Poster V2 - Challenges of Heterologous Plant Defense Receptor Expression in Yeast

Introduction: Numerous bacterial infections throughout history have devastated crop populations. To defend against such infections, plants possess an innate immune system consisting of pattern recognition receptors (PRRs). These receptors bind highly conserved microbe-associated molecular patterns (MAMPs). Upon MAMP detection, the receptor forms a signaling complex to relay pathogen perception to the rest of the cell.
Perception of pathogenic bacteria in many crop plants is facilitated by the PRR FLAGELLIN-SENSING2 (FLS2), a leucine-rich repeat receptor-like kinase (LRR-RLK). FLS2 binds flg22, a MAMP representing the most conserved domain of bacterial flagellin. However, many pathogenic bacteria evade FLS2 perception due to variations in their flagellin sequences. Different plant species have varying responses to different pathogenic bacteria. By studying these systems, we aim to identify FLS2 sequences and variants of these sequences with enhanced pathogen recognition.
However, previous research has been limited to in planta mutagenesis studies, which are time-consuming and low-throughput: it takes approximately three months to generate a few hundred variants. Yeast surface display (YSD) offers a promising alternative: in approximately two weeks, a YSD library on the order of 108 variants can be generated and evaluated. YSD could expedite the screening for FLS2 variants with improved pathogen recognition, benefiting crop defense against bacterial infections. However, the heterologous expression of FLS2 presents some challenges due to differences between plant and yeast systems. We have cloned FLS2 into a YSD expression vector and transformed this into Saccharomyces cerevisiae, achieving successful expression, to investigate the feasibility of heterologous expression of FLS2 for yeast surface display.

Materials and

Methods: We have cloned the LRR region of FLS2, which binds flg22, into the pCT-80 expression vector for yeast surface display on the C-terminus of Aga2. This vector contains HA and c-myc epitope tags—on the N- and C-termini, respectively—for measuring protein expression, along with necessary machinery for plasmid propagation in E. coli and protein expression in S. cerevisiae. The vector was transformed into S. cerevisiae via electroporation and protein expression was induced via the galactose-inducible GAL1 promoter. High-throughput flow cytometry was used to quantify the levels of protein expression and flg22 binding.

Results, Conclusions, and Discussions: Flow cytometry was used to quantify the expression and function of FLS2 when displayed on the surface of yeast. Results indicate a strong HA expression signal, whereas c-myc expression is inconsistent between samples, possibly from its attachment to FLS2. Unlike the HA tag—flanked by flexible linkers—the c-myc tag directly follows FLS2, possibly resulting in interaction with FLS2. This could impact its availability for binding with flow cytometry antibodies, explaining signal variation. Nevertheless, the HA signal provides reasonable confidence of FLS2 expression on the yeast cell surface.
Additionally, flow cytometry results show no significant flg22 binding signal, possibly due to the detection system. A biotin-conjugated flg22 was bound to FLS2, and this interaction was measured through a biotin-binding fluorophore-conjugated streptavidin. Streptavidin is a bulky protein, which may hinder it from reaching the flg22-binding pocket of FLS2. This may mean that flg22 is binding, but we are unable to measure it. To improve the flg22 binding signal, we are exploring the use of detection systems that employ small fluorescent probes in place of the bulky streptavidin.
Another potential issue is the difference in N-linked glycosylation patterns between plants and yeast. FLS2 is natively expressed in plants, in which these glycans are very small, while yeast form large, branching glycans. FLS2 has 22 putative N-linked glycosylation sites, some of which are close to the flg22 binding pocket. Hyperglycosylation from the host yeast cells may hinder binding. To overcome this, we are investigating the substitution of N-linked glycosylation sites to reduce the effects of hyperglycosylation.
Following successful demonstration of flg22 binding in yeast, a library of FLS2 variants will be generated and evaluated against a panel of pathogenic bacterial flagellins. Analyzing the relationship between FLS2 sequence and pathogen perception, in addition to providing a deeper understanding of plant defense receptor systems, holds promise for agricultural advancements by improving pathogen recognition in crops. Furthermore, due to difficulties in working with plants, these defense receptor systems are understudied. By developing a platform for the expression of plant defense receptors in yeast, we are advancing the tools that can be used to study these systems.

Acknowledgements (Optional): • Flow Cytometry Guidance and Instrumentation: MSU Flow Cytometry Core
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Funding: U.S. Department of Agriculture AFRI FASE Grant