THE KEY QUESTION IN SYMBIOTIC NITROGEN FIXATION: HOW DOES HOST MAINTAIN A BACTERIAL SYMBIONT?

The fact that plants cannot use nitrogen in the gaseous form makes them dependent on the levels of usable nitrogen forms in the soil. Legumes overcome nitrogen limitation by entering a symbiotic association with rhizobia, soil bacteria that convert atmospheric nitrogen into usable ammonia. In root nodules, bacteria are internalized by host plant cells inside an intracellular compartment called the symbiosome where they morphologically differentiate into nitrogen-fixing forms by symbiosome-secreted host proteins. In this project, I explained the host proteins required to maintain bacterial symbionts and described their delivery to the symbiosome. I showed that the SYNTAXIN 132 (SYP132) gene in the model legume Medicago truncatula undergoes alternative cleavage and polyadenylation during transcription, giving rise to two t-SNARE protein isoforms. One of the isoforms, SYP132A, is a component of a nodule-specific secretory pathway required for the delivery of host proteins to the symbiosome. Among the host proteins targeted to the symbiosome is DNF2. I discovered that DNF2 belongs to a novel class of phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes that cleaves proteins containing glycophosphatidylinositol (GPI), a glycolipid that is attached to the C-termini of proteins as membrane anchors. I demonstrated that among DNF2 substrates are GPI-anchored lysin motif (LysM) domain proteins, whose LysM domains detect microbial surface molecules (such as bacterial peptidoglycan) and initiate defense responses. The timely action of DNF2 cleaves LysM domain proteins (LYMs) off the membrane of nascent symbiosomes, suppressing defense responses and ensuring the long-term survival of intracellular rhizobia. I further discovered that the GPI-anchored arabinogalactan-like proteins: ENOD16 and ENOD20 are other DNF2 substrates. These symbiosome-localized proteins play significant roles in nodule development. My study revealed that, once bacteria are internalized, the host cell delivers the DNF2 enzyme via a SYP132A-dependent nodule-specific protein secretory pathway into symbiosomes to cleave LYMs and ENOD16/20, thereby nitrogen-fixing bacteria can survive inside host cell. My study explains the molecular mechanism of how host plant maintains intracellular bacterial symbionts, which is one of the critical steps of symbiotic nitrogen fixation