Fig 1.
Tobacco rattle virus-mediated silencing of PDS, ChlI and ChlH in eggplant leaves.
(A) Phenotypes of eggplant leaves infiltrated with different VIGS constructs: 1: Empty vector; 2: TRV-PDS, 3: TRV-ChlI, 4: TRV-ChlH. Scale bar equals 1cm. (B) Schematic depiction of virus-induced gene silencing (VIGS) treatment in eggplant (C) Confirmation of TRV infection in leaves by PCR. M, 2000 Marker; 1: Mock-infected; 2: Empty vector; 3: TRV-PDS, 4: TRV-ChlI, 5: TRV-ChlH. PDS, phytoene desaturase; CHLI, CHLH: Magnesium chelatase subunits. Oligonucleotides used are shown in Supplementary S1 Table.
Fig 2.
Chlorophyll and carotenoid levels are reduced in eggplant leaves with silenced PDS, ChlI and ChlH.
(A) Quantitative RT-PCR measurement of PDS, ChlI and ChlH mRNA. Data are the avg. ± SD of three biological replicates. (B) Quantitation of chlorophyll levels in WT, TRV, TRV-PDS, TRV-ChlI and TRV-ChlH leaves. (C) Quantitation of carotenoids levels in WT, TRV, TRV-PDS, TRV-ChlI and TRV-ChlH leaves. Student’s t-test was used to determine the statistical significance of differences between two samples (*P<0.05; **P<0.01). Duncan’s multiple range test was used to compare three samples (**P<0.01).
Fig 3.
Suppression of carotenoid and chlorophyll levels decrease glycoalkaloid biosynthesis in eggplant leaves.
Relative content of solamargine (A) and solasonine (B) in eggplant leaves mock-infected and infected with TRV, TRV-PDS, TRV-ChlI and TRV-ChlH. ISTD means Internal Standard, because metabolites have been relatively quantified using Formononetin as internal standard. Data are the avg. ± SD of three biological replicates, and Student’s t-test was used to determine the statistical significance of differences between two samples (*P<0.05; **P<0.01). Duncan’s multiple range test was used to compare three samples (**P<0.01).
Fig 4.
Relative expression levels of glycoalkaloid pathway genes in leaves infected with different constructs.
A schematic view of the SGA biosynthetic pathways. Dashed arrows represent multiple biosynthetic reactions. Graphs next to each gene name show expression levels determined by qRT-PCR. Expression levels were normalized to the Actin housekeeping gene and to the mock-infected control. The X-axis number can be expressed as follows, 1: Mock-infected; 2: Empty vector; 3: PDS-silenced, 4: ChlI-silenced, 5: ChlH-silenced. Data are the avg. ± SD of three biological replicates. Asterisks indicate a significant difference from the control in a Student’s t-test (**P < 0.01 and *P < 0.05).
Fig 5.
Metabolic analysis by LC-HESI-MS.
Heatmap of semi-polar metabolites (including glycoalkaloids) profiled in eggplant leaves. Colors represent fold-change values between each line and the control (TRV) sample. Red and green indicate up- and down-regulated metabolites, respectively. Fold-change values were log2-transformed. Both columns (samples) and rows (metabolites) were subjected to hierarchical clustering analysis (HCA).