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Previous genetic studies have revealed that a pollen-specific calmodulin-binding protein, No Pollen Germination 1 (NPG1), is required for pollen germination. However, its mode of action is unknown. Here we report direct interaction of NPG1 with pectate lyase-like proteins (PLLs). A truncated form of AtNPG1 lacking the N-terminal tetratricopeptide repeat 1 (TPR1) failed to interact with PLLs, suggesting that it is essential for NPG1 interaction with PLLs. Localization studies with AtNPG1 fused to a fluorescent reporter driven by its native promoter revealed its presence in the cytosol and cell wall of the pollen grain and the growing pollen tube of plasmolyzed pollen. Together, our data suggest that the function of NPG1 in regulating pollen germination is mediated through its interaction with PLLs, which may modify the pollen cell wall and regulate pollen tube emergence and growth.

The herpes simplex virus type 1 origin-binding protein, OBP, is a DNA helicase encoded by the UL9 gene. The protein binds in a sequence-specific manner to the viral origins of replication, two OriS sites and one OriL site. In order to search for efficient inhibitors of the OBP activity, we have obtained a recombinant origin-binding protein expressed in Escherichia coli cells. The UL9 gene has been amplified by PCR and inserted into a modified plasmid pET14 between NdeI and KpnI sites. The recombinant protein binds to Box I and Box II sequences and possesses helicase and ATPase activities. In the presence of ATP and viral protein ICP8 (single-strand DNA-binding protein), the initiator protein induces unwinding of the minimal OriS duplex (≈80 bp). The protein also binds to a single-stranded DNA (OriS*) containing a stable Box I-Box III hairpin and an unstable AT-rich hairpin at the 3'-end. In the present work, new minor groove binding ligands have been synthesized which are capable to inhibit the development of virus-induced cytopathic effect in cultured Vero cells. Studies on binding of these compounds to DNA and synthetic oligonucleotides have been performed by fluorescence methods, gel mobility shift analysis and footprinting assays. Footprinting studies have revealed that Pt-bis-netropsin and related molecules exhibit preferences for binding to the AT-spacer in OriS. The drugs stabilize structure of the AT-rich region and inhibit the fluctuation opening of AT-base pairs which is a prerequisite to unwinding of DNA by OBP. Kinetics of ATP-dependent unwinding of OriS in the presence and absence of netropsin derivatives have been studied by measuring the efficiency of Forster resonance energy transfer (FRET) between fluorophores attached to 5'- and 3'- ends of an oligonucleotide in the minimal OriS duplex. The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase. The protein induces conformation changes (bending and partial melting) of OriS duplexes and stimulates HJ formation in the absence of ATP. The antiviral activity of bis-netropsins is coupled with their ability to inhibit the fluctuation opening of АТ base pairs in the А + Т cluster and their capacity to stabilize the structure of the АТ-rich hairpin in the single-stranded oligonucleotide corresponding to the upper chain in the minimal duplex OriS. The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.

We investigated a phenomenon of ultrasonic cleavage of DNA complexed with transition metal cations Ag(I), Cu(II) and Hg(II). We found the statistically significant dependence of relative intensity of cleavage on cation type and concentration. Each cation may cause two different types of distortion in the DNA double-helix depending on whether it binds to major or minor DNA groove. The intensity of ultrasonic cleavage decreases if cation binds to the major DNA groove; the intensity of cleavage increases if cation binds to the minor DNA groove and disturbs the hydrogen bonds of complementary base pairs or it intercalates between bases. Both types of DNA distortion can affect the intensity of N-S interconversion of deoxyribose.

SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

When grown for energy production instead for smoking, tobacco can generate a large amount of inexpensive biomass more efficiently than almost any other agricultural crop. Tobacco possesses potent oil biosynthesis machinery and can accumulate up to 40% of seed weight in oil. In this work, we explored two metabolic engineering approaches to enhance the oil content in tobacco green tissues for potential biofuel production. First, an Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in triacylglycerol (TAG) biosynthesis, was expressed in tobacco under the control of a strong ribulose-biphosphate carboxylase small subunit promoter. This modification led to up to a 20-fold increase in TAG accumulation in tobacco leaves and translated into an overall of about a twofold increase in extracted fatty acids (FA) up to 5.8% of dry biomass in Nicotiana tabacum cv Wisconsin, and up to 6% in high-sugar tobacco variety NC-55. Modified tobacco plants also contained elevated amounts of phospholipids. This increase in lipids was accompanied by a shift in the FA composition favourable for their utilization as biodiesel. Second, we expressed in tobacco Arabidopsis gene LEAFY COTYLEDON 2 (LEC2), a master regulator of seed maturation and seed oil storage under the control of an inducible Alc promoter. Stimulation of LEC2 expression in mature tobacco plants by acetaldehyde led to the accumulation of up to 6.8% per dry weight of total extracted FA. The obtained data reveal the potential of metabolically modified plant biomass for the production of biofuel.

The regulation of the reporter gene activity in a single bacterial cell by means of lambda-phage C1 repressor has been described by the methods of statistical thermodynamics. The equations for the calculation of the mean production rate of the reporter protein and its standard deviation as a function of C1 repressor concentration in the cell have been obtained. The stochastic nature of C1 repressor binding with OR1 and OR2 operator sites becomes apparent when both repressor molecules and operators are present in the bacterial cell in a small number of copies. In this case, the number of repressor molecules that bind to OR1 and OR2 sites fluctuates considerably. The in vitro binding of C1 repressor to OR1 and OR2 sites, their mutant forms, and nonspecific DNA regions has been well studied. Using the binding constants of in vitro binding of C1 repressor to OR1, OR2 and nonspecific DNA regions and also the value of the cooperativity parameter for C1 repressor binding to OR1 and OR2 sites, we calculated the mean rate of synthesis of the reporter protein and its standard deviation as a function of repressor concentration in cell. The theoretical relations fit well the experimental results. The results of calculations confirm the assumption that gene expression noise in a single cell at a repressor concentration exceeding 100 nM is related to the stochastic nature of binding of repressor dimers to OR1 and OR2 sites. Other mechanisms of the generation of gene expression noise (for example, monomer-dimer balance) make a significant contribution at concentrations less than 100 nM.

The extracellular virion membrane protein B5 is a potent inducer of immune responses capable of protecting mice and primates against poxvirus infections. Here, we examined the antibody response induced in mice immunized intramuscularly (i.m.) or intranasally (i.n.) with plant-derived B5 (pB5) accompanied or not with plant total soluble protein (TSP) at various concentrations. Increasing amounts of TSP inhibited the pB5-specific response in both i.m.- and i.n.-immunized mice, with more dramatic effects in the latter. pB5 administered to mucosal surfaces induced specific IgG and IgA responses, whereas i.m. immunization produced high serum IgG titers and no IgA. A 6-fold increase in pB5 dosage administered i.n. led to an antibody response comparable to that obtained by i.m. injection. Our study addresses the quality/quantity issues of the pB5 subunit preparation and demonstrates the feasibility of mucosal administration of plant-derived smallpox subunit vaccine in obtaining a potent immune response. Overall, this work points to the practicability of needle-free mucosal administration of such vaccines in light of purity, dosage and adjuvant formulation.

The hepatitis B core antigen (HBcAg) can generate a strong immune response and is recognized as an effective carrier for foreign epitopes. The domain-4 epitope of the anthrax protective antigen (PA-D4) plays an essential role in generating protective immunity against virulent Bacillus anthracis. Here we report the successful production of a recombinant protein comprised of the antigenic PA-D4 integrated into the c/e1 loop of HBcAg in transgenic low-alkaloid Nicotiana tabacum. Sera of mice injected with the plant-derived purified HB/PA-D4 protein exhibited significant anti-PA- and anti-HBcAg-specific IgG titers; however, formation of virus-like particles (VLP) was not observed. These data support the feasibility of producing complex protein chimeras in plants.

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.

We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems.

Pectate lyases catalyse the eliminative cleavage of de-esterified homogalacturonan in pectin, a major component of the primary cell walls in higher plants. In the completed genome of Arabidopsis, there are 26 genes (AtPLLs) that encode pectate lyase-like proteins. Here, we analysed the expression pattern of all AtPLLs in different organs, at different stages of seedling development and in response to various hormones and stresses. The expression of PLLs varied considerably in different organs, with no expression of some PLLs in vegetative organs. Interestingly, all PLL genes are expressed in flowers. Several PLLs are expressed highly in pollen, suggesting a role for these in pollen development and/or function. Analysis of expression of all PLL genes in seedlings treated with hormones, abiotic stresses and elicitors of defense responses revealed significant changes in the expression of some PLLs without affecting the other PLLs. The stability of transcripts of PLLs varied considerably among different genes. Our results indicate a complex regulation of expression of PLLs and involvement of PLLs in some of the hormonal and stress responses.

Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Several tumor-associated antigens in tumor cells have been identified as targets for therapeutic mAbs. Here we describe the production of mAb BR55-2 (IgG2a) in transgenic plants that recognizes the nonprotein tumor-associated antigen Lewis Y oligosaccharide overexpressed in human carcinomas, particularly breast and colorectal cancers. Heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of low-alkaloid tobacco transgenic plants (Nicotiana tabacum cv. LAMD609). Expression levels of plant-derived mAb (mAbP) were high (30 mg/kg of fresh leaves) in T1 generation plants. Like the mammalian-derived mAbM, the plant mAbP bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAbP and mAbM showed the similar binding to FcgammaRI receptor (CD64). Comparable levels of cytotoxicity against SK-BR3 cells were also shown for both mAbs in antibody-dependent cell-mediated cytotoxicity assay. Furthermore, plant-derived BR55-2 efficiently inhibited SW948 tumor growth xenografted in nude mice. Altogether, these findings suggest that mAbP originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.

In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis.

The receptive field of a direction-selective unit of any type consists of an excitatory center 3 degrees -5 degrees in size, a near inhibitory surround and a far surround upon which illumination exerts an influence for both spontaneous activity and specific cell response.