent tool paved way for a faster and more efficient form of quantitative assay of plant DNA and its mutations. The researchers are now keen on developing an AI algorithm for the high throughput analysis so that the imaging can be done on a larger scale than manual analysis. This can give us critical information on how epigenetic players play a role in plant defense mechanisms.
Also read: Plant biostimulants: A Novel Strategy to Boost Crop Production
Reference:
- Dinesh S. Pujara et al, Imaging-based resistance assay using enhanced luminescence-tagged Pseudomonas syringae reveals a complex epigenetic network in plant defense signaling pathways, Molecular Plant-Microbe Interactions (2021). http://dx.doi.org/10.1094/MPMI-12-20-0351-TA
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