Fig 1.
Abbreviations, trivial names and side-chain structures of the glucosinolates detected in this study.
Fig 2.
Glucosinolate biosynthetic pathways in Brassicaceae.
Arrows between compounds represent the number of putative enzymatic reactions. For simplicity, only genes discussed in the text have been included. The figure is constructed following several publications [17–19].
Fig 3.
Response of BnMAM1, BnCYP83A1 and BnUGT74B1 to Sclerotinia sclerotiorum and Botrytis cinerea infection.
Relative expression levels of BnMAM1, BnCYP83A1 and BnUGT74B1 in Brassica napus were determined by qRT-PCR at 0, 6, 12, 24 and 36 h post S. sclerotiorum inoculation (A) and 0, 12, 24, 48 and 72 h post B. cinerea inoculation (B). The expression levels were relative to no inoculation (0 h) and quantified by qRT-PCR. Values are means of three replicates. Each bar represents means ± SE.
Fig 4.
Characterization of BnMAM1-, BnCYP83A1- and BnUGT74B1-overexpressing lines in Brassica napus.
(A) Diagram of the plasmids used in this study. pBI121 contain BnMAM1, BnCYP83A1 or BnUGT74BA1 cDNAs, respectively. RB, right border; NOS-pro, nopaline synthase gene promoter; NPTII, coding region of neomycin phosphotransferase II gene; CaMV-35S, cauliflower mosaic virus 35S promoter; NOS-ter, nopaline synthase gene terminator; LB, left border. (B) Molecular characterization of BnMAM1-, BnCYP83A1- and BnUGT74BA1-overexpressing lines. Structure of the BnMAM1, BnCYP83A1 or BnUGT74BA1 genes including exon and intron boundaries. Accumulation of BnMAM1, BnCYP83A1 or BnUGT74BA1 mRNA in WT (untransformed wild-type control) and corresponding two independent transgenic T2 lines were measured by RT-PCR on 7-week-old leaves. Actin 7 (ACT7, Brassica napus) gene expression was used as a constitutive control. Primers used for this study are indicated as solid black arrows (See primers in S1 Table). (C) Accumulation of BnMAM1, BnCYP83A1 or BnUGT74BA1 mRNA in 7-week-old leaves of WT control and corresponding overexpressing lines. The expression level of Actin 7 (ACT7, Brassica napus) was used as a constitutive control. Values are the means and SE of three replicates performed on cDNA dilutions obtained from three independent mRNA extractions. The significant differences is shown as ** (P < 0.05) on the bar. Primers used for this study are indicated as hollow white arrows (See primers in S1 Table).
Table 1.
GSL contents (nmol/g) in 7-week-old leaves of BnMAM1, BnCYP83A1 and BnUGT74B1 overexpressing T2 lines.
Fig 5.
Resistance of BnMAM1, BnCYP83A1 and BnUGT74B1 overexpressing T2 plants to S. sclerotiorum and B. cinerea.
(A-D) Disease responses of inoculated plants with S. sclerotiorum at 48 h post-inoculation (hpi). (E) Lesion sizes of leaves inoculated with S. sclerotiorum, which were measured for the first 48 h after inoculation. Means and SE are shown (n ≥ 12). The significant differences is shown as ** (P < 0.05) using t-tests. OE- M-1 and OE-M-2 are transgenic lines for BnMAM1, OE- C-1 and OE-C-2 are transgenic lines for BnCYP83A1, OE- U-1 and OE-U-2 are transgenic lines for BnUGT74B1. WT, untransformed wild-type control. (F-I) Disease responses of inoculated plants with B. cinerea at 96 hpi. (J) Lesion sizes of leaves inoculated with Botrytis cinerea, which were measured at 96 hpi. Means and SE are shown (n ≥ 12). The significant differences is shown as ** (P < 0.05) using t-tests.
Table 2.
The morbidity rate of S. sclerotiorum infection at early stage.
Table 3.
The morbidity rate of B. cinerea infection at early stage.