Figure 1.
(A) The scale shows the number of recombinant individuals in the BC mapping population (n = 106) at markers (M) identified in a chromosome walk (W). The walk proceeded from marker 134 towards marker 124 and was composed of BACs (grey boxes) and FPC-based BAC contigs (blue boxes). (B) Fluorescence in situ positions of markers 124, Hf5p7 and 134 on the short arm of HF polytene chromosome X2. The arrowhead indicates the position of the X2 centromere.
Figure 2.
Mapping vH13 within BAC Hf5p7.
(A) Map showing the positions of the molecular markers (a-i) that were used to refine the position of vH13 on BAC Hf5p7 (scale = kb). Predicted genes are shown below the map. Genes transcribed from left-to-right are colored dark grey and genes transcribed from right-to-left are colored light grey. Asterisks indicate genes encoding predicted signal peptides. (B) Table showing the numbers of recombinant individuals within structured mapping populations (BC and RIL) and field populations (LA, AL, GA and SC) at each of the markers (a-i) shown in A.
Figure 3.
vH13 candidate gene 13 structure and expression in H13-virulent and avirulent strains.
(A) H13-avirulent genomic DNA sequence of vH13 candidate-13 showing exons (capital letters), intron (lower case letters), PCR primer-targeted sites (bold underlining), the positions of virulence-associated insertions (triangles 1, 2 and 3) and the predicted amino acid sequence (bold letters). The predicted signal peptide is boxed and the three imperfect direct repeats are underlined with arrows. (B) Candidate-13 fragments amplified using genomic DNA template extracted from H13-virulent (v) and H13-avirulent (a) individuals. H13-virulence associated sequences correspond to the insertions (1, 2 and 3) shown in panel A. For an explanation of the band lengths, see Figure S3. (C) Candidate-13 (13) and candidate-14 (14) transcripts amplified using total RNA extracted from pools of first-instar larvae (KS-GP, lane 1; IN-L, lane 2; vH13, lane 3 and IN-vH9, lane 4). Only candidate-14 sequence was amplified using the RNA extracted from the pool of H13-virulent first-instar (vH13, lane 3). Genomic DNA extracted from a single KS-GP larva was amplified as a control (lane 5). (D) Amplification of candidate-13 (13) and HF-ubiquitin (U) gene sequences using total RNA extracted from pools of H13-avirulent first-instars (lane 1), second-instars (lane 2), third-instars (lane 3), first-instar salivary glands (lane 4), and the carcases of first-instar larvae after salivary gland removal (lane 5).
Figure 4.
vH13 knockdown allows H13-avirulent larvae to escape H13-directed ETI.
Pools of 100 H13-avirulent neonate larvae were soaked in 0.5 mg/ml of either sham-, or vH13-dsRNA for 48 h. (A) Percent transcription of vH13 in vH13-dsRNA-treated larvae (t) relative to sham-treated larvae (c) as measured using qRT-PCR. (B) Amplification of the vH13 transcript (13-1 and 13-2) and the ubiquitin transcript (U) from RNA samples extracted from sham-treated (c) and vH13-treated (t) larvae after 35 cycles of RT-PCR. Ubiquitin transcript amplification was performed using the same RNA used in 13-1. (C-H) Similarly treated larvae were transferred, five per plant, to H13-resistant (Molly), or susceptible (Newton) near-isogenic wheat seedlings. Plants shown 12 days after infestation (C, D, and E) have their leaves numbered. Stunted plants (D and E) were darker green than unstunted plants (C) and never developed a fourth leaf. HF pupae (arrows) were visible on stunted plants 20 days after infestation (F, G and H). Sham-treated larvae failed to stunt (C) and survive (F) on Molly, but did stunt (D) and survive (G) on Newton. Some candidate-gene-13-dsRNA-treated larvae also stunted (E) and survived (H) on Molly.