Plant-Encoded Sense & Response Biosensors

SCOPING BIOLOGY_DOMAIN

Species: ARATH

Genes: FLS2 EFR CERK1 BAK1 BIK1 RBOHD MPK6 NPR1 EDS1 PAD4 RPS2 RPM1

Warnings (1)

Plant-Encoded Sense & Response Biosensors

Bottom line: the SEED SFA at ORNL is building plant-encoded biosensors that detect microbes
through plant immune receptors and report or respond to them. The flagship design is a chitin
sensor that splits GFP across LYK5 and a kinase-dead CERK1, so chitin-induced receptor pairing
reconstitutes fluorescence. This page collects notes on that work and lists about 20 receptor,
signalling and NLR genes worth curating, because an engineered sensor is only as reliable as
our understanding of the receptors and pathway it borrows. Scoped, not yet started as a
curation project: none of the TODO items below is done. Twelve of the listed Arabidopsis genes
(FLS2, EFR, CERK1, BAK1, BIK1, RBOHD, MPK6, NPR1, EDS1, PAD4, RPS2, RPM1) already have reviews
in genes/ARATH/ from other work, covering 560 annotations; LYK5, LYK4, PEPR1/2, PBL27, MPK3,
WRKY33 and RPS4 do not, and the Populus orthologs have not been mapped.

Notes from SEED SFA (Secure Ecosystem Engineering and Design) at ORNL.

Key Publication

Overview

Goal: Create plant-encoded biosensors that detect microbial presence and trigger programmable responses.

Two-part system:
1. Upstream (Sense): Detect microbial ligands via receptor proteins
2. Downstream (Response): Signal transduction leading to gene expression changes

Receptor Classes (Sensing)

Pattern Recognition Receptors (PRRs)

Receptor-Like Kinases (RLKs)

Chitin Perception Pathway (Fungal Detection)

Image from: Mittendorf et al. 2024. New Phytologist. doi:10.1111/nph.20074

Key insight: Chitin perception is one of the earliest molecular events in plant response to both beneficial and pathogenic fungal colonization. A specific biosensor for chitin detection has NOT yet been developed - this is an engineering target for SEED.

Receptor Complex

Upon chitin binding, CERK1 and LYK5 form a heterodimer.

Receptor Type Function Notes
CERK1 LysM-RLK Chitin receptor, kinase-active Central signaling component
LYK5 LysM-RLK High-affinity chitin binding Kinase-inactive, co-receptor
LYK4 LysM-RLK Chitin co-receptor Redundant with LYK5

Signaling Cascade

Chitin binding
     ↓
CERK1-LYK5 heterodimerization
     ↓
Phosphorylation + Ubiquitination
     ↓
  ┌──────────────────┬────────────────────┐
  ↓                  ↓                    ↓
Endocytosis      BIK1 activation    PBL27 activation
of LYK5/LYK4          ↓                    ↓
  ↓              NADPH oxidase       MAPK cascade
Vacuolar              ↓                    ↓
degradation     ROS burst        Transcriptional
                (O₂ → ROS)        reprogramming

Key Signaling Components

Gene Function Notes
BIK1 RLCK Activates NADPH oxidase (RBOHD)
PBL27 RLCK Activates MAPK cascade
RBOHD NADPH oxidase Produces ROS burst
MPK3/MPK6 MAPKs Defense gene activation

Receptor Turnover

Receptor-Like Proteins (RLPs)

Signaling Pathway Components

Cytoplasmic Kinases

ROS Production

Intracellular Receptors (NLRs)

Transcriptional Outputs

Immunity Pathways

Phytohormone Signaling

Mobile Signals

Key Genes/Proteins to Curate

Receptors

Gene Type Ligand Notes
FLS2 LRR-RLK flagellin (flg22) Bacterial detection
EFR LRR-RLK EF-Tu (elf18) Bacterial detection
CERK1 LysM-RLK chitin Fungal detection
LYK5 LysM-RLK chitin Co-receptor with CERK1
PEPR1/2 LRR-RLK AtPep peptides Damage signals
BAK1 LRR-RLK multiple Co-receptor
PtLecRLK1 Lec-RLK fungal signals Populus - symbiosis

Signaling

Gene Function Notes
BIK1 RLCK Central hub downstream of PRRs
MPK3/6 MAPK Defense gene activation
WRKY33 TF Defense transcription factor
NPR1 SA receptor/coactivator Master regulator of SAR
EDS1 Lipase-like TNL signaling
PAD4 Lipase-like TNL signaling

NLRs

Gene Type Effector recognized Notes
RPS2 CNL AvrRpt2 Arabidopsis
RPM1 CNL AvrRpm1/AvrB Arabidopsis
RPS4 TNL AvrRps4 Arabidopsis

SEED Model Systems

Populus (poplar trees)

Bacillus velezensis EB14

Laccaria bicolor

Biosensor Engineering Strategies

  1. Reporter fusions - Link defense promoters to fluorescent/luminescent reporters
  2. Synthetic receptors - Engineer receptor specificity for novel ligands
  3. Orthogonal signaling - Rewire outputs to custom responses
  4. Tunable systems - Anti-CRISPR for controllable gene editing
  5. Split-intein biosensors - Detect protein-protein interactions via reconstitution

Split-Intein Based Biosensor System

From: Boone et al., 2025. Plant Biotechnology Journal. doi:10.1111/pbi.70523

Principle

Detects protein dimerization events using split-intein mediated protein reconstitution.

Components

Component Function
N-terminal GFP half Reporter fragment 1
C-terminal GFP half Reporter fragment 2
Split inteins Mediate protein splicing when brought together
FKBP12 Dimerization domain 1 (binds rapamycin)
FRB domain Dimerization domain 2 (binds rapamycin)
Rapamycin Small molecule inducer of dimerization

Mechanism

  1. Two fusion proteins expressed:
  2. FKBP12 - Intein(N) - GFP(N)
  3. FRB - Intein(C) - GFP(C)
  4. In absence of rapamycin: proteins separate, no GFP signal
  5. Rapamycin addition: FKBP12-FRB dimerize
  6. Intein halves brought into proximity → protein splicing
  7. Functional GFP reconstituted → fluorescence output

Key Proteins

Protein Source UniProt Notes
FKBP12 Human P62942 FK506/rapamycin binding protein
FRB Human mTOR P42345 (residues 2015-2114) FKBP-rapamycin binding domain
Split inteins Various (Npu DnaE, Cfa, etc.) - Fast-splicing preferred

Applications

Chitin Biosensor (Engineered)

From: Boone et al., 2025. Plant Biotechnology Journal. doi:10.1111/pbi.70523

Design

Applies the split-intein GFP system to detect chitin-induced CERK1-LYK5 heterodimerization.

Fusion Constructs

Construct Components
LYK5 fusion LYK5 - Intein(N) - GFP(N-terminal half)
CERK1 fusion CERK1^Y428F - Intein(C) - GFP(C-terminal half)

Note: CERK1^Y428F is a kinase-dead mutant - prevents downstream signaling, isolates the dimerization readout.

Mechanism

         LYK5                    CERK1^Y428F
           │                          │
     ┌─────┴─────┐              ┌─────┴─────┐
     │  Intein-N │              │  Intein-C │
     │  GFP-N    │              │  GFP-C    │
     └───────────┘              └───────────┘
           │                          │
           └──────── Chitin ──────────┘
                       ↓
              Heterodimerization
                       ↓
           Intein halves associate
                       ↓
         Protein trans-splicing
                       ↓
            Reconstituted GFP
                       ↓
              FLUORESCENCE

Significance

Plant RNA Vision - RNA Biosensor

From: Liu et al. (2025). Plant Biotechnology Journal. doi:10.1111/pbi.14612
2025 R&D 100 Finalist

DOE article: https://www.energy.gov/science/ber/articles/novel-biosensors-offer-vivo-rna-imaging-plants

Principle

Detects specific RNA transcripts using ribozyme-mediated transcript splicing to reconstitute sfGFP.

Genetic Design

Two expression cassettes:

Cassette 1: [35S]──[sfGFP Fragment 1]──[Ribozyme Fragment 1]──[Guide RNA 1]
Cassette 2: [35S]──[HSP]──[sfGFP Fragment 2]──[Ribozyme Fragment 2]──[Guide RNA 2]

Components

Component Function
sfGFP Fragment 1 N-terminal half of superfolder GFP
sfGFP Fragment 2 C-terminal half of superfolder GFP
Ribozyme Fragment 1 Split ribozyme (catalytic RNA)
Ribozyme Fragment 2 Split ribozyme complement
Guide RNA 1 Directs to target transcript (5' region)
Guide RNA 2 Directs to target transcript (3' region)
35S Constitutive promoter (CaMV)
HSP Heat shock promoter element

Mechanism

     sfGFP-1 ─ Ribozyme-1 ─ gRNA-1
                    │
                    ↓ (gRNA-1 binds target)
              ┌─────────────────┐
              │ Transcript Target│
              └─────────────────┘
                    ↑ (gRNA-2 binds target)
                    │
     sfGFP-2 ─ Ribozyme-2 ─ gRNA-2

                    ↓
        Guide RNAs bring ribozyme
        halves to same transcript
                    ↓
        Ribozyme assembly & activation
                    ↓
        Trans-splicing of sfGFP fragments
                    ↓
           Spliced sfGFP mRNA
                    ↓
            Translation → GFP
                    ↓
             FLUORESCENCE

Key Features

Applications

RNA Biosensor Applications Across DOE/DARPA

The SEED RNA biosensor system is being extended to multiple projects:

1. DARPA Ag x BTO - Viral Detection in Crops

Goal: Early detection of viral infections in U.S. domestic agricultural crops

Component Description
Input Viral RNA + capsid protein
Sensor Genetically encoded biosensor in crop plants
Output Fluorescence detectable by drone w/ hyperspectral camera

2. DOE Center for Bioenergy Innovation (CBI) - Cell-Type Specificity

Goal: Tool for studying cell-type specific gene expression in plants

Design:
- Ribozyme halves + GFP^UV coding sequence
- Homology regions target specific cell-type transcripts
- 1) Complementation of homology arms to target RNA
- 2) Ribozyme splicing & translation → GFP^UV output

Enables single-cell resolution imaging of gene expression in tissues.

3. Plant-Microbe Interface (PMI) SFA - Drought Stress Response

Goal: Non-destructive measures of plant gene expression responsive to drought stress

Design:
- Agrobacterium-mediated plant transformation
- RNA biosensor construct: [UTR]─[biosensor]─[GFP^UV]
- In-vivo imaging under UV light

Timing advantage of RNA biosensors:

Time after stress onset (hours)
0     100    200    300    400    500    600    700
│      │      │      │      │      │      │      │
├──────┼──────┴──────┴──────┴──────┴──────┴──────┤ Stress sensed
│      ├────┤                                      Early signal transduction
│      ├──────────┤                                Gene expression changes ← RNA BIOSENSOR DETECTS HERE
│             ├──────────────┤                     Protein translation
│                  ├────────────────┤              Protein modification (PTMs)
│                       ├──────────────────────────┤ Phenotype (stomatal closure, leaf curl)

RNA biosensors detect stress ~100-200 hours earlier than visible phenotypes (stomatal closure, cuticle changes, new organs).

TODO

Slides