PEST sequences from a cactus dehydrin regulate its proteolytic degradation

Dehydrins (DHNs) are intrinsically disordered proteins expressed under cellular dehydration-related stresses. In this study, we identified potential proteolytic PEST sequences located at the central and C-terminal regions from the Opuntia streptacantha OpsDHN1 protein. In order to evaluate these PEST sequences as proteolytic tags, we generated a translational fusion with the GUS reporter protein and OpsDHN1 coding sequence. We found a GUS degradation effect in tobacco agro-infiltrated leaves and Arabidopsis transgenic lines that expressed the fusion GUS::OpsDHN1 full-length. Also, two additional translational fusions between OpsDHN1 protein fragments that include the central (GUS::PEST-1) or the C-terminal (GUS::PEST-2) PEST sequences were able to decrease the GUS activity, with PEST-2 showing the greatest reduction in GUS activity. GUS signal was abated when the OpsDHN1 fragment that includes both PEST sequences (GUS::PEST-1-2) were fused to GUS. Treatment with the MG132 proteasome inhibitor attenuated the PEST-mediated GUS degradation. Point mutations of phosphorylatable residues in PEST sequences reestablished GUS signal, hence these sequences are important during protein degradation. Finally, in silico analysis identified potential PEST sequences in other plant DHNs. This is the first study reporting presence of PEST motifs in dehydrins

Effect of salt stress, proline, and polyamines on seed germination of Opuntia streptacantha

"In this study we conducted experiments in order to determine whether polyamines (putrescine, spermidine, and spermine) and proline treatments help to break seed dormancy of Opuntia streptacantha and also if they facilitate seed germination under salt stress. The results showed that 1mM proline stimulated a better germination percentage as compared to untreated seeds, although the difference was not statistically significant. With respect to polyamines, they did not break seed dormancy. On the other hand, salinity affected seed germination at the higher concentrations (50 and 75 mM NaCl) used, whereas, at 25 mM NaCl, results were similar to the control. The germination percentage in the treatments with proline (1 and 10 mM) and with polyamines (1μM each) diminished significantly when increasing the concentration of NaCl. The exception was spermine treatment, which alleviates seed germination at the higher concentration of NaCl used (75 mM).

The corn smut-made cholera oral vaccine is thermostable and induces long-lasting immunity in mouse

"The use of corn smut for the production of recombinant vaccines has been recently implemented by our group. In this study, the stability and immunogenic properties of the corn smut-based cholera vaccine, based on the cholera toxin B subunit (CTB), were determined in mouse. The immunogenic potential of distinct corn smut CTB doses ranging from 1 to 30 ?g were assessed, with maximum humoral responses at both the systemic (IgG) and intestinal (IgA) levels at a dose of 15 ?g. The humoral response last for up to 70 days after the third boost. Mice were fully protected against a challenge with cholera toxin after receiving three 15 ?g-doses. Remarkably, the corn smut-made vaccine retained its immunogenic activity after storage at room temperature for a period of 1 year and no reduction on CTB was observed following exposure at 50 °C for 2 h. These data support the use of the corn smut-made CTB vaccine as a highly stable and effective immunogen and justify its evaluation in target animal models, such as piglet and sheep, as well as clinical evaluations in humans.

Down-regulation of arginine decarboxylase gene-expression results in reactive oxygen species accumulation in Arabidopsis

"Arabidopsis amiR:ADC-L2 is a non-lethal line with several developmental defects, it is characterized by a drastic reduction in free polyamine content. Herein, we found that catalase application had growth-promoting effects in amiR:ADC-L2 and parental Ws seedlings. Differences in ROS content between amiR:ADC-L2 and Ws seedlings were detected. Increased H2O2 levels were found in the amiR:ADC-L2, as well as low AtCAT2 gene expression and reduced catalase activity. Estimation of polyamine oxidase activity in amiR:ADC-L2 line indicated that the over-accumulation of H2O2 is independent of polyamine catabolism. However, increments in NADPH oxidase activity and O2•? content could be associated to the higher H2O2 levels in the amiR:ADC-L2 line. Our data suggest that low polyamine levels in Arabidopsis seedlings are responsible for the accumulation of ROS, by altering the activities of enzymes involved in ROS production and detoxification.

Identification of differentially expressed genes potentially involved in the tolerance of Lotus tenuis to long-term alkaline stress

"Soil alkalinity is one of the most serious agricultural problems limiting crop yields. The legume Lotus tenuis is an important forage acknowledged by its ability to naturally grow in alkaline soils. To gain insight into the molecular responses that are activated by alkalinity in L. tenuis plants, subtractive cDNA libraries were generated from leaves and roots of these plants. Total RNAs of non-stressed plants (pH 5.8; E.C. 1.2), and plants stressed by the addition of 10 mM of NaHCO3 (pH 9.0; E.C. 1.9), were used as source of the driver and the tester samples, respectively. RNA samples were collected after 14 and 28 days of treatment. A total of 158 unigenes from leaves and 92 unigenes from roots were obtained and classified into 11 functional categories. Unigenes from these categories (4 for leaves and 8 for roots), that were related with nutrient metabolism and oxidative stress relief were selected, and their differential expression analyzed by qRT-PCR. These genes were found to be differentially expressed in a time dependent manner in L. tenuis during the alkaline stress application. Data generated from this study will contribute to the understanding of the general molecular mechanisms associated to plant tolerance under long-term alkaline stress in plants.

Arabidopsis thaliana polyamine content is modified by the interaction with different Trichoderma species

"Plants are associated with a wide range of microorganisms throughout their life cycle, and some interactions result on plant benefits. Trichoderma species are plant beneficial fungi that enhance plant growth and development, contribute to plant nutrition and induce defense responses. Nevertheless, the molecules involved in these beneficial effects still need to be identify. Polyamines are ubiquitous molecules implicated in plant growth and development, and in the establishment of plant microbe interactions. In this study, we assessed the polyamine profile in Arabidopsis plants during the interaction with Trichoderma virens and Trichoderma atroviride, using a system that allows direct plant-fungal contact or avoids their physical interaction (split system). The plantlets that grew in the split system exhibited higher biomass than the ones in direct contact with Trichoderma species. After 3 days of interaction, a significant decrease in Arabidopsis polyamine levels was observed in both systems (direct contact and split). After 5 days of interaction polyamine levels were increased. The highest levels were observed with T. atroviride (split system), and with T. virens (direct contact). The expression levels of Arabidopsis ADC1 and ADC2 genes during the interaction with the fungi were also assessed. We observed a time dependent regulation of ADC1 and ADC2 genes, which correlates with polyamine levels. Our data show an evident change in polyamine profile during Arabidopsis – Trichoderma interaction, accompanied by evident alterations in plant root architecture. Polyamines could be involved in the changes undergone by plant during the interaction with this beneficial fungus.

The Corn Smut (‘Huitlacoche’) as a New Platform for Oral Vaccines

<div><p>The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field. In this study, <i>Ustilago maydis</i>, the causal agent of common corn smut or ‘huitlacoche’has been genetically engineered to assess expression and immunogenicity of the B subunit of the cholera toxin (CTB), a relevant immunomodulatory agent in vaccinology. An oligomeric CTB recombinant protein was expressed in corn smut galls at levels of up to 1.3 mg g-1 dry weight (0.8% of the total soluble protein). Mice orally immunized with ‘huitlacoche’-derived CTB showed significant humoral responses that were well-correlated with protection against challenge with the cholera toxin (CT). These findings demonstrate the feasibility of using edible corn smut as a safe, effective, and low-cost platform for production and delivery of a subunit oral vaccine. The implications of this platform in the area of molecular pharming are discussed.</p></div

Inhibition of polyamine oxidase activity affects tumor development during the maize-Ustilago maydis interaction

Ustilago maydis is a biotrophic plant pathogenic fungus that leads to tumor development in the aerial tissues of its host, Zea mays. These tumors are the result of cell hypertrophy and hyperplasia, and are accompanied by the reprograming of primary and secondary metabolism of infected plants. Up to now, little is known regarding key plant actors and their role in tumor development during the interaction with U. maydis. Polyamines are small aliphatic amines that regulate plant growth, development and stress responses. In a previous study, we found substantial increases of polyamine levels in tumors. In the present work, we describe the maize polyamine oxidase (PAO) gene family, its contribution to hydrogen peroxide (H2O2) production and its possible role in tumor development induced by U. maydis. Histochemical analysis revealed that chlorotic lesions and maize tumors induced by U. maydis accumulate H2O2 to significant levels. Maize plants inoculated with U. maydis and treated with the PAO inhibitor 1,8-diaminooctane exhibit a notable reduction of H2O2 accumulation in infected tissues and a significant drop in PAO activity. This treatment also reduced disease symptoms in infected plants. Finally, among six maize PAO genes only the ZmPAO1, which encodes an extracellular enzyme, is up-regulated in tumors. Our data suggest that H2O2 produced through PA catabolism by ZmPAO1 plays an important role in tumor development during the maize-U. maydis interaction.Fil: Jasso Robles, Francisco Ignacio. Universidad Autónoma de San Luis Potosí. Facultad de Ciencias; MéxicoFil: Jiménez Bremont, Juan Francisco. Instituto Potosino de Investigación Científica y Tecnológica; MéxicoFil: Becerra Flora, Alicia. Instituto Potosino de Investigación Científica y Tecnológica; MéxicoFil: Juárez Montiel, Margarita. Instituto Potosino de Investigación Científica y Tecnológica; MéxicoFil: Gonzalez, Maria Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Pieckenstain, Fernando Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: García de la Cruz, Ramón Fernando. Universidad Autónoma de San Luis Potosí. Facultad de Ciencias Químicas; MéxicoFil: Rodríguez Kessler, Margarita. Universidad Autónoma de San Luis Potosí. Facultad de Ciencias; Méxic

Antibody levels of BALB/c mice immunized with corn smut galls expressing CTB protein.

<p>Detection of: (a) anti-CTB IgA antibodies in intestinal washes, and (b) anti-CTB IgG antibodies in sera. Mice immunized with different treatments (biomass from ‘huitlacoche’: FB 1 x FB2-CTB 3 and FB1 x FB2 as control) were bled at day 21, and serum was diluted 1:40. Statistical differences versus the WT group are indicated by an asterisk (<i>P</i> < 0.05).</p

Tumors induced on maize plants, after inoculation with FB2-CTB strains paired with FB1 strain.

<p>Leaf tumors induced in 8-day-old maize seedlings inoculated with (a) FB1 x FB2, (b) FB1 x FB2-CTB 3 and (c) FB1 x FB2-CTB 4 crosses. Photos were taken 8 days post-inoculation (dpi). Corn smut galls (‘huitlacoche’) produced by inoculating with (d) FB1 x FB2, (e) FB1 x FB2-CTB 3 and (f) FB1 x FB2-CTB 4 crosses. Plants were grown under greenhouse conditions and photos were taken 18–20 dpi.</p