Takeaways
- Identification of the feeding tube composition: Researchers successfully isolated the root-knot nematode feeding tube and confirmed the specific group of proteins it is made from.
- A universal weakness: Because these proteins are found across multiple species of root-knot nematodes and are essential for the parasite’s growth and reproduction, they represent a universal target for developing new agricultural pest management strategies.
- Technological breakthrough in microscopy: The team developed a new protocol to manually extract cytoplasm from the giant cells of host plants, revealing that the feeding tubes are nearly three times longer than previously estimated.
For decades, agricultural scientists have been stymied by a microscopic mystery hidden deep within the roots of the world’s most important crops. While it has long been known that root-knot nematodes cause more than $100 billion in annual global crop losses, the exact mechanism they use to siphon nutrients from plants remained a mystery.
Now a team of researchers, led by University of Georgia plant nematologist Melissa Mitchum and her predecessor, Richard S. Hussey, distinguished professor emeritus, has identified the molecular blueprints of the nematode feeding tube, a discovery that could redefine how we protect global food supplies. The findings were published in the journal Proceedings of the National Academy of Sciences.
A microscopic straw in a living vault
The root-knot nematode is a master of biological manipulation. Once it enters a plant root, it transforms ordinary cells into specialized giant cells. To survive, the nematode must extract nutrients from these cells through a delicate, self-assembling straw called a feeding tube, said Mitchum, whose lab is affiliated with the Department of Plant Pathology and Institute of Plant Breeding, Genetics, and Genomics in the College of Agricultural and Environmental Sciences (CAES).
Hussey, along with colleague Charles Mims, described the structures in a 1991 study, however the tubes are incredibly difficult to study due to their size — only about 1 micrometer, or a millionth of a meter, wide and 70 micrometers long — and because they are buried within dense cytoplasm inside root galls, making them one of the least understood aspects of plant parasitism. At Hussey’s urging, Mitchum visioned isolating the tubes while on the faculty at University of Missouri. In 2019, she was recruited by UGA and moved her research program to a new lab in the Center for Applied Genetic Technologies.
The cytoplasm breakthrough
To solve the mystery, the research team developed a new protocol to isolate the tubes. By manually extracting the cytoplasm — the gelatinous liquid inside a cell — from the giant cells of host plants like tobacco, tomato and eggplant, the team was able to isolate the structures using meticulous laboratory work. By stripping the cortex from plant galls, the team could observe the giant cells under the microscope and extract minute quantities of cytoplasm as it oozed out. This allowed them to observe the feeding tubes apart from other cellular components like nuclei and organelles for further analysis using scanning electron microscopy.
What is a plant gall?
Plant galls are abnormal growths of plant tissues, usually in response to an invading organism like the root-knot nematode.
“Peering into the giant cell cytoplasm, you can see nuclei, organelles, and an array of feeding tubes. They discovered that these tubes are far more complex than previously estimated. The researchers found tubes reaching lengths of up to 224 micrometers — three times longer than earlier estimates — with a remarkable ability to bend and coil within the host cell,” Mitchum said. “Our ability to collect that cytoplasm and develop a method to enrich for and solubilize the feeding tubes was the crux of our success,” Mitchum said.
This, coupled with mass spectrometry-based proteomics, allowed us to identify the proteins present in the feeding tube sample.
Identifying the core component
The most significant finding of the study is the identification of specific proteins that build these lifelines. The researchers narrowed down thousands of potential candidates to a group of proteins known as protein family 7.
These proteins are produced in the female nematode’s dorsal gland and injected into the plant cell through a needle-like mouthpart called a stylet from which the crystalline-like feeding tube forms. This structure interacts with the plant’s own internal membrane system to facilitate nutrient uptake.
Using microscale analysis and protein localization, the team was able to identify specific genes within the nematode that produce the proteins found in these tubes. By using antibodies, they confirmed that the feeding tube is of nematode origin.
“This provided definitive proof that the composition of the tubes is of nematode origin,” Mitchum said.
A microscope image of a two root galls created by root-knot nematodes on a tomato root.. A microscope image of two adult female root-knot nematodes feeding from giant cell cytoplasm. (Photo by Melissa Mitchum and Richard Hussey) A microscopic image of feeding tubes embedded in clumps of granular cytoplasm and clusters of enlarged nuclei . Feeding tubes lie within dense cytoplasm and the enlarged nuclei of giant feeding cells created by female root-knot nematodes to extract nutrients. (Photo by Melissa Mitchum and Richard Hussey)A universal target
The study revealed that these specific family 7 proteins are present across multiple species of root-knot nematodes. Because the feeding tube is essential for the nematode’s growth and reproduction, this protein family represents a universal weakness that can be targeted to combat the parasite.
Researchers are now focused on understanding how the feeding tube is formed and how it interacts with host membrane proteins. By understanding the building blocks of this microscopic parasite’s feeding system, scientists can develop new management and production strategies aimed at disrupting the formation of these tubes, potentially cutting off their lifeline before they can devastate a harvest.
“All root-knot nematodes have to form these tubes to feed,” Mitchum said. “From here, we need to figure out how the tube is formed and how we can engineer a broad-spectrum resistance by preventing the tube from forming. With this we can potentially target all species of root-knot nematodes simultaneously to prevent them from feeding.”
This discovery is considered a major step toward developing new agricultural strategies to disrupt nematode feeding and prevent the billions of dollars in annual crop losses they cause.
The work was supported in part by the UGA Office of the President Strategic Hiring Initiative. Current and future work is supported by a new $1.1 million award from the joint National Science Foundation and U.S. Department of Agriculture National Institute of Food and Agriculture (NSF-NIFA) Plant-Biotic Interactions Program. In addition to Mitchum and Hussey, other researchers on the project included University of Missouri biochemistry researcher Lesa Beamer; John Shields, former managing director of Georgia Electron Microscopy; former UGA postdoctoral researcher Raquel Rocha, now with the Connecticut Agricultural Experiment Station; and CAES horticulture graduate and plant pathology master’s degree student Rebekah Lee Paul.
