L-Theanine from Bacillus tequilensis metabolites inhibits Alternaria alternata via an Nrf2-dependent oxidative-stress and ABC-transporter regulatory axis
- 1Plant Protection Institute, Hebei Academy of Agriculture and Forestry Sciences, National Collection of Plant-associated Microbes (Hebei), IPM Innovation Center of Hebei, International Science and Technology Joint Research Center on IPM of Hebei
Abstract
Background: Postharvest decay caused by Alternaria alternata and contamination with tenuazonic acid, alternariol, and tentoxin pose significant threats to pear fruit quality and safety. The reliance on synthetic fungicides is becoming unsustainable, and environmentally friendly alternatives are needed.
Methods: L-Theanine was detected and quantified in crude metabolites of Bacillus tequilensis by LC-MS/MS using an authentic standard. Commercial L-theanine (purity ≥98%) was used to evaluate antifungal activity against A. alternata . The antifungal mechanism was investigated using transcriptomics, weighted gene co-expression network analysis (WGCNA), and targeted gene knockout. Antifungal assays used four technical replicates per concentration in two independent experiments. Transcriptomic and gene expression analyses used three biological replicates. Mutant phenotyping used three biological replicates with three technical replicates each.
Results: L-Theanine inhibited mycelial growth (EC ₅₀ = 1.54 μg·mL ⁻¹ ; 95% CI 1.32–1.76 μg·mL ⁻¹ ) and spore germination (EC ₅₀ = 0.61 ± 0.05 μg·mL ⁻¹ ; 95% CI 0.52–0.70 μg·mL ⁻¹ ) and significantly reduced toxin production. Transcriptomic profiling revealed time-dependent downregulation of 12 ABC transporter genes, and WGCNA indicated Nrf2 as a candidate central node within the co-expression module. Functional validation revealed that Nrf2 deletion markedly attenuated L-theanine susceptibility (EC ₅₀ increased 3.8-fold), abolished toxin suppression, and abrogated oxidative stress responses, including ROS accumulation, lipid peroxidation, and suppression of antioxidant enzyme activities. Complementation fully restored the wild-type phenotype. Mechanistically, L-theanine induced a transient transcriptional activation of antioxidant genes ( CAT , SOD , GPX ) in the wild-type strain, followed by a progressive decline in enzyme activities and subsequent oxidative damage, whereas the Δ Nrf2 mutant failed to activate this transcriptional response. In detached pear fruit, injection of L-theanine into the same wound that received the pathogen significantly reduced lesion expansion. These results support a model in which Nrf2 is required for L-theanine-induced oxidative stress and for the suppression of toxin biosynthesis. Direct promoter binding of Nrf2 to ABC transporter genes was not demonstrated, and Nrf2-dependence of L-theanine-induced ABC transporter repression was not tested in this study.
Conclusion: This study identifies Nrf2 as a pivotal molecular switch and establishes L-theanine as a promising candidate for further development in integrated management of postharvest pear diseases. The increased resistance of the Δ Nrf2 mutant is attributable to the loss of the Nrf2-dependent oxidative-stress response rather than to changes in ABC transporter expression alone. Validation was performed under laboratory and detached-fruit conditions; storage, safety, stability, phytotoxicity, and fruit-quality studies are required before practical application.
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BibTeX
@article{bi2026ltheanine,
title = {{L-Theanine from Bacillus tequilensis metabolites inhibits Alternaria alternata via an Nrf2-dependent oxidative-stress and ABC-transporter regulatory axis}},
author = {Qiuyan Bi and Fen Lu and Jie Wu and Xiangyu Liu and Jianjiang Zhao and Xiuying Han},
journal = {Frontiers in Plant Science},
year = {2026},
month = oct,
doi = {10.3389/fpls.2026.1987466},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2026.1987466/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - L-Theanine from Bacillus tequilensis metabolites inhibits Alternaria alternata via an Nrf2-dependent oxidative-stress and ABC-transporter regulatory axis
AU - Qiuyan Bi
AU - Fen Lu
AU - Jie Wu
AU - Xiangyu Liu
AU - Jianjiang Zhao
AU - Xiuying Han
PY - 2026
DA - 2026/10/14
JO - Frontiers in Plant Science
DO - 10.3389/fpls.2026.1987466
UR - https://www.frontiersin.org/articles/10.3389/fpls.2026.1987466/full
AB - Background: Postharvest decay caused by Alternaria alternata and contamination with tenuazonic acid, alternariol, and tentoxin pose significant threats to pear fruit quality and safety. The reliance on synthetic fungicides is becoming unsustainable, and environmentally friendly alternatives are needed. Methods: L-Theanine was detected and quantified in crude metabolites of Bacillus tequilensis by LC-MS/MS using an authentic standard. Commercial L-theanine (purity ≥98%) was used to evaluate antifungal activity against A. alternata . The antifungal mechanism was investigated using transcriptomics, weighted gene co-expression network analysis (WGCNA), and targeted gene knockout. Antifungal assays used four technical replicates per concentration in two independent experiments. Transcriptomic and gene expression analyses used three biological replicates. Mutant phenotyping used three biological replicates with three technical replicates each. Results: L-Theanine inhibited mycelial growth (EC ₅₀ = 1.54 μg·mL ⁻¹ ; 95% CI 1.32–1.76 μg·mL ⁻¹ ) and spore germination (EC ₅₀ = 0.61 ± 0.05 μg·mL ⁻¹ ; 95% CI 0.52–0.70 μg·mL ⁻¹ ) and significantly reduced toxin production. Transcriptomic profiling revealed time-dependent downregulation of 12 ABC transporter genes, and WGCNA indicated Nrf2 as a candidate central node within the co-expression module. Functional validation revealed that Nrf2 deletion markedly attenuated L-theanine susceptibility (EC ₅₀ increased 3.8-fold), abolished toxin suppression, and abrogated oxidative stress responses, including ROS accumulation, lipid peroxidation, and suppression of antioxidant enzyme activities. Complementation fully restored the wild-type phenotype. Mechanistically, L-theanine induced a transient transcriptional activation of antioxidant genes ( CAT , SOD , GPX ) in the wild-type strain, followed by a progressive decline in enzyme activities and subsequent oxidative damage, whereas the Δ Nrf2 mutant failed to activate this transcriptional response. In detached pear fruit, injection of L-theanine into the same wound that received the pathogen significantly reduced lesion expansion. These results support a model in which Nrf2 is required for L-theanine-induced oxidative stress and for the suppression of toxin biosynthesis. Direct promoter binding of Nrf2 to ABC transporter genes was not demonstrated, and Nrf2-dependence of L-theanine-induced ABC transporter repression was not tested in this study. Conclusion: This study identifies Nrf2 as a pivotal molecular switch and establishes L-theanine as a promising candidate for further development in integrated management of postharvest pear diseases. The increased resistance of the Δ Nrf2 mutant is attributable to the loss of the Nrf2-dependent oxidative-stress response rather than to changes in ABC transporter expression alone. Validation was performed under laboratory and detached-fruit conditions; storage, safety, stability, phytotoxicity, and fruit-quality studies are required before practical application.
SN - 1664-462X
ER -