BnGSTU12 was induced by biotic and abiotic stress and hormones
BnGSTU12 was strongly induced in B. napus infected byS. sclerotiorum . In the early stage of infection (within 12 h), the expression of BnGSTU12 decreased, and then increased significantly at 24 h after infection (Figure 3A).
GSTs have been shown to play the great role in abiotic stress (Kumar and Trivedi 2018). We investigated the expression pattern of BnGSTU12in response to PEG 6000, NaCl, H2O2 and UV-B (Figure 3A). The expression of BnGSTU12 was significantly down-regulated after treatment with 10% PEG 6000 and 0.9% NaCl.BnGSTU12 was induced rapidly at 1 h after H2O2 treatment. Under UV-B treatment, the expression of BnGSTU12 was down-regulated in the early stage, significantly up-regulated after treatment 180 min. These results suggested thatBnGSTU12was induced by H2O2and UV-B (Figure 3A).
In order to identify the hormone signaling pathway related withBnGSTU12 , we analyzed the expression pattern of BnGSTU12in response to the hormones, ABA, GA3, KT, ETH, SA and MeJA. We found that BnGSTU12 induced rapidly at 1 h after treatment for these six hormones. The expression of BnGSTU12 showed a slight increase (2-3 fold) in response to ABA, GA3 and KT (Figure 3B). However, the expression ofBnGSTU12 significantly increased 10-20 fold at 1 h after induced by SA, ETH and MeJA (Figure 3B). The results showed thatBnGSTU12was rapidly induced by SA, ETH and MeJA, and it might involve inS. sclerotiorum resistance through SA, ETH and MeJA-related pathway.
Subcellular location of BnGSTU12
To determine the subcellular localization of BnGSTU12, full length of BnGSTU12 was fused to the C-terminus of green fluorescent protein (GFP) and transiently transformed to tobacco epidermis. The results showed that BnGSTU12 localized in nuclear and cytoplasm (Figure 4).
Overexpression of BnGSTU12 in A. thaliana and transient expression ofBnGSTU12 in tobacco enhanced S. sclerotiorumresistance
In order to explore the function of BnGSTU12 , BnGSTU12overexpression A. thaliana lines were generated driven by the 35S promoter. Three homozygous lines (#1, #6, #7) were used to investigate the S. sclerotiorum resistance. Lesion area of infected leaves in BnGSTU12 -OE A. thaliana lines was significantly smaller at 12 h, 24 h and 36 h after inoculation compared to WT (Figure 5A and 5B).
To determine S. sclerotiorum resistance of BnGSTU1 2, transient expression ofBnGSTU12 was performed in tobacco. We injected the empty vector (EV) and 35S:BnGSTU12 into tobacco leaves, and then inoculated withS. sclerotiorum after 36 h transient expression. Trypan blue staining showed that lighter necrosis in the tobacco leaves expressing 35S:BnGSTU12 than EV (Figure 5C). And the lesion area of tobacco leaves (2.4 cm2) expressing 35S:BnGSTU12 was significantly smaller than EV (4.6 cm2,P <0.05) (Figure 5D). These data suggested that overexpression of BnGSTU12 inA. thaliana and tobacco could enhanceS. sclerotiorum resistance.
BnGSTU12improved S. sclerotiorum resistance in B. napus
To further investigate the function of BnGSTU12 in B. napus , BnGSTU12 was overexpressed driven by 35S promoter inB. napus . And three overexpressed lines were obtained and the expression levels were detected (Figure 6A and 6B). After inoculation with S. sclerotiorum the lesion area in BnGSTU12 -OE lines (4.35 cm2) were significantly smaller than WT (5.30 cm2), reducing by 17.8% (Figure 6C). In addition, the transient silencing of BnGSTU12 in B. napus using using VIGS. Ten days after Agrobacterium infiltration of ZS11, we detected the expression levels of BnGSTU12 using qRT-PCR (Figure 6D and 6E). Compared to the WT, the expression level of BnGSTU12in B. napus reduced, suggesting that BnGSTU12 was silenced in the TRV:BnGSTU12 lines.S. sclerotiorum resistance was evaluated using detached leaf inoculation, the results showed that the lesion area of the TRV:BnGSTU12 lines (5.13 cm2) were significantly larger than the control lines 36 h after inoculation (3.67 cm2,P <0.05) (Figure 6F). All together, BnGTU12 could enhanced the S. sclerotinia resistance in B. napus .
BnGSTU12 enhanced the S. sclerotiorum resistance through c hanging the equilibrium of ROS
GST enzyme activity was measured in WT and overexpressedA. thaliana and B. napus before and after inoculation withS. sclerotiorum. GST enzyme activity increased at 6 h after inoculation in WT and BnGSTU12 -OE A. thaliana lines (Supplementary Figure 2). The GST enzyme activity of WT lines decreased with the inoculation, whereas GST enzyme activity in OE lines increased and reached maximum at 24 h after inoculation (240.1 U/mg) (Supplementary Figure 2). In B. napus , GST enzyme activity increased at 12 h after inoculation in WT andBnGSTU12 -OE lines. The GST enzyme activity in BnGSTU12 -OE lines increased and peaked at 24 h after inoculation (84.1 U/mg), whereas it decreased at 24 and 36 h after inoculation in WT lines (Figure 7). The results showed that GST enzyme activity was significantly increased in overexpressed A. thalianaand B. napus after inoculation with S. sclerotiorum .
To further investigate the molecular mechanism that BnGSTU12involved inS. sclerotiorum resistance, the H2O2content was examined in OE and WT lines in A. thaliana andB. napus . The results showed that the H2O2 content in A. thaliana was increased in OE and WT at 6 h after inoculation withS. sclerotiorum(Supplementary Figure 2). After 12 h of inoculation, the H2O2 content in WT continue to increase (0.3 umol/g), whereas the H2O2 content in OE decreased down to 0.05 umol/g (P<0.01). After 24 h of inoculation, OE produced lower H2O2(0.04 umol/g) compared to WT (0.16 umol/g) (Supplementary Figure 2). InB. napus , H2O2 content showed the similar trend with that in A. thaliana . The H2O2 content increased at 12 h of inoculation in WT and OE B. napus lines, but it increased less in OE B. napus (31.0 umol/g) lines than WT (46.5 umol/g). After 24 h of inoculation, OE B. napus lines produced less H2O2 (25.1 umol/g) than WT (34.7 umol/g) (P<0.01) (Figure 7). In addition, we measured the antioxidant enzyme activities, POD, SOD and CAT. POD, SOD and CAT activities in OE lines were significantly higher than WT after inoculation with S. sclerotiorum (Figure 7 and Supplementary Figure 2). All together,BnGSTU12 enhanced theS. sclerotiorum resistance by changing the H2O2 levels, POD, CAT and SOD activities. The higher antioxidant enzyme activity, the lower H2O2 content in BnGSTU12 -OE plants.