Study offers new insight into mixed virus interactions in plants

UNIVERSITY PARK, Pa. — Similar to humans, plants can become infected with more than one virus at a time, opening the door for more severe infections and new disease variants. But these mixed infections are often under-studied and poorly understood.

New research led by researchers at Penn State examined what happens when two common viruses — tomato spotted wilt orthotospovirus (TSWV) and impatiens necrotic spot orthotospovirus (INSV) — infect the same plant.

The study, published in the journal Viruses, found that when plants were infected with both viruses, TSWV levels were much lower than when plants were infected with TSWV alone. These levels were also lower than INSV in mixed infected plants, suggesting an antagonistic relationship between the two viruses.

Cristina Rosa, professor of plant virology in the College of Agricultural Sciences, said that while preliminary, the findings provide a better understanding of these viruses that could help lead to better disease control in the future.

“Ours is the first study that looked at these mixed infections,” she said. “The knowledge generated here could eventually be used in management of plant viruses transmitted by vectors if the plant pathways identified in this study can be manipulated to help in control disease in plants. Theoretically this approach could result in economic benefits for growers.”

While TSWV and INSV were historically thought to be the same species, Rosa said, they are now considered separate viruses. However, they do have similarities at the genomic and ecological level. They both infect a wide range of plants, cause symptoms such as necrosis and are mainly spread by tiny insects called thrips. Thrips feed by puncturing the plant and sucking out its internal fluids, often causing visible damage and spreading disease. 

The two viruses also exist in overlapping parts of the world, making co-infections in plants theoretically common. Rosa said these mixed infections are important because every time a plant is infected with two viruses at the same time, it increases the risk for the viruses to exchange genetic segments and make new reassortant viruses.

Still, recombinations of TSWV and INSV have not been found, which Rosa said could be due to several reasons.

A previous study in Rosa’s lab found that not only can thrips tell which viruses a plant is infected with, but they actually prefer to lay their eggs in plants infected with both TSWV and INSV. But even though the first instar thrips grow up acquiring both of these viruses, they don’t transmit them equally when they get older — the thrips favor one virus over the other.

Since thrips are the main vector of the viruses, Rosa said this could be one reason recombinations have not been found, because while both viruses can infect the thrips at the same, the insects only pass one along to new plants. Another reason, she added, could be how the viruses affect each other in the plant host.

“When two microbes infect the same organism, you can have two outcomes: they either help or hurt each other,” Rosa said. “That’s important for the result of the infection, because if the viruses have a synergistic interaction, you would see a more severe disease. But if they are antagonistic, one virus could decrease the fitness of the other and you could have a reduced severity of the disease.”

Having already studied the viruses’ interactions in thrips, Rosa and her co-authors decided to study how they behave in plants and examine whether the result of coinfection is due to direct virus-virus interactions or indirectly via the plant. The team infected three groups of "Nicotiana benthamiana" or benthi plants — a close relative of tobacco — with different combinations of the viruses at different times: one group with TSWV, one with INSV and one with both, in synchronous as well as sequential times.

The team then was able to measure the different amounts of the viruses in the plants and compare between single and mixed infections. They also performed an analysis using small RNA sequencing — a lab method used to read and measure all the RNA molecules in a sample to see which genes are active, which helps the researchers better understand the molecules that carry instructions from DNA to the plants' cells to make proteins.

After analyzing the data, the researchers found that there was a lower number of co-infected plants than plants infected with a single virus. The amount of TSWV in the co-infected plants was also lower than in the plants with only one of the viruses.

Taken together, Rosa said this suggests an antagonistic relationship between the viruses, with ISNV being favored by the plant.

“When we looked at small RNA sequencing, we found that there were similar levels of ISNV small RNAs in both single and mixed infections,” she said. “So ISNV seems to be processed the same during the two types of infections, while the TSWV was processed differently. Basically, we have a much lower level of TSWV small RNAs in mixed infections compared to single infections.”

Rosa said the research team also looked at micro RNA profiles in the different infections and found that once again, ISNV profiles were similar in both single and mixed infections, while TSWV profiles were different, indicating that TSWV is processed in a different way than INSV.

In the future, the researchers said, additional studies could help identify the mechanisms behind these interactions.

Md Tariqul Islam, postdoctoral scholar Penn State; Kaixi Zhao, associate principal scientist at Merck; Nathan Johnson, assistant professor at Universidad Mayor; and Michael Axtell, professor of biology and Louis and Hedwig Sternberg Chair in Plant Biology at Penn State, also co-authored the paper.

The U.S. Department of Agriculture's National Institute of Food and Agriculture helped support this research.