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1.
Proc Natl Acad Sci U S A ; 120(48): e2312918120, 2023 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-37983505

RESUMO

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ácidos Indolacéticos/metabolismo , Mutação , Fenótipo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/metabolismo
3.
Curr Biol ; 32(8): R370-R372, 2022 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-35472426

RESUMO

Pollen grains stock up on starch to power germination and pollen tube growth upon pollination. New findings in barley show that localized auxin biosynthesis by a YUC flavin monooxygenase leads to reprogramming energy metabolism during pollen maturation.


Assuntos
Hordeum , Biologia , Hordeum/metabolismo , Ácidos Indolacéticos/metabolismo , Plantas/metabolismo , Pólen/metabolismo
4.
Plant Physiol ; 188(4): 1757-1768, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-34893903

RESUMO

Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently edit target genes without leaving any transgenes in plants. Some approaches directly address this issue by editing plant genomes with DNA-free reagents. On the other hand, DNA-based techniques require another step for ensuring plants are transgene-free. Fluorescent markers, pigments, and chemical treatments have all been employed as tools to distinguish transgenic plants from transgene-free plants quickly and easily. Moreover, suicide genes have been used to trigger self-elimination of transgenic plants, greatly improving the efficiency of isolating the desired transgene-free plants. Transgenes can also be excised from plant genomes using site-specific recombination, transposition or gene editing nucleases, providing a strategy for editing asexually produced plants. Finally, haploid induction coupled with gene editing may make it feasible to edit plants that are recalcitrant to transformation. Here, we evaluate the strengths and weaknesses of recently developed approaches for obtaining edited plants without transgene residuals.


Assuntos
Edição de Genes , Genoma de Planta , Plantas Geneticamente Modificadas , Endonucleases/genética , Edição de Genes/métodos , Genoma de Planta/genética , Plantas Geneticamente Modificadas/genética , Transgenes
5.
Genome Res ; 31(9): 1638-1645, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34285053

RESUMO

Massively parallel reporter assays (MPRAs) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream from a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag-sequence-specific effects and show that our method can significantly reduce this source of variation and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps not only to improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.


Assuntos
Regulação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico , Viés , Bioensaio , Genes Reporter
6.
Nat Commun ; 12(1): 3854, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158505

RESUMO

Sexual reproduction constrains progeny to inherit allelic genes from both parents. Selective acquisition of target genes from only one parent in the F1 generation of plants has many potential applications including the elimination of undesired alleles and acceleration of trait stacking. CRISPR/Cas9-based gene drives can generate biased transmission of a preferred allele and convert heterozygotes to homozygotes in insects and mice, but similar strategies have not been implementable in plants because of a lack of efficient homology-directed repair (HDR). Here, we place a gene drive, which consists of cassettes that produce Cas9, guide RNAs (gRNA), and fluorescent markers, into the CRYPTOCHROME 1 (CRY1) gene through CRISPR/Cas9-mediated HDR, resulting in cry1drive lines. After crossing the cry1drive/cry1drive lines to wild type, we observe F1 plants which have DNA at the CRY1 locus from only the cry1drive/cry1drive parent. Moreover, a non-autonomous trans-acting gene drive, in which the gene drive unit and the target gene are located on different chromosomes, converts a heterozygous mutation in the target gene to homozygous. Our results demonstrate that homozygous F1 plants can be obtained through zygotic conversion using a CRISPR/Cas9-based gene drive.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Sistemas CRISPR-Cas , Criptocromos/genética , Tecnologia de Impulso Genético/métodos , Edição de Genes/métodos , Cruzamentos Genéticos , DNA de Plantas/genética , Genoma de Planta/genética , Genótipo , Plantas Geneticamente Modificadas , Reparo de DNA por Recombinação/genética , Reprodução/genética , Seleção Genética , Sequenciamento Completo do Genoma/métodos
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