Plant ‘first responder’ cells warn neighbors about bacterial pathogens

WEST LAFAYETTE, Ind. — Purdue University researchers found that a subset of epidermal cells in plant leaves serves as early responders to chemical cues from bacterial pathogens and communicate this information to neighbors through a local traveling wave of calcium ions. The properties of this local wave differ from those generated when epidermal cells are wounded, suggesting that distinct mechanisms are used by plants to communicate specific types of pathogen attack, the team reported Dec. 2 in Science Signaling.

The new work from Purdue’s Emergent Mechanisms in Biology of Robustness Integration and Organization (EMBRIO) Institute highlights the importance of calcium ion signatures or patterns in the cytoplasm of cells. Plants and animals use calcium ions to transmit biologically critical sensory information within single cells, across tissues and even between organs.

“When a bacterium infects plant material, or when a fungus tries to invade plant tissue, cells and tissues recognize the presence of an attacker,” said Christopher Staiger, a professor in the Department of Botany and Plant Pathology and Distinguished Professor of Biological Sciences. “They recognize both chemical and mechanical cues. This study is largely about how the chemical cues are sensed.”

In addition to Staiger, who led the study, the co-authors include EMBRIO Institute members in the Weldon School of Biomedical Engineering, including associate professor Elsje Pienaar and David Umulis, who is co-director of the EMBRIO Institute. Funded through the National Science Foundation-Biology Integration Institutes program, EMBRIO brings together biologists and experimentalists with mathematical and computational modelers to describe how different organisms sense and respond to chemical, mechanical and electrical cues. The research is motivated mainly by its long-term practical implications.

“We want to understand how plant defense works, to find the key signaling processes or signaling pathways that could help us develop novel strategies to control plant disease,” said study co-author Weiwei Zhang.

In plants and other organisms, growing evidence suggests that calcium ion fluxes in the cytoplasm generate a unique “signature” with features that depend on the specific stimulus. Calcium levels might spike, plunge, then spike again. The signature is defined by the number, amplitude and frequency of the peaks. Downstream molecules decode these signatures, triggering an appropriate cellular response, Staiger explained.

Scientists already knew that when a leaf senses a bacterial infection, it generates a fast-moving traveling calcium wave that is transmitted to other leaves on the plant, inducing a systemic defense response. By focusing more locally on tissues and cells, the Purdue researchers found that not all cells respond at the same time, nor in the same fashion. Instead, a subset of cells generates a local traveling wave of cytosolic calcium ions to alert a small group of neighbors of the danger.

“Our study indicates there might be a subset of cells, and they are the first responders that initiate those local waves. We quantitatively characterized the features of chemically induced waves versus mechanically induced waves. They travel differently with different molecular mechanisms,” Staiger said.

The interdisciplinary EMBRIO team included senior research scientists Zhang and Nilay Kumar. Zhang provided the experimental data, which Kumar translated into a simple mathematical model.

“This work sits at the intersection of mathematics, computation and cell signaling, and it allows us to ask questions that neither experiments nor simulations could answer alone,” Kumar said. The researchers formally tested whether calcium-induced calcium release (CICR) could explain calcium waves that traveled at a constant, slow speed in response to pathogen-associated molecular patterns.

“Experimentally, the waves appeared highly coordinated, but there was still uncertainty about whether such behavior required an active propagation mechanism,” Kumar said. “The CICR-based model successfully reproduced the constant-speed wave propagation seen experimentally, demonstrating that an active process is sufficient to account for this behavior.”

On the experimental side, Zhang and her colleagues developed an imaging and quantitative analysis system, especially for calcium, from scratch. Their task was to measure its rapid and short-lived dynamics in living cells with high precision to better understand its importance in plant defense. Although some labs already can do this, she noted, the capability is not yet widely available.

The system required a special microscopy imaging chamber because calcium imaging differs from their more routine imaging, which involves image collection only once or for short time periods. “For calcium, we needed to be able to add treatments in the middle of imaging and to record the calcium signals reliably in real time before and after treatment,” Zhang said.

Other researchers had already shown that wound-induced cellular damage generates a calcium wave. Zhang generated such calcium waves in the laboratory by using a strong, focused laser to injure a single cell on the leaf epidermis. In that way, she experimentally mimicked the traveling calcium wave, which spread locally to only a few cells. 

When Zhang quantified the wave front’s properties, she found clear differences between wound-induced and chemically induced waves. The wound-generated waves started fast at high amplitude, then their speed and amplitude faded over time and distance. “Like dropping a pebble in a pond, the wave is big at first and as it progresses, it diminishes,” Staiger said.

The chemically induced waves behaved quite differently. “Weiwei noticed it isn’t perfectly radial,” Staiger said. Instead of spreading evenly with a high initial speed, the chemically triggered wave moves slowly and often asymmetrically, maintaining a nearly constant speed as it advances from its point of origin. 

“Weiwei’s great work of quantifying and carefully analyzing both the subcellular signature of initiator cells and, more importantly, the traveling wave, is an important advance for the field,” Staiger said.

The modeling insights, meanwhile, clarified the difference between a calcium flood and a calcium wave front. The latter has a trailing edge that drops in volume or amplitude. The mathematical modelers on the team showed the likely existence of a sink that removes calcium ions from the cytosol, limiting the distance traveled. That’s just one of the new testable hypotheses the predictive modeling effort generated, which also helps explain the data.

“For example, the work suggests that intracellular calcium pools may contribute to wave attenuation, which is something we can experimentally probe in future studies,” Kumar said.

Media contact: Trevor Peters, peter237@purdue.edu