Fig. 1. Interaction phenotypes of B. napus cv. Glacier cotyledons and leaves infiltrated with L. maculans. Cotyledons and leaves were inoculated by infiltration with pycnidiospores from either incompatible (PG2) or compatible (PG3) L. maculans (210⁷ spores/ml) or sterile distilled water. The inoculated lobes of cotyledons are indicated by arrows. Each treatment was repeated at least three times with consistent results. A, Infiltration with a 1-cc syringe. Inset, spread of inoculum in a lobe (left) of a cotyledon is demonstrated using red dye. B, Glacier leaves (top) and cotyledons (bottom), 10 d.p.i. (days postinoculation). C, Time course of interaction phenotype development on Glacier cotyledons. D, Live inocula vs. killed inocula on Glacier cotyledons, 12 d.p.i.

Fig. 2. Germination and hyphal growth of L. maculans in B. napus. Treatments are indicated on the figures. A-E Fluorescence microscopy using calcofluor to stain fungal pycnidiospores and hyphae (arrows). G,H Light microscopy using aniline blue to stain fungal hyphae (arrows).
 

Fig. 3. Brassica PR1 Amino acid sequences using conceptual translation from cDNA sequences. Amino acid sequences of Ypr1.2 identical to those of Ypr1.1 are presented as dots. Alignment gaps are presented as dashes. Acidic amino acid residues are marked by open diamonds and basic ones by solid diamonds.
 

Fig. 4. DNA hybridization analysis of PR1 gene family in B. napus. Westar genomic DNA (20 g) was digested with HindIII, BamHI, EcoRI or DraI. Digests were electrophoresed on a 1.0% agarose gel and blotted onto Nylon membranes. The blot was probed with [³²P]-labeled Ypr1.1 cDNA. The sizes of marker bands are in kb.
 

Fig. 5. Comparison of the induction of PR1 by live and killed inocula in Glacier cotyledons. Total RNA was extracted from cotyledons inoculated with either the live or heat-killed pycnidiospores (2 10⁷ spores/ml) of PG2, PG3 or water 48 h.p.i. and analyzed by RNA hybridization. 10 g of RNA was loaded per lane. A, RNA hybridization with Brassica Ypr1.1 cDNA as probe. B, RNA gel stained with ethidium bromide.
 

Fig. 6. Induction of PR1 mRNA in cotyledons over a 96 hr. timecourse after inoculation with compatible or incompatible L. maculans. Glacier cotyledons were inoculated with pycnidiospores (2 10⁷ spores/ml) of PG2 or PG3. Samples were harvested at various times from 0 to 96 hr.p.i. 10 g of RNA was loaded per lane. A, RNA hybridization with Brassica Ypr1.1 cDNA as probe. B, RNA gel stained with ethidium bromide. C, Relative hybridization signal from densitometry ( water, PG2, PG3, mean of 2 inoculations) with standard error of the mean (vertical lines).
 

Fig. 7. RNA hybridization analysis of induction of PR2 and defensin by PG2 and PG3 in Glacier. Total RNA was extracted from cotyledons inoculated with pycnidiospores (2 10⁷ spores/ml) of PG2, PG3, or water 36 hr.p.i. 10 g of RNA was loaded per lane. The blot was probed by [³²P]-labeled Arabidopsis -1,3-glucanase cDNA (A) and radish defensin cDNA (C). B and D, RNA gel stained with ethidium bromide.
 

Fig. 8. RNA hybridization analysis of induction of PR1 by SA, wounding, and heat shock and constitutive expression of PR1 in Glacier. Total RNA was extracted from PG2-inoculated cotyledons (48 hr.p.i.), healthy cotyledons, mature leaves, flowers, and young siliques, or cotyledons treated with SA (5 mM, 24 hr), wounding (24 hr), or heat shock (40^oC for 2 hr, sampled after 24 hr). 10 g of RNA was loaded per lane. A, RNA hybridization with Brassica Ypr1.1 cDNA as probe. B, RNA gel stained with ethidium bromide.
 

Fig. 9. RNA hybridization analysis of induction of PR1 by different blackleg pathogenicity groups PG1 - PG4 on cultivars Westar, Glacier and Quinta. Interaction phenotypes are represented as follows: -: null; NHR: non-host resistance; R: single gene resistance; S: - susceptibility. Total RNA was extracted from leaves inoculated with pycnidiospores (2 10⁷ spores/ml) of PR1, PG2, PG3, PG4 or water 36 h.p.i. 10 g of RNA was loaded per lane. Brassica Ypr1.1 cDNA was used as probe. RNA staining in ethidium bromide is shown below the autoradiogram.