Specific Bacterial Pathogen Phytosensing Is Enabled by a Synthetic Promoter-Transcription Factor System in Potato

Abstract

Phytosensors are genetically engineered plant-based sensors that feature synthetic promoters fused to reporter genes to sense and report the presence of specific biotic and abiotic stressors on plants. However, when induced reporter gene output is below detectable limits, owing to relatively weak promoters, the phytosensor may not function as intended. Here, we show modifications to the system to amplify reporter gene signal by using a synthetic transcription factor gene driven by a plant pathogen-inducible synthetic promoter. The output signal was unambiguous green fluorescence when plants were infected by pathogenic bacteria. We produced and characterized a phytosensor with improved sensing to specific bacterial pathogens with targeted detection using spectral wavelengths specific to a fluorescence reporter at 3 m standoff detection. Previous attempts to create phytosensors revealed limitations in using innate plant promoters with low-inducible activity since they are not sufficient to produce a strong detectable fluorescence signal for standoff detection. To address this, we designed a pathogen-specific phytosensor using a synthetic promoter-transcription factor system: the S-Box cis-regulatory element which has low-inducible activity as a synthetic 4xS-Box promoter, and the Q-system transcription factor as an amplifier of reporter gene expression. This promoter-transcription factor system resulted in 6-fold amplification of the fluorescence after infection with a potato pathogen, which was detectable as early as 24 h post-bacterial infection. This novel bacterial pathogen-specific phytosensor potato plant demonstrates that the Q-system may be leveraged as a powerful orthogonal tool to amplify a relatively weak synthetic inducible promoter, enabling standoff detection of a previously undetectable fluorescence signal. Pathogen-specific phytosensors would be an important asset for real-time early detection of plant pathogens prior to the display of disease symptoms on crop plants.

Performance of abiotic stress-inducible synthetic promoters in genetically engineered hybrid poplar (Populus tremula × Populus alba)

“…As high-throughput sequencing continues to advance, it will become easier to elucidate the tissue-specific or cell-specific expression of the TF complex through techniques using single-cell analysis ( Dorrity et al., 2021 ; Marand et al., 2021 ; Zhang et al., 2021 ), allowing the use of TF-modulation of synthetic promoters in plant systems ( Belcher et al., 2020 ). The synthetic neurospora-based ‘Q-system’ has previously been applied to enhance synthetic promoters to tune signal specificity and sensitivity in Arabidopsis and potato ( Persad et al., 2020 ; Persad-Russell et al., 2022 ). Modifying or identifying a similar orthogonal system could help to improve synthetic promoter activity in other species such as poplar.…”

The NF-Y Transcription Factor Family in Watermelon: Re-Characterization, Assembly of ClNF-Y Complexes, Hormone- and Pathogen-Inducible Expression and Putative Functions in Disease Resistance

“…Furthermore, the majority of the ClNF-Y genes were induced by Fon , and the pathogen-induced expression was highly evident in roots ( Figure 7 C). Among these pathogen-inducible ClNF-Y genes, the ClNF-YA1 , -YB8 , and -YB9 promoters harbor a pathogen-inducible Box S element ( Figure 4 ), which is known to confer a high level of expression of the target genes in response to elicitors, oomycetes, and bacterial pathogens [ 70 , 71 , 72 ]. Functional studies in transgenic Arabidopsis revealed that 6 ClNF-Y genes play a role in disease resistance ( Figure 9 and Figure 10 ).…”

Novel synthetic inducible promoters controlling gene expression during water‐deficit stress with green tissue specificity in transgenic poplar

“…Reads were aligned to the Populus trichocarpa v3.0 genome (https://phytozome-next.jgi.doe.gov/info/Ptrichocarpa_ v3_0), using Bowtie 2 (Langmead and Salzberg, 2012). Aligned reads mapped to genome features were counted using htseq-count function in HTSeq 2.0 (Putri et al, 2022). Differential expression gene (DEG) analysis was performed using DESeq2 (Love et al, 2014).…”

“…However, in synthetic promoters, the small number of specific CREs without non‐essential elements allows researchers to off‐target binding of transcription factors and focus on valuable responses, leading to more practical applications. In a number of recent studies, synthetic promoters have been applied to effectively manage gene editing, transgenic crop improvement, and orthogonal biosensor construction (Moreno‐Gimenez et al ., 2022; Oliva et al ., 2019; Persad et al ., 2020; Persad‐Russell et al ., 2022).…”