To stay healthy, plants balance the energy they put into growing with the amount they use to defend against harmful bacteria. The mechanisms behind this equilibrium have largely remained mysterious.
Now, engineers at Princeton have found an answer in an unexpected place: the harmless, or sometimes beneficial, bacteria that cluster around plants' roots.
In an article published Dec. 24 in the journal Cell Reports, researchers showed that some types of soil bacteria can influence a plant's balance of growth and defense. The bacteria produce an enzyme that can lower a plant's immune activity and allow its roots to grow longer than they would otherwise.
"This is trying to get at a really big biological question where there are not good answers — about how microbiomes interface with host immune systems," said senior study author Jonathan Conway , an assistant professor of chemical and biological engineering . "It's a small step in the direction of trying to understand how microbes live on hosts — either plants or humans or other animals — all the time and don't activate our immune responses constantly."
To search for immune-balancing bacteria, Conway's team turned to plants that were engineered to have heightened immune responses to a protein that makes up the threadlike appendages, called flagella, that allow bacteria to swim. The protein that makes up flagella, called flagellin, is a potent trigger of immune responses in hosts from plants to humans.
The researchers grew seedlings of Arabidopsis — a small plant in the mustard family that's commonly used in plant research — from a line that was engineered to produce high levels of flagellin-sensing immune receptor in its roots. When grown on plates containing the piece of flagellin that activates this receptor, the seedlings' roots are short and stubby, since their energy is directed toward immunity more than growth.
The experiment involved growing the seedlings on plates with flagellin as well as with 165 different bacterial species isolated from the roots of soil-grown Arabidopsis. 68 of these isolates, or 41%, suppressed the stunted growth response by tamping down the plants' immunity and allowing their roots to grow longer.
One of the bacterial species that allowed the roots to grow the best was Dyella japonica. Previous work had shown that that this species' immune-modulating activity was dependent on a bacterial secretion system — a protein complex that can move substances out of bacterial cells and into the environment, including inside plant cells or the spaces between plant cells.
A scan of D. japonica's genome revealed a gene encoding a secreted enzyme called a subtilase, with the potential ability to chop flagellin into small pieces and prevent it from activating the immune response.
The team used both genetic and biochemical methods to demonstrate that the subtilase enzyme was indeed capable of degrading the specific segment of flagellin that triggers the immune response. The degradation was sufficient to tamp down the immune response and allow for increased growth in Arabidopsis seedlings.
The researchers ran into some snags when trying to purify the subtilase enzyme, said Samuel Eastman , a co-first author of the paper and a postdoctoral research associate in Conway's lab. Obtaining pure protein is essential for definitively demonstrating an enzyme's function in a test tube.
In 2023, Eastman presented a poster on the project at a conference in Providence, Rhode Island, and was approached by Todd Naumann, a chemist at the USDA's Agricultural Research Service in Peoria, Illinois. Naumann said his experience suggested the enzyme could be purified from yeast cells, rather than bacteria.
Within a couple of months, Naumann had purified the protein and shipped it to Princeton. "Now we can do chemistry with it, and we can actually look at this in vitro," said Eastman. "We're able to achieve a level of investigation into this protein that wouldn't have been possible without that collaboration."
Naumann is a co-author on the paper, along with eight other Princeton researchers in addition to Eastman and Conway. The process of screening and verifying 165 bacterial isolates was a lengthy team effort, and six undergraduates were integral to this and other aspects of the work, said Conway. Britley Jones , a member of Princeton's Class of 2023, played a key role in screening the bacterial collection as part of her senior thesis.
Eastman shares lead authorship of the paper with postdoctoral research associate Ting Jiang and Kaeli Ficco , a 2024 Princeton graduate who is now a Ph.D. student at Cornell University. As part of her thesis, Ficco helped engineer mutant bacterial strains that demonstrated a genetic requirement for the subtilase gene in immune suppression and developed some of the experimental methods herself.
"I really liked how discovery-based the project was," said Ficco. "That definitely influenced my trajectory after Princeton." Now, she is beginning studies on the regulation of immunity by the human microbiome.
Beyond analyzing the specific enzyme produced by D. japonica, the team found that similar genes are found in many common soil bacteria, and their assays showed that dozens of bacterial isolates could suppress flagellin-induced immunity.
Now, they would like to better understand why these enzymes may be advantageous to both bacteria and their plant hosts. One hypothesis is that chopping up the flagella of pathogens prevents them from moving and invading a plant's roots.
"So, in that way it could be suppressing pathogens as well as the plant immune system," said Eastman. An alternative hypothesis is that these enzymes are "suppressing the immune system so a pathogen could maybe go undetected and cause more disease than it would otherwise."
The latter scenario would be problematic in harnessing this phenomenon to improve growth in agricultural settings, because it could make plants more vulnerable to disease. So, more study is needed, said Eastman.
"We don't want to compromise the immune system, but we also want plants to save that immune response for when it matters," he said. "We want them to keep calm and keep growing."
