Kerry Lutz

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general.

Conservation of RNA editing between rice and maize plastids: are most editing events dispensable?

Abstract. The extent of conservation of RNA editing sites in the plastid genome of rice was determined by comparing the genomic sequence with that of the cDNA. The presence of a T in the cDNA predicted to be a C by the DNA sequence of the plastid genome, indicated C to U editing. In the 11 plastid transcripts of rice a total of 21 editing sites were found. In maize, a closely related grass species, 26 editing sites have been reported in 13 plastid transcripts. Most editing sites are conserved between the two species, although differences in RNA editing were found at eight sites. In seven cases the T was already encoded at the DNA level, eliminating the requirement for RNA editing. In one case (rpoB, codon 206) the RNA sequence was conserved between the two species, but the mRNA is still not edited in rice. It appears that, although evolutionarily conserved, RNA editing is essential only for a few plastid editing sites. Information about RNA editing in rice plastids will facilitate the design of plastid vectors with broad applicability in grass species.

Plastid marker gene excision by the phiC31 phage site-specific recombinase.

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.

A Guide to Choosing Vectors for Transformation of the Plastid Genome of Higher Plants

Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3′rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3″-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3″-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.

Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system.

Incorporation of a selectable marker gene in the plastid genome is essential to uniformly alter the thousands of genome copies in a tobacco cell. When transformation is accomplished, however, the marker gene becomes undesirable. Here we describe plastid transformation vectors, the method of plastid transformation using tobacco leaves and alternative protocols for marker gene excision with the P1 bacteriophage Cre-loxP site-specific recombination system. Plastid vectors carry a marker gene flanked with directly oriented loxP sites and a gene of interest, which are introduced into plastids by the biolistic process. The transforming DNA integrates into the plastid genome by homologous recombination via plastid targeting sequences. Marker gene excision is accomplished by a plastid-targeted Cre protein expressed from a nuclear gene. Expression may be from an integrated gene introduced by Agrobacterium transformation (Transformation Protocol), by pollination (Pollination Protocol) or from a transient, non-integrated T-DNA (Transient Protocol). Transplastomic plants are obtained in about 3 months, yielding seed after 2 months. The time required to remove the plastid marker and nuclear genes and to obtain seed takes 10–16 months, depending on which protocol is used.

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing.

BackgroundHigh throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.ResultsWe describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.ConclusionsExtracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

Transformation of the Plastid Genome to Study RNA Editing

In this chapter we provide an overview of cytosine-to-uridine (C-to-U) RNA editing in the plastids of higher plants. Particular emphasis will be placed on the role plastid transformation played in understanding the editing process. We discuss how plastid transformation enabled identification of mRNA cis elements for editing and gave the first insight into the role of editing trans factors. The introduction will be followed by a protocol for plastid transformation, including vector design employed to identify editing cis elements. We also discuss how to test RNA editing in vivo by cDNA sequencing. At the end, we summarize the status of the field and outline future directions.

Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process.

The plastid genome (ptDNA) of higher plants is highly polyploid, and the 1000-10 000 copies are compartmentalized with up to approximately 100 plastids per cell. The problem we address here is whether or not a newly arising genome can be established in a developing tobacco shoot, and be transmitted to the seed progeny. We tested this by generating two unequal ptDNA populations in a cultured tobacco cell. The parental tobacco plants in this study have an aurea (yellowish-golden) leaf color caused by the presence of a bar(au) gene in the ptDNA. In addition, the ptDNA carries an aadA gene flanked with the phiC31 phage site-specific recombinase (Int) attP/attB target sites. The genetically distinct ptDNA copies were obtained by Int, which either excised only the aadA marker gene (i.e. did not affect the aurea phenotype) or triggered the deletion of both the aadA and bar(au) transgenes, and thereby restored the green color. The ptDNA determining green plastids represented only a small fraction of the population and was not seen in a transient excision assay, and yet three out of the 53 regenerated shoots carried green plastids in all developmental layers. The remaining 49 Int-expressing plants had either exclusively aurea (24) or variegated (25) leaves with aurea and green sectors. The formation of homoplastomic green shoots with the minor green ptDNA in all developmental layers suggests that the ptDNA population in a regenerating shoot apical meristem derives from a small number of copies selected through a stochastic process.

Transplastomics in Arabidopsis: progress toward developing an efficient method.

Protocols developed for plastome engineering in Nicotiana tabacum rely on biolistic delivery of the transforming DNA to chloroplasts in intact leaf tissue; integration of the foreign DNA into the plastid genome by homologous recombination via flanking plastid DNA (ptDNA) targeting regions; and gradual dilution of non-transformed ptDNA during cultivation in vitro. Plastid transformation in Arabidopsis was obtained by combining the tobacco leaf transformation protocol with Arabidopsis-specific tissue culture and plant regeneration protocols. Because the leaf cells in Arabidopsis are polyploid, this protocol yielded sterile plants. Meristematic cells in a shoot apex or cells of a developing embryo are diploid. Therefore, we developed a regulated embryogenic root culture system that will generate diploid tissue for plastid transformation. This embryogenic culture system is created by steroid-inducible expression of the BABY BOOM transcription factor. Plastid transformation in Arabidopsis will enable the probing of plastid gene function, and the characterization of posttranscriptional mechanisms of gene regulation and the regulatory interactions of plastid and nuclear genes.

Tobacco Chloroplasts as a Platform for Vaccine Production

Initial developments in the field of plant-based vaccines have been limited by low level of immunogen expression from nuclear genes. An alternative method is to express vaccine antigens from the chloroplast genome. Recently, we developed a production system for expression of recombinant proteins in tobacco chloroplasts including vectors, expression cassettes, and a system for marker gene elimination. This development is reviewed here. For general reviews on plastid transformation see (Bock, 2001; Maliga, 2002; Staub, 2002).