High-throughput sequencing of small RNAs associated with Chalcone Synthase A (CHS-A) sense cosuppression in petunia
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ABSTRACT:
ORGANISM(S): Petunia x hybrida
PROVIDER: GSE13764 | GEO | 2009/09/28
SECONDARY ACCESSION(S): PRJNA141675
REPOSITORIES: GEO
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Project description:We report the application of sequencing-by-synthesis technology for high-throughput profiling of small RNAs involved in Chalcone synthase A (CHS-A) sense cosuppression in petunia. Sense cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a transgene overexpressing the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. Though sense cosuppression is recognized as an RNA silencing mechanism, little evidence has been yet provided demonstrating its association with the generation of individual small interfering RNAs (siRNAs) that are the assumed determinants. In this work, the deep sequencing of small RNAs in cosuppressed transgenic petunia and WT petunia respectively allowed for the identication of siRNAs that vastly predominate in the silenced flower and guide prominent cleavage events in CHS-A mRNA. Examination of 2 small RNA populations from WT and cosuppressed petunia petals respectively
2009-09-27 | E-GEOD-13764 | biostudies-arrayexpress
Project description:Background: Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. Results: We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. Conclusions: The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena. Small RNAs isolated from petal tissues in two petunia lines were analyzed.
2013-12-14 | E-GEOD-42965 | biostudies-arrayexpress
Project description:Background: Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. Results: We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. Conclusions: The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena.
2013-12-14 | GSE42965 | GEO
Project description:We present results from deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the seed coats of the yellow varieties having either the dominant I or i-i alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or i-i alleles thus demonstrating the novelty of an endogenous inverted repeat region of CHS genes driving RNA silencing in trans of non-linked CHS family members in a tissue-specific manner. The phenomenon results in inhibition of a metabolic pathway by siRNAs in one tissue allowing expression of the flavonoid pathway and synthesis of secondary metabolites in other organs as the chalcone synthase small RNAs are found in the seed coats of yellow seeded soybean varieties but not in the cotyledons of the same genotype. In order to compare the population of chalcone synthase related small RNAs, we sequenced 3 to 6 million small RNAs using the Illumina Genome Analyzer from the following four soybean cultivars and tissues with specific genotypes at the I locus: Richland immature seed coats (homozygous for the dominant I allele that specifies yellow seed coat); Williams immature seed coats (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum) Williams (i-i/i-i yellow) immature cotyledons (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum); Williams 55 immature seed coats (a Williams isogenic line homozygous for the recessive i allele that specifics pigmented seed coats. All seed coats and cotyledons were dissected from green stage immature seeds within the fresh weight range of 50-75 mg.
2010-05-13 | E-GEOD-21825 | biostudies-arrayexpress
Project description:We present results from deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the seed coats of the yellow varieties having either the dominant I or i-i alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or i-i alleles thus demonstrating the novelty of an endogenous inverted repeat region of CHS genes driving RNA silencing in trans of non-linked CHS family members in a tissue-specific manner. The phenomenon results in inhibition of a metabolic pathway by siRNAs in one tissue allowing expression of the flavonoid pathway and synthesis of secondary metabolites in other organs as the chalcone synthase small RNAs are found in the seed coats of yellow seeded soybean varieties but not in the cotyledons of the same genotype.
2010-05-14 | GSE21825 | GEO
Project description:The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat. High-throughput sequencing using Genome Analyzer II and Illumina HiSeq 2000 was performed with two biological replicates (Some stages don't have replicate).
2014-02-13 | E-GEOD-43348 | biostudies-arrayexpress
Project description:The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat.
2014-02-13 | GSE43348 | GEO
Project description:The soybean (Glycine max) seed coat has distinctive, genetically programmed patterns of pigmentation and the recessive k1 mutation can epistatically overcome the dominant I and i-i alleles, which inhibit seed color by producing small interfering RNAs (siRNAs) targeting chalcone synthase (CHS) mRNAs. Small RNA sequencing of dissected regions of immature seed coats demonstrated that CHS siRNA levels cause the patterns produced by the i-i and i-k alleles of the I locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. To identify the K1 locus, we compared RNA-Seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by CHS siRNAs but different genotypes (homozygous i-k K1 versus homozygous i-i k1). By examining differentially expressed genes, mapping information, and genome resequencing, we identified a 129-bp deletion in Glyma.11G190900 encoding Argonaute5 (AGO5), a member of the Argonaute family. Amplicon sequencing of several independent saddle pattern mutants from different genetic backgrounds revealed independent lesions affecting AGO5, thus establishing Glyma.11G190900 as the K1 locus. Non-functional AGO5 from k1 alleles leads to altered distributions of CHS siRNAs, thus explaining how the k1 mutation reverses the phenotype of the seed coat regions from yellow to pigmented, even in the presence of the normally dominant I or i-i alleles.
2017-03-30 | GSE89126 | GEO
Project description:The soybean (Glycine max) seed coat has distinctive, genetically programmed patterns of pigmentation and the recessive k1 mutation can epistatically overcome the dominant I and i-i alleles, which inhibit seed color by producing small interfering RNAs (siRNAs) targeting chalcone synthase (CHS) mRNAs. Small RNA sequencing of dissected regions of immature seed coats demonstrated that CHS siRNA levels cause the patterns produced by the i-i and i-k alleles of the I locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. To identify the K1 locus, we compared RNA-Seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by CHS siRNAs but different genotypes (homozygous i-k K1 versus homozygous i-i k1). By examining differentially expressed genes, mapping information, and genome resequencing, we identified a 129-bp deletion in Glyma.11G190900 encoding Argonaute5 (AGO5), a member of the Argonaute family. Amplicon sequencing of several independent saddle pattern mutants from different genetic backgrounds revealed independent lesions affecting AGO5, thus establishing Glyma.11G190900 as the K1 locus. Non-functional AGO5 from k1 alleles leads to altered distributions of CHS siRNAs, thus explaining how the k1 mutation reverses the phenotype of the seed coat regions from yellow to pigmented, even in the presence of the normally dominant I or i-i alleles.
2017-03-30 | GSE43347 | GEO
Project description:Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript amplifying the silencing signal in plants. 22nt siRNAs are under-represented due to competition with DCL4, while only a small number of 22nt miRNAs exist due to the rare occurrence of an asymmetric bulge in the precursor miRNA stem. Here we report a strategy to produce abundant 22nt siRNAs and other desired siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges periodically into the antisense strand of hpRNA, we successfully shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. We showed that the asymmetric hpRNA constructs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes targeting both GUS and EIN2 showed increased amount of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. Consistent with the production of secondary siRNAs, the 22nt asymmetric hpRNA constructs increased gene silencing phenotypes and enhanced virus resistance in plants compared to the traditional hpRNA constructs.
2024-06-27 | GSE243296 | GEO