Controlled hydroxylations of diterpenoids allow for plant chemical defense without autotoxicity. 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. Overall design: 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.