Plastid Molecular Genetics

Plastids of higher plants are genetically semi-autonomous: some of the plastid functions are encoded in the organelle's genome while most are encoded in nuclear genes which target their products to plastids. Our long-term interest is to learn how plastid function and development depend on coordinated expression of genes in the two genetic compartments. We are also interested in biotechnological applications, which exploit the plastid’s unique transcription and translation machinery.

Regulation of Plastid Gene Transcription

Plastid genes in higher plants are transcribed by two distinct RNA polymerases: the plastid-encoded eubacterial-type multisubunit enzyme (PEP) and the nuclear-encoded phage-type enzyme (NEP), the latter of which evolved by duplication of the mitochondrial transcription machinery. Understanding the contribution of the two RNA polymerases to plastid function and development has been the subject of research during the past several years. Recently, we His-tagged the tobacco PEP a subunit and reconstituted a PEP holoenzyme from the affinity-purified core and sigma factors expressed in E. coli. In higher plants a small family of nuclear genes encodes the plastid sigma factors. The homologous in vitro transcription system will enable us to determine the role of individual plastid sigma factors in plastid transcription.

Site-specific Recombinases to Manipulate the Plastid Genome of Higher Plants

Recently, we developed new tools to manipulate the tobacco plastid genome with phage site-specific recombinases. We have shown that the P1 phage CRE-loxP site-specific recombination system is efficient for the removal of marker genes. Now INT, the phiC31 phage integrase, has been adapted for incorporation of transforming DNA in the plastid genome. INT mediates recombination between attB and attP sequences. Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome and a nuclear-encoded, plastid-targeted INT inserts an attP vector into the attB site. Since the system does not rely on the plastid’s homologous recombination machinery we expect that the phage integrase will enhance transformation efficiency in recalcitrant species such as Arabidopsis, in which homologous recombination rarely yields transplastomic clones.

The Role of Plastid ClpP1 Protease in Plant Development

The CRE-lox marker gene elimination system has been applied to study plastid gene function. We have shown that deletion of all clpP1 copies, encoding the ClpP1 subunit of an ATP-dependent Clp protease, can be linked to ablation of the shoot system of tobacco plants. Excision was accomplished with CRE, a site-specific recombinase derived from the P1 bacteriophage, which excises any DNA sequence between two directly oriented lox sites. Within the chloroplast, the Clp protease is thought to be responsible for most protein degradation. In Arabidopsis, in addition to the plastid-encoded ClpP1, there are at least four proteolytic and two regulatory Clp subunits encoded by nuclear genes. Lack of shoot development in the clpP1 deletion seedlings suggests that the nuclear-encoded catalytic subunit genes in tobacco cannot replace the plastid clpP1 gene. An intriguing possibility is that the lack of shoot development is caused by the lack of degradation of a regulatory protein that is a specific substrate for the ClpP1 isoform, the subject of future research.

Plant Biotechnology

We explore the feasibility of high-level expression of recombinant proteins in tobacco leaves. The examples include production of an oral tetanus vaccine, which was shown to induce immunity in mice to a tetanus challenge after immunization with a crude protein extract as a nasal spray. Currently, we are testing the production of a hepatitis B surface antigen in the chloroplast expression system to be used as a vaccine.

Lab Members:

Dr. Pal Maliga

Professor

Dr. Hiroshi Kuroda

Research Associate

Dr. Jon Suzuki

Research Associate

Arun Azhagiri

Graduate Fellow

Kerry Lutz

Graduate Fellow

Tarinee Tungsuchat

Graduate Fellow

Benjawan Lertwirijawong

Graduate Fellow

Massimo Bosacchi

Laboratory Researcher

Dr. Zora Svab

Volunteer