Fig 1.
(A) Schematic map of the BN1703 RNAi cassettes within the pHELLSGATE8 vector. The intron-hairpin interfering cassette is under the control of the 35S promotor of Cauliflower mosaic virus. The 259 bp fragment of the ABHYDROLASE-3 gene coding sequence from S. sclerotiorum (SS1G_01703) was directionally cloned to generate sense and antisense arms flanking an intronic spacer. NOS (nopaline synthase) provides selection against kanamycin. (B) Estimated number of BN1703 copies inserted into B. napus transformants. Copy number was determined by comparing relative abundance measured by qPCR to the single copy gene, High Mobility Group protein HMG-I/HMG-Y. Data for nucleic acid quantifications represents three biological replicates with error bars representing standard error. Statistical differences calculated using ANOVA (with significance of p<0.05). (C) Relative expression of BNDCL2A in B. napus transformants relative to untransformed plants and normalized to the housekeeping reference BNATGP4. Data for nucleic acid quantifications represents three biological replicates with error bars representing standard error. Statistical differences calculated using ANOVA (with significance of p<0.05). (D) Schematic diagram of the amplification of sRNA1 from 1703 hpRNA using linear variable hairpin qPCR through the reverse transcribed extension of a hairpin primer. (E) Transcript abundance of sRNA1 using linear variable hairpin qPCR of B. napus transformants. Data for nucleic acid quantifications represents three biological replicates with error bars representing standard error. Statistical differences calculated using ANOVA (with significance of p<0.05). (F) Relative expression of SS1G_01703 in infected untransformed and BN1703.2 B. napus stems 1, 2, 4, and 7 days post-inoculation demonstrating transcript knockdown within BN1703.2 stems. Asterisks represent statistical differences from the untransformed control (one-tailed t-test with Bonferroni correction, p<0.05 from three biological replicates).
Fig 2.
(A) Representative images of infected untransformed and BN1703.2 B. napus leaves and stem cross-sections 3 and 7 days post-inoculation respectively. Scale bars represent 1 cm for stem cross sections. (B) Relative leaf lesion area of infected B. napus transformants compared to untransformed leaf lesions. Data represents at least ten leaf lesions per line with bar representing the median. Statistical differences were tested with a one-way ANOVA (with significance of p<0.05), where significant differences are denoted with differing letters. (C) Relative fungal load of infected stem lesions 2, 4, and 7 days post inoculation. 18S rDNA abundance was quantified for untransformed and T2 BN1703.2 stem lesions. Asterisks represent statistical differences from the untransformed control (one-tailed t-test with Bonferroni correction, p<0.05 from 3 biological replicates). Lactophenol blue staining of stem transverse-sections 7 days post-inoculation of untransformed (D) and BN1703.2 (E) stems. Untransformed stems show extensive colonization and degradation of the epidermis (E), cortex (C) and secondary phloem (2°P) and penetration into the secondary xylem (2°X) through medullary rays. BN1703.2 stems show colonization of the epidermis (E) and cortex (C), although they remain intact. Secondary phloem (2°P) and xylem (2°X) remain uncolonized. Black arrowheads indicate S. sclerotiorum hyphae highlighted by lactophenol blue stain and white arrowheads indicate medullary rays. Scale bars represent 250 μm and micrographs are representative images of at least three biological replicates from different stems and ten cross sections from each.
Fig 3.
BN1703.2 confers whole protection up to plant maturity.
(A) Relative abundance of sRNA1 in BN1703.2 uninfected stems, leaves and flowers to demonstrate transgene expression in these organs. Statistical differences were tested with a one-way ANOVA (with significance of p<0.05), where significant differences are denoted with differing letters. (B) Total petal forming lesions per plant were quantified after 7 days of incubation in a humidity chamber after spraying. Plants were then allowed to mature fully before total sclerotia and seed mass were quantified per plant. Data represents at least ten plants per cultivar, bars represent the median and asterisks represent statistical differences from the untransformed control (one-tailed t-test with Bonferroni correction, p<0.05). (C) Representative images of ascospore spray infected untransformed and transgenic BN1703.2 plants after seven days of incubation in a humidity chamber after spraying. Untransformed plants show extensive infection on flowers and leaves and several sites of lesion progression into the stem. BN1703.2 plants show infection of flowers and leaves however, minimal lesion progression upon the leaves and stem.
Fig 4.
Transcriptomic characterization of plant defense to S. sclerotiorum in BN1703.2.
(A) Stacked bar plot displaying the number of B. napus genes differentially expressed in untransformed and transgenic BN1703.2 infected stems 1 and 7 days post-inoculation and compared to the uninfected control (false discovery rate<0.01). Darker shades of color represent higher numbers of patterns shared between treatments. (B) Upset diagram showing the sizes of gene sets induced by each combination of patterns. The bars for the set sizes are coloured by the deviation from the size predicted by random mixing. (C) Heatmap of significantly enriched GO terms of untransformed and BN1703.2 days 1 and 7 compared to uninfected from total DEG. GO terms are considered statistically significant if the hypergeometric P-value<0.001. (D) Heatmap of a subset of genes encompassing significantly enriched GO terms within BN1703.2 during infection. GO terms are considered statistically significant if the hypergeometric P-value<0.001.
Fig 5.
Structural differences between BN1703.2 and untransformed lead to enhanced protection from S. sclerotiorum.
(A) Heatmap of genes involved in suberin and callose biosynthesis, which were enriched GO terms for BN1703.2. GO terms are considered statistically significant if the hypergeometric P-value<0.001. (B) A diagram of the biosynthesis pathway of suberin in plant cells. The enzyme font color corresponds to the B. napus gene ID in the heatmap in (A). Scanning electron micrographs of infected B. napus stems 7 days post-inoculation in untransformed (C) and BN1703.2 (D) genotypes. The untransformed stem shows extensive colonization throughout, particularly the pith (P) region. The BN1703.2 stem shows no colonization of the pith and a vascular coating within the secondary xylem (2°X). Scale bars represent 1 mm for untransformed and 0.5 mm and 50 μm for BN1703.2. Black arrowheads indicate Sclerotinia hyphae and white arrowheads indicate the vascular coating. Hand cut stem transverse sections stained with Sudan IV of untransformed (E) and BN1703.2 (F) stems 7 days post-inoculation. Red staining indicates deposition of suberin. The untransformed stem shows sparse Sudan staining while BN1703.2 has extensive deposition within the 2°X. Black arrowheads indicate Sudan IV stain and scale bars represent 150 and 50 μm for the lower and higher magnification respectively. Micrographs of stem transverse sections 7 days post-inoculation of untransformed (G) and BN1703.2 (H) stems stained with aniline blue. Aniline blue stains callose depositions dark blue-purple with the Periodic Acid-Schiff’s reagent stain staining plant and fungal cell walls bright pink. Untransformed stems show low levels of aniline blue stain in the secondary phloem (2°P) while BN1703.2 stems show heavy staining. Black arrowheads indicate Sclerotinia hyphae, white indicate callose, empty arrowheads indicate the vascular cambium and grey indicate the medullary ray. Scale bars represent 50 μm. All micrographs are representative of at least three biological replicates showing similar phenotypes.