Abstract：
BACKGROUND: As an important herbivore-induced plant volatile, (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) is known for its defensive role against multiple insect pests, including attracting natural enemies. A terpene synthase (GhTPS14) and two cytochrome P450 (GhCYP82L1, GhCYP82L2) enzymes are involved in the de novo synthesis of DMNT in cotton. We conducted a study to test the potential of manipulating DMNT-synthesizing enzymes to enhance plant resistance to insects.
OBJECTIVE: To manipulate DMNT emissions in cotton and generate cotton lines with increased resistance to mirid bug Apolygus lucorum.
METHODS: Biosynthesis and emission of DMNT by cotton plants were altered using CRISPR/Cas9 and overexpression approaches. Dynamic headspace sampling and GC-MS analysis were used to collect, identify and quantify volatiles. The attractiveness and suitability of cotton lines against mirid bug and its parasitoid Peristenus spretus were evaluated through various assays.
RESULTS: No DMNT emission was detected in knockout CAS-L1L2 line, where both GhCYP82L1 and GhCYP82L2 were knocked out. In contrast, gene-overexpressed lines released higher amounts of DMNT when infested by A. lucorum. At the flowering stage, L114 (co-overexpressing GhCYP82L1 and GhTPS14) emitted 10-15-fold higher amounts than controls. DMNT emission in overexpressed transgenic lines could be triggered by methyl jasmonate (MeJA) treatment. Apolygus lucorum and its parasitoid were far less attracted to the double edited CAS-L1L2 plants, however, co-overexpressed line L114 significantly attracted bugs and female wasps. A high dose of DMNT, comparable to the emission of L114, significantly inhibited the growth of A. lucorum, and further resulted in higher mortalities.
CONCLUSIONS: Turning down DMNT emission attenuated the behavioral preferences of A. lucorum to cotton. Genetically modified cotton plants with elevated DMNT emission not only recruited parasitoids to enhance indirect defense, but also formed an ecological trap to kill the bugs. Therefore, manipulation of DMNT biosynthesis and emission in plants presents a promising strategy for controlling mirid bugs.
OBJECTIVE: To manipulate DMNT emissions in cotton and generate cotton lines with increased resistance to mirid bug Apolygus lucorum.
METHODS: Biosynthesis and emission of DMNT by cotton plants were altered using CRISPR/Cas9 and overexpression approaches. Dynamic headspace sampling and GC-MS analysis were used to collect, identify and quantify volatiles. The attractiveness and suitability of cotton lines against mirid bug and its parasitoid Peristenus spretus were evaluated through various assays.
RESULTS: No DMNT emission was detected in knockout CAS-L1L2 line, where both GhCYP82L1 and GhCYP82L2 were knocked out. In contrast, gene-overexpressed lines released higher amounts of DMNT when infested by A. lucorum. At the flowering stage, L114 (co-overexpressing GhCYP82L1 and GhTPS14) emitted 10-15-fold higher amounts than controls. DMNT emission in overexpressed transgenic lines could be triggered by methyl jasmonate (MeJA) treatment. Apolygus lucorum and its parasitoid were far less attracted to the double edited CAS-L1L2 plants, however, co-overexpressed line L114 significantly attracted bugs and female wasps. A high dose of DMNT, comparable to the emission of L114, significantly inhibited the growth of A. lucorum, and further resulted in higher mortalities.
CONCLUSIONS: Turning down DMNT emission attenuated the behavioral preferences of A. lucorum to cotton. Genetically modified cotton plants with elevated DMNT emission not only recruited parasitoids to enhance indirect defense, but also formed an ecological trap to kill the bugs. Therefore, manipulation of DMNT biosynthesis and emission in plants presents a promising strategy for controlling mirid bugs.
摘要：
背景：作为重要的草食动物引起的植物挥发性，（3E）-4,8-二甲基-1,3,7-壬三烯（DMNT）因其对多种害虫的防御作用而闻名，包括吸引天敌。萜烯合酶（GhTPS14）和两种细胞色素P450（GhCYP82L1，GhCYP82L2）酶参与棉花DMNT的从头合成。我们进行了一项研究，以测试操纵DMNT合成酶以增强植物对昆虫的抗性的潜力。
目的：操纵棉花中的DMNT排放，并产生对mirid虫Alighighuslucorum抗性增强的棉花品系。
方法:使用CRISPR/Cas9和过表达方法改变棉花植物的DMNT的生物合成和发射。采用动态顶空进样和GC-MS分析,识别和量化挥发物。通过各种试验评估了棉花品系对低毛虫及其寄生类Peristenusspretus的吸引力和适用性。
结果：在敲除CAS-L1L2品系中未检测到DMNT发射，其中GhCYP82L1和GhCYP82L2均被敲除。相比之下,当被A.lucorum感染时，基因过表达的品系释放出更大量的DMNT。在开花阶段，L114（共同过表达GhCYP82L1和GhTPS14）的释放量比对照高10-15倍。过表达的转基因品系中的DMNT发射可由茉莉酸甲酯处理(MeJA)触发。A.lucorum及其寄生虫对双重编辑的CAS-L1L2植物的吸引力要小得多，然而,共同过表达的品系L114显着吸引了虫子和雌性黄蜂。高剂量的DMNT,与L114的排放相当，显着抑制了A.lucorum的生长，并进一步导致更高的死亡率。
结论：降低DMNT的排放会减弱A.lucorum对棉花的行为偏好。转基因棉花植物与高DMNT排放不仅招募寄生虫增强间接防御,而且还形成了一个生态陷阱来杀死虫子。因此,植物中DMNT生物合成和排放的工程操作为控制微生物虫提供了有希望的策略。
目的：操纵棉花中的DMNT排放，并产生对mirid虫Alighighuslucorum抗性增强的棉花品系。
方法:使用CRISPR/Cas9和过表达方法改变棉花植物的DMNT的生物合成和发射。采用动态顶空进样和GC-MS分析,识别和量化挥发物。通过各种试验评估了棉花品系对低毛虫及其寄生类Peristenusspretus的吸引力和适用性。
结果：在敲除CAS-L1L2品系中未检测到DMNT发射，其中GhCYP82L1和GhCYP82L2均被敲除。相比之下,当被A.lucorum感染时，基因过表达的品系释放出更大量的DMNT。在开花阶段，L114（共同过表达GhCYP82L1和GhTPS14）的释放量比对照高10-15倍。过表达的转基因品系中的DMNT发射可由茉莉酸甲酯处理(MeJA)触发。A.lucorum及其寄生虫对双重编辑的CAS-L1L2植物的吸引力要小得多，然而,共同过表达的品系L114显着吸引了虫子和雌性黄蜂。高剂量的DMNT,与L114的排放相当，显着抑制了A.lucorum的生长，并进一步导致更高的死亡率。
结论：降低DMNT的排放会减弱A.lucorum对棉花的行为偏好。转基因棉花植物与高DMNT排放不仅招募寄生虫增强间接防御,而且还形成了一个生态陷阱来杀死虫子。因此,植物中DMNT生物合成和排放的工程操作为控制微生物虫提供了有希望的策略。
