Another active area of research in our group is to understand the regulation of PCD induced during the immune response. We discovered that macroautophagy (hereinafter referred to as autophagy) plays an important role in delimiting the PCD to the infection site (PMID:15907470; PMID:17932459). Autophagy is a dynamic process conserved across eukaryotes that entails the engulfment of cellular components in double membrane vesicles called autophagosomes that are then targeted to the vacuole/lysosome for degradation or recycling. Autophagosome formation and cargo delivery is regulated by a series of Autophagy (Atg) proteins that are conserved from yeast to higher eukaryotes including plants. To understand the interplay between autophagy and PCD during immunity, we have been studying Atg-interacting proteins and different cargoes carried by autophagosomes during immune response. Atg8 plays an important role in autophagosome biogenesis and recruitment of cargo to autophagosomes. Biogenesis of autophagosomes requires processing of Atg8 by Atg4, a cysteine protease, and conjugation of PE (phosphatidylethanolamine) to Atg8. While yeast has only one Atg4 and one Atg8, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a–AtAtg8i) homologs.
Using a novel BRET-based synthetic AtAtg8 substrate combined with biochemical studies, we showed that AtAtg4a is predominantly involved in processing and production of mature AtAtg8 isoforms (PMC3896200). This hints at a potential selective function for the different AtAtg8 isoforms in planta during different biological processes. Our recent sequence and phylogenetic analyses from 17 sequenced plant genomes indicate that Atg4 and Atg8 proteins are highly conserved in plant lineages. Our analyses suggested that the Atg8 expansion in plants might be attributed to whole genome duplication, segmental and dispersed duplication, and purifying selection. Our cross-kingdom biochemical analyses of Atg8 processing by Atg4 indicated that human Atg4 has broad substrate specificity and cleaves Atg8s from yeast, plant and human; whereas, plant and yeast Atg4s fails to process human Atg8 (also known as LC3a). Molecular modeling indicated that the lack of processing of HsLC3A is due to structural differences between HsLC3A and yeast and plant Atg8s (PMC5103345).
