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Cell Wars

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OSU scientists examine a world of molecular missiles and cellular smart bombs to learn how viruses invade plants and how plants fight back.

Viral invaders are a plant’s phantom menace. Leaving characteristic fingerprints—mosaic mottling or ringspot stamps—viruses can inflict severe deformities on their plant victims.

But deformed leaves and warty fruit simply may be combat wounds from a war that’s been waged over millennia. In these cell wars, plants and viruses slug it out on a molecular battlefield.

Stationary plants are not as defenseless as they seem, although until recently scientists had little idea that the conflict between plant and virus was so complex. Groundbreaking research at OSU has revealed that plants fight back by muzzling the genetic code of the invading virus.

With warp speed, this breakthrough in basic science has found practical application. Recent discoveries put some 500 known plant viruses directly in the scientific cross-hairs.

James Carrington, a plant biologist and director of OSU’s Center for Gene Research and Biotechnology, is leading the charge. Conducting experiments on the plant equivalent of the lab rat—a mustard relative known as Arabidopsis—Carrington is not only accelerating the advances in plant research, but could help speed the discovery of potential disease therapies in humans as well.

As we celebrate the 50th anniversary of the discovery of DNA’s double-helix structure, scientists are decoding the genetic blueprints, called genomes, of many plants and animals, including humans. Carrington’s work has helped crack open plant genomes to reveal a secret command center at work, masterminded by tiny forms of RNA molecules within the cell. These and other discoveries related to small RNAs garnered the coveted distinction of "scientific breakthrough of the year" by Science magazine in 2002.

Overshadowed in the cellular world by the more famous double helix of DNA, RNA was known as the dutiful go-between—assembling amino acids according to DNA’s blueprint into a dynamic group of proteins. Proteins do most of the cellular work that is essential to life. RNAs were thought simply to transmit a section of the DNA blueprint in order to direct the production of proteins.

Man working with a large green plant. One tall and healthy plant and one small and sickly plant.

Jim Carrington, director of OSU's Center for Gene Research and Biotechnology, uses Arabidopsis plants to study how viruses move through host plants, how plants respond to viruses, and how viruses counter-respond to the plant's defenses. Photo: Steve Dodrill

 

Viral infection has caused severe developmental effects in the Arabidopsis plant on the right. Photo: Kristin Kasschau

But RNA’s role in cell function would turn out to be much more than that of a drone conveying DNA’s instructions. Lurking in the spaces between the well-known protein-coding genes are other tiny genes that do not code for proteins. In fact, they orchestrate the formation of tiny RNA molecules, called microRNAs. Up to this point, these tiny genes had been overlooked. Like the enormous amount of "dark matter" that fills most of the universe, these microRNA genes were throughout the genome, but no one knew they were there. They were so small that researchers overlooked them without ever knowing they existed.

"Scientists never knew to look, so they didn’t," said Carrington.

One of the first clues to this hidden force came from an unlikely source—petunias. In the early 1990s, scientists seeking a deeper purple flower added extra copies of a pigment-producing gene to petunias in the laboratory. To everyone’s surprise, many of the flowers that resulted were white. Contrary to everyone’s expectations, neither the extra copies of the pigment-producing genes nor the original pigment gene were expressed.

What these researchers stumbled across was a powerful mechanism that plants use to combat foreign invaders. The plant perceived the added pigment gene as a foreign virus, and somehow silenced both the additional and original pigment genes. Scientists later found that gene silencing occurs naturally when viruses invade plants.

"Gene silencing is the basis of the plant’s immune system against a virus," said Carrington. "And, like immunization that we use in humans to protect against disease-causing viruses, we can use gene silencing to pre-program plants to resist viruses."

Healthy Arabidopsis flower. Sickly Arabidopsis flower.

Scanning electron micrographs show a developing Arabidopsis flower that is healthy (left) and a developing Arabidopsis flower infected with a virus. Photo: Kristin Kasschau

 

Photo: Kristin Kasschau

In recent years, scientists have harnessed this mechanism to silence genes for a variety of specific purposes. From creating tomatoes with delayed ripening to virus-resistant vegetables, the technology has become robust and reliable. "We can take almost any virus and produce plants that will be resistant to that virus," says Carrington.

However, researchers didn’t understand how silencing worked until they discovered the tiny world of small RNAs and their big role in plant immune systems.

One of the first discoveries was the tiny molecule called small interfering RNA, which plants produce during the process of silencing genes. Small interfering RNAs guide the destruction of virus genomes, and without this blueprint for life, a virus simply can’t survive.

Parts of the gene-silencing mystery were beginning to come to light. The formation of double-stranded RNA seemed to signal the plant to launch its silencing immune response. Many kinds of virus must form a double-stranded sequence in order to replicate. Plants have evolved to know that "things that make double-stranded RNA are harmful," says Carrington.

Defensive silencing starts when a plant cell sends out a dicing enzyme to chop up the double strands of viral RNA. The bits left over from all that dicing are the small interfering RNAs. These bits then are incorporated into a molecular missile that is guided by the small interfering RNA to the genome of the invading virus. In this way, the small interfering RNA functions as the software for cellular smart bombs.

As researchers examined this genetic silencing, they discovered another tiny molecule, microRNA. It is roughly the same length as small interfering RNA, and produced by similar dicing enzymes. But its role is very different in the battle against viruses. While small interfering RNAs are a plant’s storm troopers battling viral invasion, microRNAs are the Jedi knights of cell expression.

Man in greenhouse holding plant.

Valerian Dolja, professor of botany and plant pathology, screens experimental plants for effects of RNA silencing on a viral infection. Photo: Steve Dodrill

Within the Arabidopsis genome, scientists estimate that there are about 25,000 genes. However, wedged in between the conventional genes were volumes of genetic material that had been written off as useless junk by most researchers. However, some of the junk in the genome’s trunk turned out to be genes for microRNAs.

"Unlike small interfering RNAs, microRNAs function as guides to control the expression of the plant’s own genes," said Carrington. "They shut down specific sets of genes that control key steps in development, such as formation of leaves and flowers."

Like a complex computer program, a genome has a huge amount of code and not everything in the code is expressed at the same time. If it were, the computer program would crash. MicroRNAs are the master controllers over certain parts of the genetic code. For example, certain microRNAs tell the plant how to make a proper flower. Other microRNAs control what the leaves should look like. Turning off specific genes at a precise moment assures that the right information gets through at the right time in order for the plant to develop normally.

The discovery of microRNAs and their role in plant development ignited Carrington and his colleagues at OSU. By 2002, all the pieces were coming together to connect the function of microRNAs and why viruses cause disease.

"This was an exciting time for the lab," said Carrington. "We had found all these microRNAs in Arabidopsis and we’d been studying how viruses evade the silencing process. It became clear to us that anti-viral silencing and microRNAs were connected. And this connection results in many of the deformities we see in virus-infected plants."

First, Carrington’s group found that viruses are able to suppress a plant’s silencing immune response with their own counter-defense. Through the course of evolution, viruses had figured out a way to break the silence.

"The virus comes up with a solution—they always do," says Vallerian Dolja, a plant virologist at OSU. "Viruses evolve very fast, and they’ve developed a counter-defensive mechanism to suppress the small interfering RNA defense," he adds. That is why some viruses are so virulent even though the plant has an effective immune response. They are able to suppress the silencers.

Illustration of hand holding DNA double helix as a sword.

Illustration: Tom Weeks

More discoveries soon followed.

It turns out that when small interfering RNA takes aim at replicating viruses or when microRNA regulates flower or leaf development, they use a common set of cellular functions. So, when the virus suppresses a plant’s silencing response, it also obstructs gene regulation controlled by microRNA, causing collateral damage to normal plant growth and development.

Carrington’s work revealed that the bizarre plant deformities sometimes found among infected plants in the field were the result of misfiring of developmental genes that normally are controlled by microRNAs. "This discovery puts a molecular face on why we see many types of symptoms in the field when they are infected with viruses," says Carrington.

Many viral disease symptoms are therefore developmental defects inadvertently caused by the virus as it ducks the plant’s defenses. The ramifications of these developmental defects are enormous. If gene expression is not regulated properly, plants suffer abnormalities such as stunted growth, deformed leaves, sterility, and low fruit set.

So far, about 25 microRNAs have been found in Arabidopsis. As the search expands, that number could end up being as high as 100, according to Carrington. These fractions of the genetic code, inadvertently thrown out in experiment after experiment, year after year, have become the center of a new field of discovery.

Carrington’s work has helped revolutionize the war against viral diseases in plants. His future work will focus on understanding the role of microRNAs in plant development. "This once esoteric field of biology has enormous relevance to how plants and animals develop, and how we fight disease," says Carrington. "Undoubtedly, there will be new angles and twists that are entirely unexpected."

New Ways to Treat Diseases Without Drugs

Sometimes an advance in plant science can lead to a new drug to treat a particular disease, but rarely do insights from studying plants open up an entirely new approach to treating disease.

Fast-evolving research by OSU’s Jim Carrington and other scientists around the world suggests that small RNAs could be used to treat human diseases. These tiny molecules could potentially recognize a specific gene target and shut down the production of disease-causing proteins. Rather than blocking an existing protein the way drugs do, small RNAs would be able to prevent the protein from being made.

RNA interference may have more applications beyond fighting human diseases such as HIV, hepatitis C and cancer. Recently, researchers studying the process of fat storage used RNA interference to create 15,000 mutant nematodes—each one with just one of its 15,000 genes suppressed by small RNAs. Several hundred of the gene-suppressed nematodes accumulated less fat. Just imagine if scientists could interfere with a fat storage gene in humans.

Published in: People, Health