Controlled hydroxylations of diterpenoids allow for plant chemical defense without autotoxicity

Disturbing the biosynthetic pathway of 17-hydroxylgeranyllinalool diterpene glycosides (HGL-DTGs), including silencing NaCYP736A, caused severe plant autotoxicity. This autotoxicity can be restored by co-silencing the upstream biosynthetic genes, like geranyllinalool synthase (GLS). Through comparing the transcriptome data of EV, VIGS-GLS, VIGS-CYP736A and VIGS-GLS&CYP736A, we tried to figure out the potential mechanism of this autotoxicity. Virus-induced gene silencing (VIGS) was performed on Nicotiana attenauta plants, in which NaCYP736A and NaGLS were silenced individually or together, with empty vector (EV) in as controls. Leaves of rossete stage plants were harvested after treated by 150 µg MeJA per leaf for 3 days. Four replicate samples per genotype were used for the analysis. Total RNA was extracted from 30 mg leaf samples using the Plant RNeasy kit (Qiagen) and quality checked by spectrophotometry (NanoDrop). Genomic DNA was removed by DNAse treatment (Ambion) following manufacturer instructions. RNA was cleaned up with RNeasy MinElute columns (Qiagen) and the RNA quality was checked with the RNA 6000 Nano kit (Agilent) in the Agilent 2100 Bioanalyzer (Agilent). RNA was labeled with Cyanine-3 (Cy3) with the Quick Amp labeling kit (Agilent) according to manufacturer instructions and followed by RNAeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with a NanoDrop ND-1000 Spectrophotometer. Samples were hybridized in a N. attenuata whole genome single color array (Agilent 8×60K; GPL19764). Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting. Probe feature extraction was performed with the feature extraction software (Agilent). Quality control, data filtering and normalization were performed in Genespring GX (Agilent). Probe filtering was performed in Genespring GX using the flag (gIswellAboveBG). Probes that did not pass the condition in all replicates for one of the conditions were filtered out. Probe signals were tressholded to 1, log2 transformed and normalized to the 75th percentile. Differential expression analysis between peduncles of EV and irZTL-314 plants at ZT0 was performed with a moderated t-test in Genespring GX (Agilent). Probe to gene association and gene annotations were based in the N. attenuata genome assembly v2.5.

干扰17-羟基香叶基芳樟醇二萜糖苷（17-hydroxylgeranyllinalool diterpene glycosides，HGL-DTGs）的生物合成通路，包括沉默NaCYP736A基因，会引发严重的植物自毒反应。该自毒反应可通过共沉默上游生物合成基因，如香叶基芳樟醇合酶（geranyllinalool synthase，GLS），得以恢复。本研究通过对比空载体（empty vector，EV）、VIGS-GLS、VIGS-CYP736A及VIGS-GLS&CYP736A的转录组数据，旨在解析该自毒反应的潜在分子机制。本研究以渐狭烟草（Nicotiana attenuata）为材料，采用病毒诱导基因沉默（Virus-induced gene silencing，VIGS）技术分别或共同沉默NaCYP736A与NaGLS基因，并以空载体（EV）处理作为对照。莲座期植株的叶片在每叶喷施150 µg 茉莉酸甲酯（methyl jasmonate，MeJA）处理3天后收获，每个基因型设置4个生物学重复样本用于后续分析。称取30 mg叶片样本，采用Plant RNeasy试剂盒（Qiagen）提取总RNA，并通过分光光度法（NanoDrop）检测RNA质量；按照制造商说明书，采用DNA酶（Ambion）处理以去除基因组DNA。随后采用RNeasy MinElute吸附柱（Qiagen）对RNA进行纯化，并通过Agilent 2100生物分析仪（Agilent）搭配RNA 6000 Nano试剂盒（Agilent）检测RNA质量。按照制造商说明书，采用Quick Amp标记试剂盒（Agilent）以氰基-3（Cyanine-3，Cy3）对RNA进行标记，随后通过RNAeasy吸附柱（Qiagen）完成纯化。采用NanoDrop ND-1000分光光度计检测染料掺入量及cRNA产量。将样本与渐狭烟草全基因组单通道芯片（Agilent 8×60K；GPL19764）进行杂交，芯片洗涤后立即采用Agilent DNA微阵列扫描仪（G2565BA）以单通道扫描参数进行成像。采用特征提取软件（Agilent）完成探针信号提取，在Genespring GX（Agilent）中完成质量控制、数据过滤及标准化处理：在Genespring GX中采用标记（gIswellAboveBG）完成探针过滤，将在某一处理条件下的所有重复中均未达到阈值的探针予以剔除；将探针信号阈值设为1，进行log2转换，并以75百分位数进行标准化。在Genespring GX（Agilent）中采用校正t检验完成空载体（EV）与irZTL-314植株花序梗在计时零点（Zeitgeber Time 0，ZT0）的差异表达分析。探针-基因关联及基因注释均基于渐狭烟草基因组组装版本v2.5完成。