Publications

We have identified and genetically characterized an Arabidopsis thaliana gene encoding a homolog of the Cleavage and Polyadenylation Specificity Factor (CPSF). This gene, named AtCPSF73-II, has been found to have a critical role in development by loss-of-function analysis using a Dissociation (Ds) insertion line SGT1922. The homozygous SGT1922 plants were lethal, but the heterozygous plants, while retaining their normal vegetative growth, displayed empty seed spaces as well as aborted seeds with embryos arrested at the globular stage. Genetic analysis indicated that the disruption of the AtCPSF73-II gene in SGT1922 plants caused severe reduction in genetic transmission of female gametes due to a loss of fertility, while the transmission of male gametes was normal. Two independent heterozygous lines with T-DNA insertion on the AtCPSF73-II gene also showed the similar phenotype. Gene expression analysis demonstrated that AtCPSF73-II was preferentially expressed in flowers. Protein sequence analysis revealed a group of AtCPSF73-II homologs with unknown function in animals, but not in yeast, which suggested a potential important function of this group of genes in the development of multicellular organisms.

Helper component-protease (HC-Pro) is a plant viral suppressor of RNA silencing, and transgenic tobacco expressing HC-Pro has increased susceptibility to a broad range of viral pathogens. Here we report that these plants also exhibit enhanced resistance to unrelated heterologous pathogens. Tobacco mosaic virus (TMV) infection of HC-Pro-expressing plants carrying the N resistance gene results in fewer and smaller lesions compared to controls without HC-Pro. The resistance to TMV is compromised but not eliminated by expression of nahG, which prevents accumulation of salicylic acid (SA), an important defense signaling molecule. HC-Pro-expressing plants are also more resistant to tomato black ring nepovirus (TBRV) and to the oomycete Peronospora tabacina. Enhanced TBRV resistance is SA-independent, whereas the response to P. tabacina is associated with early induction of markers characteristic of SA-dependent defense. Thus, a plant viral suppressor of RNA silencing enhances resistance to multiple pathogens via both SA-dependent and SA-independent mechanisms.

We have identified and genetically characterized an Arabidopsis thaliana gene encoding a homolog of the Cleavage and Polyadenylation Specificity Factor (CPSF). This gene, named AtCPSF73-II, has been found to have a critical role in development by loss-of-function analysis using a Dissociation (Ds) insertion line SGT1922. The homozygous SGT1922 plants were lethal, but the heterozygous plants, while retaining their normal vegetative growth, displayed empty seed spaces as well as aborted seeds with embryos arrested at the globular stage. Genetic analysis indicated that the disruption of the AtCPSF73-II gene in SGT1922 plants caused severe reduction in genetic transmission of female gametes due to a loss of fertility, while the transmission of male gametes was normal. Two independent heterozygous lines with T-DNA insertion on the AtCPSF73-II gene also showed the similar phenotype. Gene expression analysis demonstrated that AtCPSF73-II was preferentially expressed in flowers. Protein sequence analysis revealed a group of AtCPSF73-II homologs with unknown function in animals, but not in yeast, which suggested a potential important function of this group of genes in the development of multicellular organisms.

RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. A novel gene, RIE1, encoding a RING-H2 zinc-finger protein was identified in Arabidopsis thaliana and is characterized in this paper. RIE1 encodes a predicted protein product of 359 amino acids residues with a molecular mass of 40 kDa, with a RING-H2 zinc-finger motif located at the extreme end of the C-terminus. Characterization of a Dissociation (Ds) insertion line (SGT4559) and a T-DNA insertion line (SRIE1) demonstrated that disruption of RIE1 is embryo-lethal. SGT4559 heterozygous plants produced seeds with embryo development arrested from globular to torpedo stages. Some mutant seeds were rescued by embryo culture, and the mutant (rie1) plants seemed to grow normally compared to wild-type plants, except that the mutants produced only abnormal seeds. However, RIE1 was expressed in different tissues throughout the whole plant as revealed by northern blot analysis and gene fusion assay of RIE1 promoter with the beta-glucuronidase (GUS) gene. Our results indicated that RIE1 plays an essential role in seed development.

RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. A novel gene, RIE1, encoding a RING-H2 zinc-finger protein was identified in Arabidopsis thaliana and is characterized in this paper. RIE1 encodes a predicted protein product of 359 amino acids residues with a molecular mass of 40 kDa, with a RING-H2 zinc-finger motif located at the extreme end of the C-terminus. Characterization of a Dissociation (Ds) insertion line (SGT4559) and a T-DNA insertion line (SRIE1) demonstrated that disruption of RIE1 is embryo-lethal. SGT4559 heterozygous plants produced seeds with embryo development arrested from globular to torpedo stages. Some mutant seeds were rescued by embryo culture, and the mutant (rie1) plants seemed to grow normally compared to wild-type plants, except that the mutants produced only abnormal seeds. However, RIE1 was expressed in different tissues throughout the whole plant as revealed by northern blot analysis and gene fusion assay of RIE1 promoter with the beta-glucuronidase (GUS) gene. Our results indicated that RIE1 plays an essential role in seed development.

A 13-residue synthetic peptide (Rev4) was designed based on indolicidin, an antimicrobial peptide from bovine neutrophils. The synthetic peptide retains high antimicrobial activity. When tested for its stability in tobacco leaf extracts, Rev4 was highly stable compared to another antimicrobial peptide, magainin. When mixed with Rev4, magainin was protected from degradation by the leaf extract. Our results show that Rev4 is a potent protease inhibitor which selectively inhibits three out of four different types of proteases. Four other synthetic peptides were tested and the results were suggestive of no correlation between their antimicrobial and protease inhibitory activities.

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system.

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system.

Magainins are a group of short peptides originally isolated from frog skin and thought to function as a natural defense mechanism against infection due to their antimicrobial properties. The engineered magainin analog peptide Myp30 was found to inhibit spore germination of the oomycete, Peronospora tabacina (Adam) in vitro, and the growth of a bacterial pathogen Erwinia carotovora subsp. carotovora (Jones). Transgenic tobacco (Nicotiana tabacum L.) plants expressing Myp30 were evaluated for resistance to these pathogens. The expression of the peptide only to an extracellular location resulted in significant reduction in sporulation and lesion size due to P. tabacina infection. A significant increase in resistance to the bacterial pathogen was also observed regardless of the targeting location of the peptide.

The hrmA gene from Pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium P. syringae pv. tabaci in all examined tobacco cultivars. We expressed this gene in tobacco plants under the control of the tobacco Delta0. 3 TobRB7 promoter, which is induced upon nematode infection in tobacco roots (Opperman et al. 1994, Science, 263, 221-223). A basal level of hrmA expression in leaves of transgenic plants activated the expression of pathogenesis-related genes, and the transgenic plants exhibited high levels of resistance to multiple pathogens: tobacco vein mottling virus, tobacco etch virus, black shank fungus Phytophthora parasitica, and wild fire bacterium Pseudomonas syringae pv. tabaci. However, the hrmA transgenic plants were not significantly more resistant to root-knot nematodes. Our results suggest a potential use of controlled low-level expression of bacterial avr genes, such as hrmA, in plants to generate broad-spectrum resistance to bacterial, fungal and viral pathogens.