Dataset Information

Transcript profiling of common bean (Phaseolus vulgaris L.) using the GeneChip Soybean Genome Array: optimizing analysis by masking biased probes.

ABSTRACT: BACKGROUND: Common bean (Phaseolus vulgaris L.) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. This suggests that the GeneChip(R) Soybean Genome Array (soybean GeneChip) may be used for gene expression studies using common bean. RESULTS: To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and accuracy of measuring differential gene expression in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and accuracy of measuring differential gene expression. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR (qRT-PCR) analysis of 20 randomly selected genes and purine-ureide pathway genes demonstrated an increased accuracy of measuring differential gene expression after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The sequence divergence pattern analysis suggested that the genes for basic cellular functions and metabolism were highly conserved between soybean and common bean. Additionally, our results show that some classes of genes, particularly those associated with environmental adaptation, are highly divergent. CONCLUSIONS: The soybean GeneChip is a suitable cross-species platform for transcript profiling in common bean when used in combination with the masking protocol described. In addition to transcript profiling, CSH of the GeneChip in combination with masking probes in the ISV regions can be used for comparative ecological and/or evolutionary genomics studies.

SUBMITTER: Yang SS

PROVIDER: S-EPMC3017814 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Transcript profiling of common bean (Phaseolus vulgaris L.) using the GeneChip Soybean Genome Array: optimizing analysis by masking biased probes.

Yang S Samuel SS Valdés-López Oswaldo O Xu Wayne W WW Bucciarelli Bruna B Gronwald John W JW Hernández Georgina G Vance Carroll P CP

BMC plant biology 20100507

<h4>Background</h4>Common bean (Phaseolus vulgaris L.) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. This suggests that the GeneChip(R) Soybean Genome Array (soybean GeneChip) may be used for gene expression studies using common bean.<h4>Results</h4>To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate ...[more]

PMID: 20459672

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Project description:BACKGROUND: The GeneChip(R) Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa [Medicago sativa (L.) subsp. sativa]. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression. RESULTS: Transcript profiling using the Medicago GeneChip(R) was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose and lignin. A protocol was developed that masked probes targeting inter-species variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold was selected that optimized both sensitivity and accuracy. After masking for both ISV regions and previously identified single-feature polymorphisms (SFPs), the number of differentially expressed genes between the two genotypes in both ES and PES internodes was approximately 2-fold greater than the number detected prior to masking. Regulatory genes, including transcription factor and receptor kinase genes that may play a role in development of secondary xylem, were significantly over-represented among genes up-regulated in 252 PES internodes compared to 1283 PES internodes. Several cell wall-related genes were also up-regulated in genotype 252 PES internodes. Real-time quantitative RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000 genes that were differentially expressed in ES and PES internodes of genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of the differentially expressed genes suggested co-expression of some neighbouring genes on Medicago chromosomes. CONCLUSIONS: The problems associated with transcript profiling in alfalfa stems using the Medicago GeneChip as a CSH platform were mitigated by masking probes targeting ISV regions and SFPs. Using this masking protocol resulted in the identification of numerous candidate genes that may contribute to differences in cell wall concentration and composition of stems of two alfalfa genotypes.

Project description:TIFY is a large plant-specific transcription factor gene family. A subgroup of TIFY genes named JAZ (Jasmonate-ZIM domain) has been identified as repressors of jasmonate (JA)-regulated transcription in Arabidopsis and other plants. JA signaling is involved in many aspects of plant growth/development and in the defense responses to biotic and abiotic stresses. Here we identified the TIFY genes (designated as PvTIFY) from the legume common bean (Phaseolus vulgaris) and functionally characterized PvTIFY10C as a transcriptional regulator. Twenty-three genes from the PvTIFY gene family were identified through whole genome sequence analysis. Most of these were induced upon methyl-JA elicitation. We selected PvTIFY10C as a representative JA-responsive PvTIFY gene for further functional analysis. Transcriptome analysis via microarray hybridization using the designed Bean Custom Array 90K was performed in transgenic roots of composite plants with modulated -RNAi-silencing or over-expression- PvTIFY10C gene expression. Data were interpreted using Mapman adapted to common bean. Microarray differential gene expression data were validated by real-time qRT-PCR expression analysis. Comparative global gene expression analysis revealed opposite regulatory changes in processes such as RNA and protein regulation, stress response and metabolism in silenced vs. over-expressing roots. These data point to transcript reprogramming -mainly repression- orchestrated by PvTIFY10C. In addition we found that several PvTIFY genes as well as genes from the JA biosynthetic pathway responded to P-deficiency. Relevant P-responsive genes that participate in carbon metabolic pathways, cell wall synthesis, lipid metabolism, transport, DNA, RNA and protein regulation, signaling, were oppositely-regulated in control vs. PvTIFY10C silenced roots. These data indicate that PvTIFY10C regulates, directly or indirectly, gene expression of some P-responsive genes something that could be mediated by JA-signaling.

Project description:BackgroundTIFY is a large plant-specific transcription factor gene family. A subgroup of TIFY genes named JAZ (Jasmonate-ZIM domain) has been identified as repressors of jasmonate (JA)-regulated transcription in Arabidopsis and other plants. JA signaling is involved in many aspects of plant growth/development and in defense responses to biotic and abiotic stresses. Here, we identified the TIFY genes (designated PvTIFY) from the legume common bean (Phaseolus vulgaris) and functionally characterized PvTIFY10C as a transcriptional regulator.ResultsNineteen genes from the PvTIFY gene family were identified through whole-genome sequence analysis. Most of these were induced upon methyl-JA elicitation. We selected PvTIFY10C as a representative JA-responsive PvTIFY gene for further functional analysis. Transcriptome analysis via microarray hybridization using the newly designed Bean Custom Array 90 K was performed on transgenic roots of composite plants with modulated (RNAi-silencing or over-expression) PvTIFY10C gene expression. Data were interpreted using Gene Ontology and MapMan adapted to common bean. Microarray differential gene expression data were validated by real-time qRT-PCR expression analysis. Comparative global gene expression analysis revealed opposite regulatory changes in processes such as RNA and protein regulation, stress responses and metabolism in PvTIFY10C silenced vs. over-expressing roots. These data point to transcript reprogramming (mainly repression) orchestrated by PvTIFY10C. In addition, we found that several PvTIFY genes, as well as genes from the JA biosynthetic pathway, responded to P-deficiency. Relevant P-responsive genes that participate in carbon metabolic pathways, cell wall synthesis, lipid metabolism, transport, DNA, RNA and protein regulation, and signaling were oppositely-regulated in control vs. PvTIFY10C-silenced roots of composite plants under P-stress. These data indicate that PvTIFY10C regulates, directly or indirectly, the expression of some P-responsive genes; this process could be mediated by JA-signaling.ConclusionOur work contributes to the functional characterization of PvTIFY transcriptional regulators in common bean, an agronomically important legume. Members from the large PvTIFY gene family are important global transcriptional regulators that could participate as repressors in the JA signaling pathway. In addition, we propose that the JA-signaling pathway involving PvTIFY genes might play a role in regulating the plant response/adaptation to P-starvation.

Project description:Common bean (Phaseolus vulgaris L.) is a staple food in Brazil with both nutritional and socioeconomic importance. As an orphan crop, it has not received as much research attention as the commodity crops. Crop losses are strongly related to virus diseases transmitted by the whitefly Bemisia tabaci, one of the most important agricultural pests in the world. The main method of managing whitefly-transmitted viruses has been the application of insecticides to reduce vector populations. Compared to chemical vector control, a more sustainable strategy for managing insect-borne viruses is the development of resistant/tolerant cultivars. RNA interference has been applied to develop plant lines resistant to the whitefly in other species, such as tomato, lettuce and tobacco. Still, no whitefly-resistant plant has been made commercially available to date. Common bean is a recalcitrant species to in vitro regeneration; therefore, stable genetic transformation of this plant has been achieved only at low frequencies (<1%) using particle bombardment. In the present work, two transgenic common bean lines were obtained with an intron-hairpin construct to induce post-transcriptional gene silencing against the B. tabaci vATPase (Bt-vATPase) gene, with stable expression of siRNA. Northern blot analysis revealed the presence of bands of expected size for siRNA in leaf samples of the line Bt-22.5, while in the other line (11.5), the amount of siRNA produced was significantly smaller. Bioassays were conducted with both lines, but only the line Bt-22.5 was associated with significant mortality of adult insects (97% when insects were fed on detached leaves and 59% on the whole plant). The expression of the Bt-vATPase gene was 50% lower (p < 0.05) in insects that fed on the transgenic line Bt-22.5, when compared to non-transgenic controls. The transgenic line did not affect the virus transmission ability of the insects. Moreover, no effect was observed on the reproduction of non-target organisms, such as the black aphid Aphis craccivora, the leafminer Liriomyza sp. and the whitefly parasitoid Encarsia formosa. The results presented here serve as a basis for the development of whitefly-tolerant transgenic elite common bean cultivars, with potential to contribute to the management of the whitefly and virus diseases.

Dataset Information

Transcript profiling of common bean (Phaseolus vulgaris L.) using the GeneChip Soybean Genome Array: optimizing analysis by masking biased probes.

Publications

Transcript profiling of common bean (Phaseolus vulgaris L.) using the GeneChip Soybean Genome Array: optimizing analysis by masking biased probes.

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