A recent study in The Plant Cell found that plants release extracellular vesicles carrying a new class of RNA molecules, named “tiny RNAs.” The findings are among the first to reveal the contents of these vesicles, and may hint at the ways plants can relay RNA messages beyond their own cells.

About four years ago, plant biologist Roger Innes of Indiana University was studying electron micrographs of plant cells when he noticed tiny vesicles budded off from cells appeared to cluster around sites of fungal infection. In animal studies, researchers had previously found that cells can pack messenger RNAs (mRNAs) and micro-RNAs (miRNAs) into extracellular vesicles. And plants and their fungal pathogens were known to communicate by exchanging RNA messages—although how they do so is not well understood. Could plant vesicles carry similar cargo as animal vesicles? If so, such transport could be a mechanism for cross-species RNA conversations.

Innes, together with plant biologist Blake Meyers of Donald Danforth Plant Science Center in St. Louis, set out to investigate the vesicles’ contents. They found that in healthy plants, extracellular vesicles are loaded with a new class of tiny RNAs that are just 10-17 nucleotides long.

The study “is a first attempt to look at plant extracellular vesicles and see what small RNAs are in there,” says biologist Michael Axtell of Pennsylvania State University, who was not involved with the study. “The small RNAs that we know to be functional in plants are usually 21-24 nucleotides long, so all of these short things are quite unexpected.”

Innes, Meyers, and their colleagues began by isolating extracellular vesicles from leaves of healthy Arabidopsis thaliana plants and sequencing the enclosed RNA. They also extracted and sequenced RNA that was released outside cells’ plasma membranes but not packaged into vesicles. But when they looked at the data, they were puzzled.

When postdoctoral researcher Patricia Baldrich began her analysis, she found surprisingly few RNA sequences. Typically, researchers trim very short or poor-quality sequences from a dataset prior to analysis, losing 1 or 2 percent of their data in the process. When Baldrich did so this time, a whopping 80 percent of RNA sequences were missing.

But when the researchers re-examined the data—including all the sequences shorter than 18 nucleotides that they had removed—they found the lost fragments. “We expected from the literature on animal cells that these vesicles might have abundant siRNAs as well as microRNAs,” Innes says. “We were surprised to find that so many of our reads were in this 10-17 nucleotide range.”

In addition to this new class of RNAs, the vesicles also harbored proteins, metabolites, microRNAs, and siRNAs. To understand the genomic source of these tiny RNAs, the researchers compared their sequences to different features of the plant genome to identify where these sequences originated. Most tiny RNAs originated from messenger RNAs and transposable elements. Of those formed from micro-RNAs, the majority appeared to be formed by parts of precursor miRNA molecules—specifically, an internal loop that’s removed in mature miRNAs. Precursor miRNAs carry characteristic tags at their 3’ and 5’ ends that are cleaved and removed by specific enzymes during maturation. But it’s not clear how the loop is processed.

“The loop is really a piece of DNA that we don’t have an easy way to capture, and in plants the size of that can be anything from less than 15 nucleotides up to 400,” Meyers explains. “It’s a pretty understudied region of the miRNA precursor.”

The methods the researchers used to identify this new class of RNAs are “very robust,” says plant pathology researcher Wenbo Ma of the University of California Riverside, who was not involved with the current study. Comparing vesicular RNA contents to other extracellular RNA is “a very convincing comparison” to show that the enrichment of tiny RNAs is unlikely to be an experimental artefact, she adds.

While the tiny RNAs seem likely to be decay products of processing this loop, precisely what they do is still a mystery. Do plant cells pack them into vesicles as a way of taking out the trash, or are these tiny RNAs some form of message being mailed to other cells?

Deciphering their function is difficult because the sequences are so short, Meyers says. But the fact that they’re highly enriched in vesicles “tells us we can’t ignore them,” he adds. “The cell is accumulating them—whether the benefit is elimination or some other function is yet to be seen.”

So far, extracellular vesicles have been primarily described in plant defense responses. Because this initial study was performed with healthy plants, both Ma and Axtell say follow-up experiments in infected individuals will help figure out whether the contents of vesicles change when a pathogen is present.

Nonetheless, these preliminary results offer a new perspective on plant extracellular vesicles. “Before this paper, I’d have assumed that they just carried microRNAs and siRNAs,” Axtell says. “This work has certainly changed how I think about extracellular vesicles.”