Science news from the Bond LSC

Gene points to entirely new resistance mechanism against pathogen

October 15, 2012 | Roger Meissen

It’s an unseen hazard of the field.

Soybean cyst nematode plagues soybeans in at least 25 states, costing U.S. farmers more than $1 billion in lost yields each year.

For years, natural resistance in some soybean varieties kept this pathogen in check when coupled with crop rotation. But now 90 percent of resistant soybeans originate from one source, and this monoculture has caused growing resistance in cyst nematode populations.

Researchers at the University of Missouri Bond Life Sciences Center are now trying to figure out how this natural defense works, with an eye toward creating future varieties that can beat cyst nematode.

Melissa Mitchum, a plant scientist and molecular nematologist at MU, has taken a major step forward by cloning the first gene linked to natural soybean cyst nematode resistance. This gene points to an entirely new, previously unknown cell process for nematode resistance.

Melissa Mitchum is a researcher at the Bond Life Sciences Center and an associate professor of plant sciences with MU’s College of Agriculture, Food and Natural Resources.

“We cloned a resistance gene for the most important pathogen on soybean. In the plant world, it’s like cloning the gene for Alzheimer’s or Parkinson’s disease,” said Mitchum. “You can’t come up with a new approach to combat the disease problem until you know what the gene is. This is why it’s such a major breakthrough, because it’s our first glimpse into the underlying mechanism of nematode resistance.”

The journal Nature published Mitchum’s research October 15 in its online edition.

The life of soybean cyst nematode

Though you can’t see it with the naked eye, the microscopic soybean cyst nematode, technically known as Heterodera glycines, has a larger-than-life impact.

First detected in North Carolina in 1954, it has been a growing problem ever since. Many farmers discover they have an infestation in their soil only when a soybean field that appears healthy yields low at harvest.

Cyst nematodes hatch from eggs in the soil in the spring and quickly migrate to the roots of nearby soybean plants. Once inside the root, they use a needle-like stylet to inject a single host cell with nematode spit full of effector proteins and the nematode soon loses all its mobility to become dependent on the single soybean cell. These effector proteins change the cell, essentially reprogramming it to serve the nutritional needs of the parasite.

Cell walls dissolve, multiple cells combine and the nuclei of these cells enlarge. Instead of looking like a normal, rectangular plant cell, this monster cell looks more like a giant fried egg with multiple yolks. This monster cell – called a syncytium – works as a nutrient sink, fattening up the nematode. This is a complete retransformation of soybean root cells and only happens in plant-nematode interactions.

“Natural resistance leads this feeding cell to die, depriving the nematode of life-sustaining nutrients,” Mitchum said. “But we don’t know what’s going on, whether in resistant soybeans the feeding cell dies first then the nematode dies, or whether the nematode dies and that leads to the degeneration of the feeding cell.”

As a female nematode grows, her fattened body eventually protrudes out of the side of a root. Once pregnant and when her eggs mature, the female dies. Her dead body hardens to protect her eggs, creating the characteristic cyst on the root it is named after.

Fields with damaging cyst nematode numbers contain 500 to more than 10,000 eggs per cup of soil tested.

Building off nature

Mitchum’s research starts where soybean evolution left off. Her lab worked with Khalid Meksem, a plant geneticist at Southern Illinois University Carbondale to set out to pinpoint exactly where this nematode resistance begins.

By comparing the genome of cyst nematode resistant and susceptible soybean varieties they mapped the location of the Rhg4 gene, which stands for “resistance to Heterodera glycines 4.” They found two specific genes in this location. and Mitchum later proved one of these is integral to resistance.

Two polymorphisms, or mutations, in the serine hydroxymethyltransferase (SHMT) gene were found in the soybean varieties with resistance to cyst nematodes. This gene surprised researchers, because it did not look like any previously known plant resistance gene.

The SHMT gene encodes an enzyme that plays a role in producing folate and other processes, like making a plant’s DNA. Folate is a necessary nutrient for plants and animals alike, contributing to cell processes and important to prevent birth abnormalities in humans. Parasites like the microscopic nematodes are no exception, also needing this nutrient once latched onto a host cell in a soybean root.

Meksem’s team made soybean plants with mutations in the SHMT gene in the resistant variety.

“Our lab examined these mutant plants and found they were more susceptible to cyst nematodes, which provided the first indication that this gene plays a role in resistance,” said Mitchum.

Mitchum’s lab cloned this gene and used two gene-silencing techniques to test if the gene actually caused resistance. Both techniques reduced resistance in the soybean variety and, consequently, cyst nematode thrived in these plants.

As the final proof, they inserted the resistance gene into the susceptible plant - a process called complementation - and saw that it kept cyst nematodes at bay.

These processes are done in Petri dishes in the lab. Pramod Kandoth, an MU post-doctoral research associate in Mitchum’s lab and co-lead author of the study, grows the transgenic soybean roots and performed the tests multiple times. While each experiment took months to complete, repetition was necessary to definitively confirm the connection between this gene and resistance.

“When we silence genes in the resistant variety, we then count the number of cysts that appear on the root to measure its susceptibility,” Kandoth said. “The cysts look like tiny cream-colored lemons that you can see under the microscope.”

By collaborating with MU computer scientist Dmitry Korkin, they mapped the exact location of the differences in SHMT and found differences in binding sites important for the enzyme’s activity. That leads researchers to wonder how these mutations alter the enzyme’s interaction with nutrients like folate and vitamin B-6.

These nutrients bond at different rates in resistant and susceptible varieties.

“In lab tests, we see reactions with the SHMT enzyme from the susceptible variety peak and slow down, whereas reactions with the SHMT enzyme from the resistant variety plateaus” said Kandoth.

That somehow is contributing to resistance, and scientists will focus on answering this question.

Future research will aim to pinpoint the exact mechanism leading to the death of the feeding cell and nematode in resistant varieties; whether the soybean cell dies first, whether the gene triggers a defense mechanism in the plant that kills the nematode or whether a different scenario is in play to cause nematode death.

“The key finding for us is that this SHMT gene plays a role in resistance and is somehow tied into one-carbon folate metabolism,” Mitchum said. “For us that’s the next 10 years, trying to figure out how resistance is actually working through this pathway.”

This work was supported by the United Soybean Board, Illinois-Missouri Biotechnology Alliance, the National Science Foundation Plant Genome Research Program, USDA Plant Genome Program, the National Science Foundation CAREER Program, Missouri Soybean Merchandising Council, Illinois Soybean Association, North Central Soybean Research Program, Iowa Soybean Association, and a Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship. Nine years of research and about $1.2 million supported Mitchum’s work.

Melissa Mitchum is a researcher at the Bond Life Sciences Center and an associate professor of plant sciences with MU’s College of Agriculture, Food and Natural Resources.