Systematic analysis of Arabidopsis organelles and a protein localization database for facilitating fluorescent tagging of full-length Arabidopsis proteins

Cells are organized into a complex network of subcellular compartments that are specialized for various biological functions. Subcellular location is an important attribute of protein function. To facilitate systematic elucidation of protein subcellular location, we analyzed experimentally verified protein localization data of 1,300 Arabidopsis (Arabidopsis thaliana) proteins. The 1,300 experimentally verified proteins are distributed among 40 different compartments, with most of the proteins localized to four compartments: mitochondria (36%), nucleus (28%), plastid (17%), and cytosol (13.3%). About 19% of the proteins are found in multiple compartments, in which a high proportion (36.4%) is localized to both cytosol and nucleus. Characterization of the overrepresented Gene Ontology molecular functions and biological processes suggests that the Golgi apparatus and peroxisome may play more diverse functions but are involved in more specialized processes than other compartments. To support systematic empirical determination of protein subcellular localization using a technology called fluorescent tagging of full-length proteins, we developed a database and Web application to provide preselected green fluorescent protein insertion position and primer sequences for all Arabidopsis proteins to study their subcellular localization and to store experimentally verified protein localization images, videos, and their annotations of proteins generated using the fluorescent tagging of full-length proteins technology. The database can be searched, browsed, and downloaded using a Web browser at http://aztec.stanford.edu/gfp/. The software can also be downloaded from the same Web site for local installation.     

A recombineering-based gene tagging system for Arabidopsis

One of the most information-rich aspects of gene functional studies is characterization of gene expression profiles at cellular resolution, and subcellular localization of the corresponding proteins. These studies require visualization of the endogenous gene products using specific antibodies, or, more commonly, generation of whole-gene translational fusions with a reporter gene such as a fluorescent protein. To facilitate the generation of such translational fusions and to ensure that all cis-regulatory sequences are included, we have used a bacterial homologous recombination system (recombineering) to insert fluorescent protein tags into genes of interest harbored by transformation-competent bacterial artificial chromosomes (TACs). This approach has several advantages compared to other classical strategies. First, the researcher does not have to guess what the regulatory sequences of a gene are, as tens of thousands of base pairs flanking the gene of interest can be included in the construct. Second, because the genes of interest are not amplified by PCR, there are practically no limits to the size of a gene that can be tagged. Third, there are no restrictions on the location in which the fluorescent protein can be inserted, as the position is determined by sequence homology with the recombination primers. Finally, all of the required strains and TAC clones are publically available, and the experimental procedures described here are simple and robust. Thus, we suggest that recombineering-based gene tagging should be the gold standard for gene expression studies in Arabidopsis.     

High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Secretion trap tagging of secreted and membrane-spanning proteins using Arabidopsis gene traps

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a beta-glucuronidase reporter enzyme that is inhibited by N-linked glycosylation specific to the secretory pathway. Treatment of seedlings with tunicamycin inhibits glycosylation, resulting in increased activity of secreted beta-glucuronidase fusions that result from gene trap integration downstream of exons encoding signal peptides. In the 2,059 gene trap lines that we screened, 32 secretion trap expression patterns were identified in a wide variety of tissues including embryos, meristems, and the developing vasculature. Genes disrupted by the secretion traps encode putative extracellular signaling proteins, membrane transport proteins, and novel secreted proteins of unknown function missed by conventional mutagenesis and gene prediction. Secretion traps provide a unique reagent for gene expression studies and can guide the genetic combination of loss of function alleles in related genes.     

High-throughput screening for in planta characterization of VOC biosynthetic genes by PTR-ToF-MS

Journal of Plant Research - Functional characterization of plant volatile organic compound (VOC) biosynthetic genes and elucidation of the biological function of their products often involve the...     

High-throughput genetic mapping in Arabidopsis thaliana

To facilitate rapid determination of the chromosomal location of novel mutations, we have improved current approaches to gene mapping using microsatellite length polymorphisms. The high-throughput linkage analysis method described here allows a novel gene to be tested for linkage against the whole genome of a multicellular eukaryote, Arabidopsis thaliana, in a single polyacrylamide gel. The procedure is based on the simultaneous co-amplification of 21 microsatellites in a single tube, using a multiplex PCR mix containing 21 primer pairs, each including one oligonucleotide labeled with one of three fluorescent dyes that have different emission wavelengths. The amplification products, which range in number from 21 to 42, depending on the genotype of the individual being tested, are electrophoresed in a single lane on a polyacrylamide gel. The use of an automated fragment analyzer makes it possible to perform linkage analysis on a one gel-one gene basis using DNA samples from 19 F₂ individuals obtained from an outcross involving a mutant and a wild-type that is genetically polymorphic with respect to the ecotype in which the mutant was generated. Discrimination of the amplification products is facilitated not only by labeling with different fluorochromes, but also by prior testing of different sequences for the ability to prime the amplification of each microsatellite, in order to ensure that multiplex PCR yields compatible amplification products of non-overlapping size. The method is particularly useful in large-scale mutagenesis projects, as well as for routine mapping of single mutants, since it reveals the map position of a gene less than 24 h after the F₂ individuals to be analyzed have become available. The concepts employed here can easily be extended to other biological systems. Received: 24 September 1998 / Accepted: 9 December 1998     

Cloning of an Arabidopsis patatin-like gene, STURDY, by activation T-DNA tagging

Activation T-DNA tagging can generate dominant gain-of-function mutants by overexpression of a particular endogenous gene. We identified an activation-tagged mutant, sturdy, exhibiting a stiff inflorescence stem, thicker leaves, shorter siliques, larger seeds, round-shaped flowers, and delayed growth. It is most important that unlike its wild-type counterpart, this mutant is less prone to lodging. Cloning of STURDY revealed that in sturdy, there is an open reading frame containing a single intron encoding a patatin-like homolog. The T-DNA is inserted into the 3' region of the second exon. The mutant phenotype was shown to be the result of overexpression of STURDY by mRNA analysis and transgenic studies. Preliminary histological studies have revealed an increase in cell number in the inflorescence stem of mutant plants; however, additional studies are needed to better understand the overexpression phenotype.     

Light-induced conformational changes in full-length Arabidopsis thaliana cryptochrome

Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This inter-domain modulation would be critical for CRYs' ability to transduce a blue-light signal into altered protein-protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.     

Establishment of a gene tagging system in Arabidopsis thaliana based on the maize transposable element Ac

Summary An Ac-derived, two-component transposable element system has been developed and analyzed with respect to its use in Arabidopsis thaliana. This system consists of an immobilized Ac element (Ac clipped wing, Ac_cl) as the source of transactivating transposase and a nonautonomous Ds element, Ds_A, which is inserted into a chimaeric neomycinphosphotransferase gene used as excision marker. After separate introduction of Ac_c1 and Ds_A into Arabidopsis thaliana, progeny analysis of crosses between five different Ac_cl lines and seven different Ds_A lines shows that: (1) different Ac_cl lines differ greatly in their capacity to transactivate Ds_A; (2) different Ds_A lines do not differ significantly with respect to Ds_A transactivation by one Ac_cl line; (3) reintegration of excised Ds_A elements, both at (genetically) linked and unlinked sites, occurs in about 50% of the excision events; and (4) plants with a high rate of somatic excisions can be used as source of new Ds_A transpositions, allowing the creation of a large number of independent Ds_A insertions.     

Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in planta

Stable genetic transformation of Arabidopsis thaliana was achieved by simple in planta inoculation of Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBl121. The transformation procedure, which we call in planta transformation, involves severing of apical shoots at their bases, inoculation with Agrobacterium at the severed sites, and in planta generation of shoots from the severed sites. On average, 5.5% of the newly formed shoots produced transformed progenies. These progenies (T₂ generation) contained T-DNA in the genome as examined by assaying the T-DNA encoded β-glucuronidase and kanamycin resistance and by genomic Southern blot analysis, the copy number of the T-DNAs in the Arabidopsis genome being single (33%) or multiple. The genetic behavior of the transformants examined at the T₃ and T₄ generations or with the F₂ progenies of the outcrosses between transformants and wild-type plants showed that most of the inserted T-DNA are inherited in a Mendelian fashion. This procedure provides a new approach for simple and efficient transformation of A. thaliana, obviating the need for plant regeneration from tissue explants in vitro.     

Activation tagging of an Arabidopsis SHI-RELATED SEQUENCE gene produces abnormal anther dehiscence and floral development

The tapetum is a layer of cells covering the inner surface of pollen sac wall. It contributes to anther development by providing enzymes and materials for pollen coat biosynthesis and nutrients for pollen development. At the end of anther development, the tapetum is degenerated, and the anther is dehisced, releasing mature pollen grains. In Arabidopsis, several genes are known to regulate tapetum formation and pollen development. However, little is known about how tapetum degeneration and anther dehiscence are regulated. Here, we show that an activation-tagged mutant of the S HI-R ELATED S EQUENCE 7 (SRS7) gene exhibits disrupted anther dehiscence and abnormal floral organ development in addition to its dwarfed growth with small, curled leaves. In the mutant hypocotyls, cell elongation was reduced, and gibberellic acid sensitivity was diminished. Whereas anther development was normal, its dehiscence was suppressed in the dominant srs7-1D mutant. In wild-type anthers, the tapetum disappeared at anther development stages 11 and 12. In contrast, tapetum degeneration was not completed at these stages, and anther dehiscence was inhibited, causing male sterility in the mutant. The SRS7 gene was expressed mainly in the filaments of flowers, where the DEFECTIVE-IN-ANTHER-DEHISCENCE 1 (DAD1) enzyme catalyzing jasmonic acid (JA) biosynthesis is accumulated immediately before flower opening. The DAD1 gene was induced in the srs7-1D floral buds. In fully open flowers, the SRS7 gene was also expressed in pollen grains. It is therefore possible that the abnormal anther dehiscence and floral development of the srs7-1D mutant would be related with JA.