Limits...
Infection and genotype remodel the entire soybean transcriptome.

Zhou L, Mideros SX, Bao L, Hanlon R, Arredondo FD, Tripathy S, Krampis K, Jerauld A, Evans C, St Martin SK, Maroof MA, Hoeschele I, Dorrance AE, Tyler BM - BMC Genomics (2009)

Bottom Line: However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge.We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%.Our results are consistent through two different normalization methods and two different statistical analysis procedures.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. lzhou@vbi.vt.edu

ABSTRACT

Background: High throughput methods, such as high density oligonucleotide microarray measurements of mRNA levels, are popular and critical to genome scale analysis and systems biology. However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge. Many researchers still use an arbitrary cut off such as two-fold in order to identify changes that may be biologically significant. We have used a very large-scale microarray experiment involving 72 biological replicates to analyze the response of soybean plants to infection by the pathogen Phytophthora sojae and to analyze transcriptional modulation as a result of genotypic variation.

Results: With the unprecedented level of statistical sensitivity provided by the high degree of replication, we show unambiguously that almost the entire plant genome (97 to 99% of all detectable genes) undergoes transcriptional modulation in response to infection and genetic variation. The majority of the transcriptional differences are less than two-fold in magnitude. We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%. Our results are consistent through two different normalization methods and two different statistical analysis procedures.

Conclusion: Our findings demonstrate that the entire plant genome undergoes transcriptional modulation in response to infection and genetic variation. The pervasive low-magnitude remodeling of the transcriptome may be an integral component of physiological adaptation in soybean, and in all eukaryotes.

Show MeSH

Related in: MedlinePlus

Distribution of gene expression changes for different contrasts. Only genes with significant changes (TST-FDR-adjusted p ≤ 0.01) are included. A negative fold-change indicates a reduction by that factor. (A-D) Infection responses in resistant genotype V71-370 and susceptible genotype Sloan. "Upper" and "lower" denote infection courts as described in the text and the methods. Distributions of gene expression changes for VPRIL9 infection responses are shown [see Additional file 4]. (E) Gene expression differences between V71-370 and Sloan following mock inoculation; genes with higher mRNA levels in V71-370 or Sloan are shown as having a positive or negative differences, respectively. For (A)-(E), the fold changes were calculated using LMMA contrast analysis using GC-RMA normalized data. (F) Differences among 4,453 genes with significant responses to time of harvest in mock-inoculated Sloan plants: genes with higher mRNA levels in 12 noon samples or 9 am samples are shown as having a positive or negative differences, respectively. The fold changes were calculated from the average difference of the two treatments using the GC-RMA normalized data. The mean, median and mode of each distribution were calculated using the log-fold changes. The bin size of the mode was 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2662884&req=5

Figure 2: Distribution of gene expression changes for different contrasts. Only genes with significant changes (TST-FDR-adjusted p ≤ 0.01) are included. A negative fold-change indicates a reduction by that factor. (A-D) Infection responses in resistant genotype V71-370 and susceptible genotype Sloan. "Upper" and "lower" denote infection courts as described in the text and the methods. Distributions of gene expression changes for VPRIL9 infection responses are shown [see Additional file 4]. (E) Gene expression differences between V71-370 and Sloan following mock inoculation; genes with higher mRNA levels in V71-370 or Sloan are shown as having a positive or negative differences, respectively. For (A)-(E), the fold changes were calculated using LMMA contrast analysis using GC-RMA normalized data. (F) Differences among 4,453 genes with significant responses to time of harvest in mock-inoculated Sloan plants: genes with higher mRNA levels in 12 noon samples or 9 am samples are shown as having a positive or negative differences, respectively. The fold changes were calculated from the average difference of the two treatments using the GC-RMA normalized data. The mean, median and mode of each distribution were calculated using the log-fold changes. The bin size of the mode was 0.01.

Mentions: Many researchers use two-fold change as an arbitrary cutoff. Our results show that 13.1 to 23.5% of the significantly changed genes in the upper infection court and 29.8% to 36.8% in the lower infection court changed by two-fold or greater in the three tested genotypes (Figure 2) [see Additional file 4]. However, 8.2 to 15.0% of the genes with significantly changes showed only subtle changes (fold change of less than 1.2-fold). To examine the functional significance of gene expression changes of various magnitudes, we plotted the distribution of changes for genes in six functional categories relevant to infection, namely defense and disease, signal transduction, transcription, intracellular trafficking, cell structure and metabolism (Figure 3) [see Additional files 5 to 7]. In the lower infection court of the susceptible cultivar, Sloan, which had the greatest level of infection, 23,556 genes showed significant changes in response to infection. Compared to the entire set of modulated genes, the distribution of changes was significantly different for all six categories (p ≤ 0.01, Kolmogorov-Smirnov test for two samples) (Figure 3). The disease and defense category and to a lesser extent metabolism showed a bias towards strongly up-regulated genes, whereas transcription and to a lesser extent signal transduction and intracellular trafficking, showed a bias towards down-regulation of genes. Similar patterns were observed in the upper infection court of Sloan and in both courts of the resistant cultivar V71-370 [see Additional files 5 to 8]. To test if significant changes in distribution were present among genes showing low magnitude modulation, the distribution comparison was restricted to genes showing less than two-fold or less than 1.5-fold modulation. Five of the categories (all except disease and defense), and three categories (transcription, cell structure and metabolism), respectively, showed significantly different distributions from the overall gene set (p ≤ 0.01). Even among genes with less than a 1.2-fold change, those in the transcription and metabolism categories still showed significantly different distributions (p ≤ 0.01) (Table 6) [see Additional file 8].


Infection and genotype remodel the entire soybean transcriptome.

Zhou L, Mideros SX, Bao L, Hanlon R, Arredondo FD, Tripathy S, Krampis K, Jerauld A, Evans C, St Martin SK, Maroof MA, Hoeschele I, Dorrance AE, Tyler BM - BMC Genomics (2009)

Distribution of gene expression changes for different contrasts. Only genes with significant changes (TST-FDR-adjusted p ≤ 0.01) are included. A negative fold-change indicates a reduction by that factor. (A-D) Infection responses in resistant genotype V71-370 and susceptible genotype Sloan. "Upper" and "lower" denote infection courts as described in the text and the methods. Distributions of gene expression changes for VPRIL9 infection responses are shown [see Additional file 4]. (E) Gene expression differences between V71-370 and Sloan following mock inoculation; genes with higher mRNA levels in V71-370 or Sloan are shown as having a positive or negative differences, respectively. For (A)-(E), the fold changes were calculated using LMMA contrast analysis using GC-RMA normalized data. (F) Differences among 4,453 genes with significant responses to time of harvest in mock-inoculated Sloan plants: genes with higher mRNA levels in 12 noon samples or 9 am samples are shown as having a positive or negative differences, respectively. The fold changes were calculated from the average difference of the two treatments using the GC-RMA normalized data. The mean, median and mode of each distribution were calculated using the log-fold changes. The bin size of the mode was 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662884&req=5

Figure 2: Distribution of gene expression changes for different contrasts. Only genes with significant changes (TST-FDR-adjusted p ≤ 0.01) are included. A negative fold-change indicates a reduction by that factor. (A-D) Infection responses in resistant genotype V71-370 and susceptible genotype Sloan. "Upper" and "lower" denote infection courts as described in the text and the methods. Distributions of gene expression changes for VPRIL9 infection responses are shown [see Additional file 4]. (E) Gene expression differences between V71-370 and Sloan following mock inoculation; genes with higher mRNA levels in V71-370 or Sloan are shown as having a positive or negative differences, respectively. For (A)-(E), the fold changes were calculated using LMMA contrast analysis using GC-RMA normalized data. (F) Differences among 4,453 genes with significant responses to time of harvest in mock-inoculated Sloan plants: genes with higher mRNA levels in 12 noon samples or 9 am samples are shown as having a positive or negative differences, respectively. The fold changes were calculated from the average difference of the two treatments using the GC-RMA normalized data. The mean, median and mode of each distribution were calculated using the log-fold changes. The bin size of the mode was 0.01.
Mentions: Many researchers use two-fold change as an arbitrary cutoff. Our results show that 13.1 to 23.5% of the significantly changed genes in the upper infection court and 29.8% to 36.8% in the lower infection court changed by two-fold or greater in the three tested genotypes (Figure 2) [see Additional file 4]. However, 8.2 to 15.0% of the genes with significantly changes showed only subtle changes (fold change of less than 1.2-fold). To examine the functional significance of gene expression changes of various magnitudes, we plotted the distribution of changes for genes in six functional categories relevant to infection, namely defense and disease, signal transduction, transcription, intracellular trafficking, cell structure and metabolism (Figure 3) [see Additional files 5 to 7]. In the lower infection court of the susceptible cultivar, Sloan, which had the greatest level of infection, 23,556 genes showed significant changes in response to infection. Compared to the entire set of modulated genes, the distribution of changes was significantly different for all six categories (p ≤ 0.01, Kolmogorov-Smirnov test for two samples) (Figure 3). The disease and defense category and to a lesser extent metabolism showed a bias towards strongly up-regulated genes, whereas transcription and to a lesser extent signal transduction and intracellular trafficking, showed a bias towards down-regulation of genes. Similar patterns were observed in the upper infection court of Sloan and in both courts of the resistant cultivar V71-370 [see Additional files 5 to 8]. To test if significant changes in distribution were present among genes showing low magnitude modulation, the distribution comparison was restricted to genes showing less than two-fold or less than 1.5-fold modulation. Five of the categories (all except disease and defense), and three categories (transcription, cell structure and metabolism), respectively, showed significantly different distributions from the overall gene set (p ≤ 0.01). Even among genes with less than a 1.2-fold change, those in the transcription and metabolism categories still showed significantly different distributions (p ≤ 0.01) (Table 6) [see Additional file 8].

Bottom Line: However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge.We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%.Our results are consistent through two different normalization methods and two different statistical analysis procedures.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. lzhou@vbi.vt.edu

ABSTRACT

Background: High throughput methods, such as high density oligonucleotide microarray measurements of mRNA levels, are popular and critical to genome scale analysis and systems biology. However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge. Many researchers still use an arbitrary cut off such as two-fold in order to identify changes that may be biologically significant. We have used a very large-scale microarray experiment involving 72 biological replicates to analyze the response of soybean plants to infection by the pathogen Phytophthora sojae and to analyze transcriptional modulation as a result of genotypic variation.

Results: With the unprecedented level of statistical sensitivity provided by the high degree of replication, we show unambiguously that almost the entire plant genome (97 to 99% of all detectable genes) undergoes transcriptional modulation in response to infection and genetic variation. The majority of the transcriptional differences are less than two-fold in magnitude. We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%. Our results are consistent through two different normalization methods and two different statistical analysis procedures.

Conclusion: Our findings demonstrate that the entire plant genome undergoes transcriptional modulation in response to infection and genetic variation. The pervasive low-magnitude remodeling of the transcriptome may be an integral component of physiological adaptation in soybean, and in all eukaryotes.

Show MeSH
Related in: MedlinePlus