L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a flavoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.

中文翻译：

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哈茨木霉L-氨基酸氧化酶在烟草中的表达赋予对核盘菌和灰葡萄孢菌的抗性

哈茨木霉 ETS323 分泌的 L-氨基酸氧化酶 (ThLAAO) 是一种具有抗菌特性的黄素酶。在这项研究中，我们将 ThLAAO 基因转化到烟草中，以阐明 ThLAAO 是否可以激活防御机制并赋予对植物病原体的抗性。过量表达 ThLAAO 的转基因烟草对核盘菌和灰葡萄孢的抗性增强，并激活了防御相关基因和参与水杨酸、茉莉酸和乙烯生物合成的基因的表达，伴随着叶绿体、叶绿体周围细胞质和转基因烟草的细胞膜。用抗坏血酸清除 H2O2 消除了对 B. cinerea 感染的抗病性，并降低了防御相关基因的表达。ThLAAO-FITC 在烟草原生质体上的应用或 ThLAAO-GFP 在烟草中的过表达揭示了 ThLAAO 在叶绿体中的定位。通过 ThLAAO-ConA 亲和层析分离叶绿素 a/b 结合蛋白 (CAB)。下拉测定结果证实了 ThLAAO-CAB 结合。ThLAAO-Cy5.5 在甘蓝根上的应用迅速转移到叶子。在甘蓝根上处理 ThLAAO 可诱导对 B. cinerea 的系统抗性。总体而言，这些结果表明 ThLAAO 可能靶向叶绿体并通过 H2O2 信号激活防御机制，从而赋予对核盘菌和灰霉病菌的抗性。下拉测定结果证实了 ThLAAO-CAB 结合。ThLAAO-Cy5.5 在甘蓝根上的应用迅速转移到叶子。在甘蓝根上处理 ThLAAO 可诱导对 B. cinerea 的系统抗性。总体而言，这些结果表明 ThLAAO 可能靶向叶绿体并通过 H2O2 信号激活防御机制，从而赋予对核盘菌和灰霉病菌的抗性。下拉测定结果证实了 ThLAAO-CAB 结合。ThLAAO-Cy5.5 在甘蓝根上的应用迅速转移到叶子。ThLAAO 对甘蓝根的处理诱导了对 B. cinerea 的系统抗性。总体而言，这些结果表明 ThLAAO 可能靶向叶绿体并通过 H2O2 信号激活防御机制，从而赋予对核盘菌和灰霉病菌的抗性。