Arabidopsis GDSL1 overexpression enhances rapeseed Sclerotinia sclerotiorum resistance and the functional identification of its homolog in Brassica napus

Abstract

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a devastating disease of rapeseed (Brassica napus L.). To date, the genetic mechanisms of rapeseed' interactions with S. sclerotiorum are not fully understood, and molecular-based breeding is still the most effective control strategy for this disease. Here, Arabidopsis thaliana GDSL1 was characterized as an extracellular GDSL lipase gene functioning in Sclerotinia resistance. Loss of AtGDSL1 function resulted in enhanced susceptibility to S. sclerotiorum. Conversely, overexpression of AtGDSL1 in B. napus enhanced resistance, which was associated with increased reactive oxygen species (ROS) and salicylic acid (SA) levels, and reduced jasmonic acid levels. In addition, AtGDSL1 can cause an increase in lipid precursor phosphatidic acid levels, which may lead to the activation of downstream ROS/SA defence-related pathways. However, the rapeseed BnGDSL1 with highest sequence similarity to AtGDSL1 had no effect on SSR resistance. A candidate gene association study revealed that only one AtGDSL1 homolog from rapeseed, BnaC07g35650D (BnGLIP1), significantly contributed to resistance traits in a natural B. napus population, and the resistance function was also confirmed by a transient expression assay in tobacco leaves. Moreover, genomic analyses revealed that BnGLIP1 locus was embedded in a selected region associated with SSR resistance during the breeding process, and its elite allele type belonged to a minor allele in the population. Thus, BnGLIP1 is the functional equivalent of AtGDSL1 and has a broad application in rapeseed S. sclerotiorum-resistance breeding.

Keywords: Sclerotinia sclerotiorum; GDSL lipases; disease resistance; jasmonic acid; phosphatidic acid; plant breeding; rapeseed (Brassica napus L.); reactive oxygen species; salicylic acid.

Figure 1

Subcellular localization of AtGDSL1. (a) Schematic diagram of constructs used for the subcellular localization analysis. RB: right border; LB: left border; p35S: cauliflower mosaic virus 35S promoter; T35S: terminator; NPTII: neomycin phosphotransferase. (b) Subcellular localization of AtGDSL1 by the transient expression of the 35S::AtGDSL1‐eGFP and 35S::eGFP fusion constructs in N. benthamiana leaves. The columns from the left are as follows: bright field, eGFP fluorescence in green, chlorophyll fluorescence in red, and the merged image. Bars = 20 µm.

Figure 2

Arabidopsis T‐DNA insertion mutant lines of AtGDSL1 showed enhanced susceptibilities to S. sclerotiorum infections. (a) qRT‐PCR detection of AtGDSL1 expression in WT (Col‐0) and T‐DNA insertion mutant lines. Values are means ± SDs from three replicates. (b) Disease symptoms (upper) and lesion area (down) measurements in WT and AtGDSL1 mutants 24 h after S. sclerotiorum infection. Bars = 1.0 cm. (c) Disease symptoms (upper) and lesion area (down) measurements in WT and BnGDSL1‐RNAi T₂ transgenic lines 24 h after S. sclerotiorum infection. Bars = 1.0 cm. Data are means ± SDs from three independent experiments. The significant differences from WT are indicated (Student’s t‐test: ***, P < 0.001).

Figure 3

Transformation of rapeseed with AtGDSL1 and BnGDSL1 and the disease symptoms of transgenic plants inoculated with S. sclerotiorum. (a) Phenotypes of WT and T₂ transgenic plants (BnGDSL1 T28 and AtGDSL1 T18) 36 h after inoculation with S. sclerotiorum. Bars = 1.0 cm. (b) Leaf lesion area measurements from 24 to 60 h after S. sclerotiorum infection. Differences in susceptibility between WT and AtGDSL1 transgenic lines were significant (P < 0.001) from 36 to 60 h. Data are means ± SDs from three independent experiments, each with 15 leaves. (c) The growth of S. sclerotiorum examined by trypan blue staining. a1, a2, a3 and a4: S. sclerotiorum ‐infected WT and transgenic leaves. Photographs were taken at 20 h post‐inoculation; b1, b2, b3 and b4: Inoculated leaves stained with trypan blue. Photographs were taken at 4 h after staining; c1, c2, c3 and c4: mycelial growth on stained leaves, visualized using a fluorescence microscope. Bars indicate 1.0 cm in a1‐a4 and b1‐b4 panels and 1 mm in c1‐c4 panels. (d) Lipase activity assay in leaves of ‘NY12’ and AtGDSL1/BnGDSL1/‐OE plants after 24 h of S. sclerotiorum treatment. Data are means ± SD from three independent experiments. The significant differences between treated and untreated (control) samples in each line are indicated (Student’s t‐test: *, P < 0.05; **, P < 0.01). (e) Lesion phenotypes (upper) and lesion lengths (down) in WT and T₂ transgenic plant stems 7 days after inoculation with S. sclerotiorum. Bars = 1.0 cm. Significant differences between WT and transgenic plants are indicated (Student’s t‐test: *, P < 0.05). The experiment was repeated three times with similar results.

Figure 4

AtGDSL1 overexpression increased ROS accumulation. (a) DAB staining in the leaves of ‘NY12’ and AtGDSL1‐OE plants after treatment for 24 h with H₂O or S. sclerotiorum. Bars = 400 μm. (b) Quantification of ROS levels in leaves of ‘NY12’ and AtGDSL1‐OE plants after 24 h of treatment. (b) Expression profiles of NADPH oxidase and PAO in ‘NY12’ and AtGDSL1 transgenic rapeseed plants after S. sclerotiorum infection. The relative expression levels were analysed by qRT‐PCR and normalized using BnTIP41 as the internal control. The experiment was repeated three times with similar results. Data represent the means ± SD from three independent experiments. Significant differences between AtGDSL1 transgenic lines and WT at each time point are indicated (Student’s t‐test) as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 5

AtGDSL1 affected SA‐ and JA‐mediated signalling pathways. (a) AtGDSL1 positively regulated SA‐related genes PAL, PR2 and NPR1. (b) AtGDSL1 negatively regulated JA‐related genes KAT4, LOX2 and PR3. The relative expression levels were analysed by qRT‐PCR and normalized using TIP41 as an internal control. Values are means ± SDs from three replicates. (c and d) AtGDSL1 affects endogenous SA (c) and JA (d) levels in ‘NY12’ and transgenic rapeseed plants after the infection. (e) Quantification of SA and JA contents in WT and AtGDSL1 insertion mutant lines. Data are means ± SDs from three independent experiments. The significant differences between ‘NY12’ and AtGDSL1‐OE at each time point, or between Col‐0 and AtGDSL1 insertion lines, are indicated (Student’s t‐test) as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 6

AtGDSL1 contributed to rapseed resistance to S. sclerotiorum in association with SA, JA and PA. (a) S. sclerotiorum disease symptoms (left) and the lesion area (right) of four‐week‐old Arabidopsis insertion mutants treated with 1 mm SA and/or 0.1 mm MeJA. Bars = 0.5 cm. (b) S. sclerotiorum disease symptoms (left) and the lesion area (right) of four‐leaf‐stage rapeseed OE plants treated with 1 mm SA and/or 0.1 mm MeJA. Bars = 1.0 cm. (c) S. sclerotiorum disease symptoms (left) and the lesion area (right) of four‐leaf‐stage rapeseed OE plants treated with 100 μM PAC. Pathogen inoculation was performed 24 h after spraying the respective plants with SA, MeJA or PAC. Lesion areas were measured 24 h after S. sclerotiorum infection. Bars = 1.0 cm. (d) Expression of AtGDSL1, PR2 and NPR1 in AtGDSL1‐OE plants 24 h after treatments with H₂O or 100 μm PAC. (d) PA contents in ‘NY12’ and transgenic rapeseed plants after infection. (e) PA contents in Col‐0 and insertion lines. The data represent the means ± SDs from three independent experiments. Significant differences between the control and AtGDSL1 OE or insertion lines are indicated (Student’s t‐test) as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 7

Candidate gene association analysis of AtGDSL1 homologs in a natural B. napus population. (a) Manhattan plots of the disease index and incidence from association analyses. The abscissa represents genes, separated by the black and red coloured intervals. Each point represents a SNP, and the SNP that exceeds the threshold (red line) −log₁₀ ^{( P/n )} (P = 0.05; n = 926) = 4.27 is significant. (b) QQ plots for the disease index and disease rate from association analyses. The red line indicates the unbiased estimates of the expected and observed values. The farther the SNP deviates from the grey area, the more significant is the correlation.

Figure 8

BnGLIP1 confers resistance to S. sclerotiorum in N. benthamiana. (a–b) Time‐course expression analyses of BnGLIP1 after S. sclerotiorum infection (a) and phytohormone (ACC, MeJA and SA) treatment (b) in rapeseed. The expression levels at 0 h (no treatment) were quantified by qPCR and served as the control. The data represent the means ± SDs from three independent experiments. The significant differences in gene expression levels between each time point and the control are indicated (Student’s t‐test) as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05. (c) Disease‐resistance effects of transient expression of AtGDSL1 or BnGLIP1 in N. benthamiana. Photographs were taken at 24 h post‐inoculation with S. sclerotiorum on leaves inoculated with A. tumefaciens carrying 35S::00, 35S::AtGDSL1 or 35S::BnGLIP1. Bars = 1.0 cm. The data represent the means ± SDs from three independent experiments, with each containing 15 leaves. Significant differences in lesion size between transgenic lines and WT are indicated (Student’s t‐test) as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 9

Selective sweep signals on chromosome C07 in resistant (R) and susceptible (S) rapeseed subgroups. (a) Comparisons of disease index between the R and S subgroups. (b) Comparisons of disease rates between the R and S subgroups. The π ratios (π R/π S) and F_ST values were calculated in 20 kb sliding windows with a step size of 2 kb. The vertical red lines correspond to the 5% left and right tails of the empirical π ratio distribution, where the π ratios are 0.52 and 1.47 for the disease index (a), and 0.49 and 1.51 for the disease rate (b) between R and S subgroups, respectively. The horizontal red lines correspond to the 5% right tail of the empirical F_ST distribution, where F_ST is 0.087 in (a) and 0.083 in (b). The data points located to the left and right of the vertical red lines, and above the horizontal red line, are the selective sweeps related to the disease index and disease rate for the R (blue dots) and S (green dots) groups, respectively. Red dots indicate regions including the BnGLIP1 gene.

Figure 10

Allelic variation analyses for BnGLIP1. (a) LD heatmap of SNPs in the BnGLIP1 gene. The numbers indicate LD values that were calculated by Haploview software, the deeper the red colour, the stronger the linkage relationship. (b) Boxplots for disease index (upper) and disease rate (down) based on the three different allele types (CC, CT and TT) of the SNP, GDSL_chrC07_37987173. Eight rapeseed accessions with missing genotypes were excluded. The phenotypic differences between different groups were tested using a two‐tailed t‐test (**P < 0.01). The centre lines indicate the median.