Structure-Function Analysis of Type II Metacaspases to Reveal Distinct Activation Mechanisms

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Plant stresses caused by diseases, insect pests, heat, cold or drought are major causes of crop loss globally. Understanding how plants respond and recover from stress can greatly facilitate the effort to improve crop productivity through improved breeding and bioengineering approaches. Highly conserved proteases, enzymes that can cleave other proteins, are important in mediating stress resistance as well as developmental pathways in plants. This research will address how plant metacaspases, one type of these conserved proteases, contribute and control the developmental processes associated with stress response. Using advances in visualization techniques of crystallography and cryo-Electron Microscopy techniques, the structures for key members of the plant metacaspase family will be clarified to atomic resolution. In parallel, studies of variants for these metacaspases in transgenic plants will inform on their effects on stress response pathways. Together, these advances will likely have important impacts on agriculture through engineering crops in the future to enhance pathogen resistance and stress tolerance by modifying metacaspase functions. This project will provide diversified, interdisciplinary training to postdoctoral researchers and graduate students to enrich the scientific workforce. In addition, involvement of undergraduate students in experiential training in our multi-disciplinary project should contribute to science education at large. Genetic studies have shown that the metacaspase (MC) proteases are involved in abiotic and biotic stresses-induced cell death in higher plants. Two classes of Type II MCs are exemplified by MC4 and MC9 from Arabidopsis with their activity dependent on calcium ion and mildly acidic pH, respectively. The crystal structure of AtMC4 has been reported at 2.8 Å resolution and uncovered evidence for a multi-step autolytic process in the activation and maturation of the AtMC4 zymogen upon calcium induction. In this project, the structure for AtMC9 will be solved by using crystallography and cryo-Electron Microscopy (cryo-EM), which will enable discovery of the molecular determinants that underlie its activation at acidic pH and independence from calcium. In addition, the structure will be solved for a chimeric protein created from swapping the linker domain between AtMC4 and AtMC9 that resulted in an active proteases which no longer show autolytic cleavage upon its activation by calcium. These new protein structures should help rationalize their observed biochemical differences. To complement these structural studies, in vivo function of mutated and chimeric Type II MCs that have novel combinations of regulatory characteristics will be examined using atmc4 and atmc9 knockout mutants as genetic backgrounds. Lastly, proposed active translocation of AtMC9 zymogen to the acidic apoplastic space upon stresses will be tested by using fusions with acid-stable GFP variants. Together, these in vitro and in vivo trait analysis will reveal the detailed molecular basis for activation of these two metacaspases to induce plant stress response.This award is co-funded by the Cellular Dynamics and Function cluster in the Division of Molecular and Cellular Biosciences and the Plant, Fungal and Microbial Developmental Mechanisms Program in the Division of Integrative Organismal Systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.