Supporting Information Results
Soybean MTI may be regulated through alternative splicing
In order to investigate the possible role of alternative splicing in controlling the soybean MTI response, an exon-alternative splicing analysis was performed. Based on the MIDAS p-value < 10E-10, 229 potential spliced exons present in 336 genes were identified (Figure 1). Likewise, a clear variation in the splicing events (both skipped- and retained-exons) among the four genotypes was detected. However, only a few of these examples appeared to be modified by MAMP treatment (Figure 1), whereas the remainders were genotype dependent (Figure 1). Three kind of splicing events were detected by comparing all four genotypes: 1) the spliced-exon was retained only in the susceptible genotypes and skipped in the resistant genotypes; 2) the opposite case to that of #1, and 3) the spliced-exon was retained or skipped in all genotypes (Figure 1). Among the genes with spliced-exons, different receptors (LRR, NB-ARC), protein kinases, calmodulins and transcription factors, mainly members of the WRKY and MYB family were indentified.
´ In order to validate the predictions of the MIDAS software, the expression of three randomly-selected genes was analyzed via RT-PCR (Figure 2). For this analysis, primers were designed to the flanking constitutive exons of each selected gene (Figure 2A). In all cases, the putative splicing event predicted for the selected genes was independently confirmed (Figure 2B-C). Interestingly, this analysis also revealed the presence in one TIR-gene of additional alternative splicing events, one of which that was MAMP responsive (Figure 2B). Although not investigated further, these data suggest that MTI induced changes in specific gene splicing could play a significant role in determining genotypic differences in the basal immunity response.
Figure 1: Alternative Splicing events in four soybean genotypes that have divergent MTI response. A) Alternative splicing triggered by MAMP treatment. One of the exon of the gene Glyma14g05350 is absent (MIDAS P-Value 3.48 E-11) after MAMP treatment. B-E) Alternative splicing dependent of the genotype. B) One exon of the gene Glyma18g43510 is absent (MIDAS P-value: 3.71 E-19) only in LDX01-1-65 (LX). C) One exon from the gene is present only (MIDAS P-value 4.45 E-17) in LDX01-1-65. D) One exon from the gene Glyma18g04980 is present only (MIDAS P-Value 1.006 E-13) in LD00-2817P (LD). E) One exon is absent (MIDAS P-Value 2.06 E-22) in LD00-2817 and LDX01-1-65, but present in Ripley (Ri) and EF 59 (EF). Red lines represent statistically significant alternative-spliced exons, whereas gray represent non alternative-spliced exon from the same gene. C30: Mock treatment at 30 min. P30: MAMP treatment at 30 minutes. Each figures show three biological replicates for each genotype.
Fig. 2 RT-PCR validation of potential splicing events in LD00-2817P (LD) and LDX01-165 (LX), two of the most contrasting soybean genotypes at the transcriptional level. (A) Schematic representation of the strategy used to for experimental validation of the potential splicing variant due to retained- or skipped-exons. Primers (in arrows) were designed into the border constitutive exons (white boxes). Alternative spliced exons are represented in red box; dotted line represents the expected results using this strategy. In B-C, each leaf panel shows the predicted expression pattern of constitutive- (gray lines) and spliced-exons (red lines) belong to the randomly selected genes [LRR (B), Unknown protein (C)], whereas the right panel shows the experimental validation by RT-PCR. The PCR product size predicted was 750 bp and 850 bp in panel B and C, respectively. Each panel shows the RT-PCR results from two independently biological replicates.