Figure 1.
Identification of SNPs linked to Avr1c by bulked segregant analysis and deep sequencing.
A, The procedure for discovery of candidate SNPs linked to Avr1c is shown. Selected F2 progeny from a cross of P. sojae strains ACR10×P7076 were pooled according to their virulence phenotype. The avirulent (A) Pool1 and virulent (V) Pool2 composite DNA samples were deeply sequenced. Sequence reads were aligned against the reference genome, and SNPs were identified and filtered based upon quality scores. High quality SNPs were further filtered according to the predicted SNP frequencies for Pool1 and Pool2. After processing, only three SNPs passed all requirements. B, Genome location of three candidate SNPs. These three candidate SNPs occur in close physical proximity in the reference genome assembly. All three sites fall within a 92 kb segment on Scaffold_7 (V5.0). Reference (Ref) allele and alternate (Alt) alleles for three SNPs are shown. The SNP frequencies (Freq) in each pool are also shown.
Table 1.
Whole genome re-sequencing of composite and strain specific P. sojae libraries.
Figure 2.
Genetic and physical mapping of Avr1c region.
A, Physical map of Avr1c region. The position of the three identified SNP markers, Avr1a, and 25 predicted Avh genes are shown. B, Genetic analysis of the Avr1c region. The position of DNA markers and their recombination frequency (RF%) with Avr1c in a segregating F2 population (n = 28) is indicated; Mbp, mega base pair. C, Predicted arrangement of the Avr1a locus in P. sojae reference strain P6497.
Figure 3.
Genomic DNA blot hybridization showing P. sojae strain-specific deletions of Avr1a and Avr1c.
Selected strains of P. sojae genomic DNA were digested with PmlI and separated by electrophoresis prior to blotting and hybridization. Virulence phenotype of the P. sojae strains on Rps1c or Rps1a plants is shown as virulent (V) or avirulent (A). The positions and sizes of the PmlI segments of Avr1a, Avr1c, and Avh72 genes are indicated on the right. The sizes of DNA markers are shown on the left; kb, kilo base pair. The Avr1c gene is deleted from parental strain P7076.
Figure 4.
Sequence read coverage of the Avr1a/Avr1c region from re-sequencing of P. sojae strains ACR10 and P7076.
An assembly of the Avr1a region from reference strain P6497 was used to align sequence reads from parental strains ACR10 and P7076. The DNA segment corresponding to the Avr1c gene lacks sequence reads in parental strain P7076. The longest segment of identity between the Avr1a and Avr1c DNA sequences is 98 nucleotides, which is shorter than the read length of 100 nucleotides.
Figure 5.
Amino acid sequence alignment of the predicted proteins for Avr1a and each of the three alleles of Avr1c.
The residues are colored according to their physicochemical properties. Signal peptide, RXLR and dEER motifs, and WY-domain are shown, and polymorphic residues among the three Avr1c alleles are underlined. An asterisk (*) indicates positions which have a single, fully conserved residue; a colon (:) indicates conservation between groups of strongly similar properties; a period (.) indicates conservation between groups of weakly similar properties.
Figure 6.
Analysis of alleles of Avr1c, and transcripts of Avr1c and Avr1a in P. sojae strains.
Shown at the top of the figure are the Avr1c alleles present in each of the selected P. sojae strains. Virulence phenotype of the P. sojae strains on Rps1c or Rps1a plants is shown as virulent (V) or avirulent (A). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed using mRNA from mycelia cultures, and Avr1a and Avr1c specific primers, to test for transcripts of these two genes. Results from RT-PCR of the control gene Actin are also shown.
Figure 7.
Transient expression of Avr1c and Avr1a triggers cell death in Rps1c soybean plants.
Measurement of cell death in soybean leaves by co-bombardment and transient expression of a GUS reporter together with a test gene. Test genes that cause cell death reduce GUS expression and blue staining. A, Leaves of Williams (rps) and the isoline L75-3735 (Rps1c) tested with each of the three Avr1c alleles, and Avr1a. B, Leaves of Williams (rps) and the isoline L75-6141 (Rps1a) tested with each of the three Avr1c alleles. Control test gene in each experiment corresponds to a synthetic Avr1a sequence with a frame-shift mutation. Results show means and standard errors of three independent biological replicates, with a minimum of three leaves per treatment, per replicate.
Figure 8.
Photographs of leaves of Williams (rps) and the isoline L75-3735 (Rps1c) tested with each of the three Avr1c alleles, and Avr1a.
Results from a representative co-bombardment experiment are shown, after staining for GUS expression.
Figure 9.
Photographs of leaves of Williams (rps) and the isoline L75-6141 (Rps1a) tested with each of the three Avr1c alleles.
Results from a representative co-bombardment experiment are shown, after staining for GUS expression.
Table 2.
Rps1c is the most prevalent known source of P. sojae resistance in soybean lines entered in Ontario variety trials1.
Figure 10.
A model for the structure and expression of the Avr1a/1c locus in P. sojae strains.
Results from the present study are summarized in this illustration of the Avr1a/1c locus. Bold arrows indicate predicted genes; Avh72 is shown with a dashed line because this is predicted to be a pseudogene due to a frame shift mutation. For Avr1c and Avr1a, an asterisk (*) indicates gene-silencing. Disease outcome on Rps1c and Rps1a soybean plants is shown: avirulent (A); or virulent (V). Gene silencing or gene deletion can account for gain of virulence in the P. sojae strains shown here, except for strain ACR9. It is not known how ACR9 evades Rps1c recognition, but an epistatic effector that suppresses the Avr1c-Rps1c interaction could explain the result.