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.