A team of researchers at the CSIR-National Botanical Research Institute (CSIR-NBRI), Lucknow, has investigated the functional activity of the plant protein Tma12, which is toxic to whiteflies. The study demonstrates that Tma12 kills whiteflies by degrading chitin. The findings of this research can be utilised by biorefineries in the production of biofuels and for expanding the horizons of biopesticides.
Infecting a wide variety of economically important crops, Bemisia tabaci, commonly known as whitefly, is globally regarded as one of the most destructive insect pests. Whitefly infestation is primarily overcome by the administration of chemical pesticides. These pesticides are widely used because of their cost-effectiveness, accessibility, and immediate results. However, their overuse leads to health and environmental risks, creating an urgency for environment-friendly substitutes for managing field pests. Pradhyumna Kumar Singh, Chief Scientist, CSIR-National Botanical Research Institute (CSIR-NBRI), Lucknow, works on the development of safer pest management strategies and crop improvement.
In 2016, Singh’s group developed a sustainable and eco-friendly alternative for controlling whitefly infestation. They reported that Tma12, a protein from the fern Tectaria macrodonta, is toxic to the whitefly population.
The team’s latest study reveals how Tma12 kills whiteflies.
Proteins involved in biological functions are known as functional proteins, as compared to the ones that are involved in providing the structure to a living organism or cell (structural proteins). Exploring the functional details of a protein is referred to as the “functional activity analysis of a protein.” In their study, the authors have explored the functional activity of Tma12 and described the distinctive features of its chitin-degrading property. Furthermore, they have located the exact chitin-binding site in the structure of Tma12.
Previous studies indicate that Tma12 might be capable of converting complex sugars like chitin into simpler compounds. Chitin is responsible for structural support in insects — their growth and development are strictly dependent on chitin. For that purpose, insects repeatedly produce chitin in different tissues. Exposure to chitinolytics (chitin-degrading substances) disrupts their normal structure, altering the life cycle of these insects and consequentially killing them.
Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that can break down complex sugars into simpler forms. Although Tma12 was known to bind chitin, a detailed characterisation of its enzymatic activity has not been done before. To check whether Tma12 has an LPMO-like chitin-degrading activity, the team provided chitin fibres to Tma12 and analysed the digested chitin oligosaccharides using liquid chromatography – tandem mass spectrometry (LC−MS/MS), which is an analytical chemistry technique that can help in identifying molecules based on the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. They found that Tma12 could indeed degrade chitin into simpler compounds.
The research group argued that the chitin-degrading activity of Tma12 is associated with its insecticidal activity. To check this, they abolished the LPMO activity of Tma12. This abolition was performed by removing the copper ions (required for the proper functioning of Tma12) present at the catalytic site of the enzyme, followed by treatments with a chemical called diethylpyrocarbonate (DEPC) that is known to modify amino acid residues of proteins. This led to the enzyme losing its chitin-degrading ability along with its insecticidal activity. The researchers used a technique called circular dichroism (a spectroscopic technique based on circularly polarised light used to determine the structure of proteins) to compare the structure of the modified and unmodified proteins. The group found that copper ions were essential for the function of Tma12 and that the DEPC treatment did not change the structure of the protein. Even after the DEPC treatment, the protein was functional.
One of the by-products generated after Tma12-based digestion of chitin is methanol, which can be utilised as a biofuel. Jyoti Singh, Senior Research Fellow and first author of this study, is optimistic about its potential industrial applications. Highlighting its dual purpose, she says, “The chitinolytic function of Tma12 protein can be exploited by industrial biorefineries in the production of biofuels and biopesticides.” Therefore, research exploring Tma12’s enzymatic activity holds a promising future.
Gauri Saxena, Professor, Department of Botany, University of Lucknow, who was not associated with the study, was intrigued by its findings. She mentions,
These tiny flies are continuously in the news for affecting agricultural productivity. It is fascinating to know that NBRI is addressing this global threat using a natural insecticidal protein (Tma12). Understanding its functionality in detail will help in developing potential management approaches against the mighty whitefly.