Diversity of genetic lesions characterizes new Arabidopsis flavonoid pigment mutant alleles from T-DNA collections

Jiang, Nan; Lee, Yun Sun; Mukundi, Eric; Gómez-Cano, Fabio; Rivero, Luz; Grotewold, Erich

doi:10.1016/j.plantsci.2019.110335

Cited by 10 publications

(16 citation statements)

References 88 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Expression of a 35S:GFP‐IRR transgene in the SALK_015201 background failed to complement the long hypocotyl phenotype (Figure 1c,d), leading us to suspect that SALK_015201 may harbor an unlinked second‐site mutation in addition to the annotated T‐DNA insertion in IRR . Such mutations have been observed in numerous other T‐DNA lines (David et al., 2019; Enders et al., 2015; Gao et al., 2015; Jiang et al., 2020; Yoshida et al., 2018) and are thought to be an unintended consequence of the T‐DNA integration process (Castle et al., 1993; Laufs et al., 1999; Nacry et al., 1998; Negruk et al., 1996; Tax & Vernon, 2001).…”

Section: Resultsmentioning

confidence: 75%

“…Additional mutant alleles unassociated with T‐DNA insertions (also called untagged T‐DNA mutants) have been reported on numerous occasions and can be associated with duplications/translocations, insertion/deletions or other complex genetic changes (Tax & Vernon, 2001). Notable examples include second‐site mutations in the abp1‐5 and phyb‐9 backgrounds (Enders et al., 2015; Gao et al., 2015; Yoshida et al., 2018), a new set of transparent testa alleles (Jiang et al., 2020), and a mutation in nrpd1a‐3 associated with ROOT HAIR DEFECTIVE 6 (RHD6) (David et al., 2019).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

slim shady is a novel allele of PHYTOCHROME B present in the T‐DNA line SALK_015201

Dash

McEwan²,

Montes

et al. 2021

Plant Direct

View full text Add to dashboard Cite

Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin, rapid changes in transcript and protein abundance occur in hypocotyls, and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin‐regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady , in an annotated insertion line in IMMUNOREGULATORY RNA‐BINDING PROTEIN ( IRR ). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T‐DNA allele of IRR found that it had a wild‐type (WT) hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid‐auxin‐phytochrome (BAP) regulatory module compared to WT, including transcription factors that regulate brassinosteroid, auxin, and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence data and genetic complementation analysis with SALK_015201C, we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase‐related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb‐9 confirmed that slim shady is a mutant allele of PHYB . This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin‐dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T‐DNA stocks.

show abstract

Section: Resultsmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

slim shady is a novel allele of PHYTOCHROME B present in the T‐DNA line SALK_015201

Dash

McEwan²,

Montes

et al. 2021

Plant Direct

View full text Add to dashboard Cite

show abstract

“…Expression of a 35S:GFP-IRR transgene in the SALK_015201 background failed to complement the long hypocotyl phenotype (Figure 1C, D), leading us to suspect that SALK_015201 may harbor an unlinked second-site mutation in addition to the annotated T-DNA insertion in IRR . Such mutations have been observed in numerous other T-DNA lines (Jiang et al, 2020; Enders et al, 2015; Gao et al, 2015; Yoshida et al, 2018; David et al, 2019) and are thought to be an unintended consequence of the T-DNA integration process (Tax and Vernon, 2001; Castle et al, 1993; Negruk et al, 1996; Nacry et al, 1998; Laufs et al, 1999).…”

Section: Resultsmentioning

confidence: 97%

“…Additional mutant alleles unassociated with T-DNA insertions (also called untagged T-DNA mutants) have been reported on numerous occasions and can be associated with duplications/translocations, insertion/deletions or other complex genetic changes (Tax and Vernon, 2001). Notable examples include second-site mutations in the abp1-5 and phyb-9 backgrounds (Enders et al, 2015; Gao et al, 2015; Yoshida et al, 2018), a new set of transparent testa alleles (Jiang et al, 2020), and a mutation in nrpd1a-3 associated with ROOT HAIR DEFECTIVE 6 (RHD6) (David et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

slim shadyis a novel allele of PHYTOCHROME B present in the T-DNA line SALK_015201

Dash

McEwan²,

Montes

et al. 2021

Preprint

View full text Add to dashboard Cite

Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin rapid changes in transcript and protein abundance occur in hypocotyls and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin-regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T-DNA allele of IRR found that it had a wild-type hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid-auxin-phytochrome (BAP) regulatory module compared to wild-type, including transcription factors that regulate brassinosteroid, auxin and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence and transcriptomics data for SALK_015201C we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase-related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb-9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T-DNA stocks.

show abstract

“…Several studies already harnessed high throughput sequencing technologies to investigate T-DNA insertion and other mutant lines [46, 47]. Oxford Nanopore Technologies (ONT) provides a cost-effective and fast approach to study A. thaliana genomes, since a single MinION/GridION Flow Cell delivers sufficient data to assemble one genotype [48].…”

Section: Introductionmentioning

confidence: 99%

Large scale genomic rearrangements in selectedArabidopsis thalianaT-DNA lines are caused by T-DNA insertion mutagenesis

Pucker¹,

Kleinboelting²,

Weißhaar³

2021

Preprint

View full text Add to dashboard Cite

Background Experimental prove of gene function assignments in plants is heavily based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. Results We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, suggesting that the number of T-DNA insertions per line was underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10% of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. Conclusion Long read sequencing was demonstrated to be a very effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.

show abstract

Diversity of genetic lesions characterizes new Arabidopsis flavonoid pigment mutant alleles from T-DNA collections

Cited by 10 publications

References 88 publications

slim shady is a novel allele of PHYTOCHROME B present in the T‐DNA line SALK_015201

slim shady is a novel allele of PHYTOCHROME B present in the T‐DNA line SALK_015201

slim shadyis a novel allele of PHYTOCHROME B present in the T-DNA line SALK_015201

Large scale genomic rearrangements in selectedArabidopsis thalianaT-DNA lines are caused by T-DNA insertion mutagenesis

Contact Info

Product

Resources

About