Plant-Encoded Sense & Response Biosensors

IN_PROGRESS BIOLOGY_DOMAIN

Warnings (1)

Plant-Encoded Sense & Response Biosensors

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