A Novel Set of SNARE Partners Facilitating Bacterial Pathogen Defence

Plant microbial pathogens destroy some 15% of crop production worldwide, inflicting major agricultural and socio-economic losses. Thus, understanding plant immunity is at the centre of efforts to mitigate the challenges in food production facing human society in the coming decades. Although plants have evolved defence systems, immunity comes at a cost to plant growth; crop bred to maximize growth-related traits, by contrast, often compromise on defense. To strategically maximize plant disease resistance, knowledge of the mechanisms underlying plant defences is vital to minimize reductions in yield.

Stomatal pores on the leaf surface exchange gas and water with the environment and are primary entry points for microbial pathogen. The initial defence against bacterial pathogen is stomatal closure, but pathogens commonly manipulate these defences and force stomatal opening. At a cellular level, these manipulations include commandeering ion transporters and their regulatory proteins to prevent stomata closure. Microbial pathogens also hijack cellular vesicle traffic to suppress secretion of defence-related molecules to the cell wall. Secretion at the plant plasma membrane is mediated by so-called SNARE proteins that assemble to drive the final stages of membrane vesicle fusion and deliver the vesicle contents to the cell wall and space outside the cell. Yet, the knowledge of molecular basis of these processes during plant pathogenesis is sparse and virtually nothing is known of their coordination.

The plasma membrane SNARE SYP132 has been associated with the secretion of antimicrobial peptides. Recently, I found that its expression and traffic within the cell are tied directly to bacterial infection. SYP132 expression, I observed, affects stomatal responses to bacterial pathogens. Furthermore, SYP132 interacts physically with the plasma membrane ion transporters and regulatory proteins that are essential for pathogen defence, and traffic of the SNARE appears to co-opt the ion transport proteins during pathogen infection. These findings point to an unexpected and central role for this SNARE as a key regulator of in stomatal defence and immunity.

My hypothesis is that SYP132 endocytosis and vesicle membrane recycling are critical for early stages of bacterial pathogenesis. SYP132 traffic and functions in immunity are particularly regulated by its interactions with the ion transporters and regulatory proteins at the plasma membrane and they allow for a co-ordination between defence signalling, antimicrobial secretion and stomatal responses to fight off disease and to regulate plant-microbe interactions.

I propose to elucidate the mechanisms underlying SYP132 traffic and its impact on the transporter binding partners to resolve the impact on plant immunity. These studies will make use of established plant and pathogen models as a backdrop for the work. I will determine how interactions of SYP132, particularly with the ion transporter and regulatory proteins identified to date, change during the progression of bacterial disease. I will expand the studies with proteomic analysis of the SYP132 interactome and measurement of quantitative changes in SYP132 interactions with new and known partners during bacterial pathogenesis to assess their roles in SYP132-mediated immunity. The knowledge gained will inform future efforts in approaches to engineering crops with enhanced defence systems in sustainable agriculture.

This proposal is based on my observations with SNARE SYP132 which suggest an entirely new paradigm for the roles of SYP132 traffic and interactions in coordinating pathogen defence-related secretion and stomatal closure in plants. In eukaryotes, cellular mechanisms underlying SNARE traffic and vesicle membrane turnover are sparsely defined. The aim of this project is to elucidate the molecular basis of SNARE traffic, to test SYP132 interactions, notably with a plasma membrane ion transporter and a regulatory protein for implications in immunity and to study effects on stomatal responses during pathogenesis. Techniques in cell biology including fluorophore tagging, membrane lipid-binding dyes and antibody labelling of proteins and biochemical analysis of membranes will be used in high-resolution image analysis to study spatial regulation of SYP132 traffic and to measure kinetics of vesicle membrane turnover. Post-endocytic fate(s) of SYP132 will be determined using co-localization with endosomal markers including treatment with traffic-inhibiting drugs. Model pathogen P. syringae triggered changes in SYP132 interactions including those that I have recently discovered will be quantitively resolved using Tandem Mass Tagging and mass spectrometry. In vitro and in vivo analysis of SYP132 binding with the ion transporter and regulatory protein will include yeast SUS, pull-down and CO-IP assays on membrane fractions. Binding motifs will be identified using site-directed mutagenesis. The readouts following manipulation of SYP132 traffic and interactions will include: bacterial growth assays, defense-molecule secretion, qRT-PCR of SNARE transcripts, measurement of plant growth, stomatal aperture and gas exchanges in Arabidopsis stable transformants. Thus, through a culmination of technical approaches in cell biology, proteomics and plant physiology, mechanisms for regulation of SNARE traffic in immunity will be elucidated with potentials for future agro-biotech applications.

This proposal is for fundamental research on SNARE regulation to co-ordinate plant immune responses through stomatal regulation defence-related secretion. The proposed research culminates advanced technologies in plant cell biology, biochemistry plant physiology and proteomics and sits squarely within emerging interests of the international plant and cell biology communities, promoting training and education. This research should impact concepts in SNARE biology and help to initiate a paradigm shift in approach. The cellular processes underlying plant immune responses and their impact on membrane transport, plant growth and development will be investigated, and novel players identified and tested for impact on plant defence. Knowledge gained will feed into methodologies for development of pathogen resilient, yet productive crop engineering. Thus, the research is expected to benefit fundamental researchers through development and optimization of experimental technologies and, in the longer term, to benefit agriculture and industry through the understanding of plant defence vs growth strategies and plant productivity. This research will support training through capacity building at undergraduate, postgraduate and postdoctoral levels. Additional impact is proposed through public outreach and communication of research outcomes to the scientific community and members of the public to promote their interest in Plant Science. Student teaching resources will be developed. Guided future efforts in applications of this research in agricultural/industrial systems are anticipated using the experience of the applicant in the pharma agro-biotech industry. Additional impacts will be found in Part 1 of the Case for Support and in the attached Impact Plan.