Functional diversification of a plant protein tafficking pathway for nutrient storage and pathogen defense

Abstract

All living organisms growing under ever-changing environmental conditions must have mechanisms to cope with such changes. The molecular machineries for responses to the environmental challenge are different from or sometime even incompatible with those for the normal growth. Animals have developed strategy using specialized cell-types, for the responses, for example, immune-specific cells for biotic stress, a major environmental challenge. In contrast, in plants with no immune-specific cells, the vegetative cells undergoing growth have to respond when challenged by biotic stress. However, it remains unknown how the plant cell efficiently readjust its molecular machineries between growth and environmental stress responses at the cellular level. Here, I provide evidence that plant cells use a strategy of bifurcated vacuolar protein trafficking system consisting of the NtSS (N-terminal sorting signal) and CtSS (C-terminal sorting signal) pathways for effective distribution of the molecular machineries required for growth and defense response, respectively. There are two vacuolar sorting pathways, NTPP (N-terminal propeptide) and CTPP (C-terminal propeptide) in plant cells. The NTPP pathway is mainly involved in sorting of proteins into lytic vacuoles for degradation in adult tissues, while the CTPP pathway mediates transport of proteins into Protein Storage Vacuole (PSV) for storage in seeds. NTPP sorting sequence has NPIR-like motif and CTPP is composed of hydrophobic amino acids but not conserved. In this study, I reveal that two potential CTPP motifs are identified by using sequence-based computational tool. Interestingly, potential CTPP motifs are highly enriched in pathogen-related (PR) proteins among vacuolar and secretory proteins. Especially, putative CTPP motifs are mainly discovered in defensins (PR-12 family), thionins (PR-13 family), lipid transfer proteins (LTP/PR-14 family) and chitinase. Next, I show that CtSS-containing PR genes, PDF1:1, PDF3:1 and CHIA1, are stored in certain compartment where GFP:CT24, a representative seed CTPP marker, is accumulated. Interestingly, SYP22, a Q-SNARE surrounds membrane of GFP:CT24-positive compartments in vegetative tissue. Using vesicle trafficking assay with vesicle trafficking-defective mutant plants, I reveal that VTI11, EpsinR1, AP1 adaptor complex and VPS35A coordinate with NtSS pathway, but VTI12, EpsinR2, SYP22, AP-3 adaptor complex and VPS35B/C are involved in CtSS pathway. Besides, I discover that CtSS – containing CHIA1 and GFP:CT24 are strongly accumulated and secreted into infection site via vesicle-mediated pathway upon G. orontii fungal inoculation. Interestingly, I provide that vps35 mutants required for CtSS pathway display more susceptibility than WT against B.g.h and E. PISI penetration. Taken together, the vegetative NtSS pathway is largely responsible for the vacuolar trafficking, leading to protein degradation during normal developmental growth. By contrast, the CtSS pathway is largely responsible for trafficking of the pathogen-related proteins such as defensins and acidic chitinase to a novel compartment, named vCPC (vegetative CtSS protein compartment). The vCPC normally fuses to the lytic vacuole for steady-state regulation of proteins. However, upon fungal infection, PR proteins stored in the vCPC are secreted specifically to the encasement of haustorium. Therefore, I propose that the strategy for biotic stress responses is drastically different between plants and animals at the whole organism level but shows striking similarities at the cellular level, which is resulted from convergent evolution.