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Plant-made biopharmaceuticals are biotechnological valuable tools in aquaculture. Herein, the immunogenic potential of an oral corn smut (‘huitlacoche’)-made antigen (CS) was assessed in Pacific red snapper. The B subunit of the cholera toxin (CTB) was used as an immunostimulant antigen model due to its well-proven mucosal immunostimulatory activity. Test fish received corn smut-based CTB (CS-CTB) or wild-type (CS-WT) as control on day 1 and 7. Changes in humoral and cellular innate immune parameters were evaluated at 7 and 14 dpi (days post-first immunostimulation). Interestingly, total IgM and anti-CTB IgM levels in skin mucus increased in all the assessed time points in the group treated with CS-CTB. Peroxidase activity significantly increased in both serum and skin mucus from immunostimulated fish. The SOD and CAT activities were significantly increased in mucus from stimulated fish with the CS-CTB. The test fish that received CS-CTB showed enhanced phagocytic ability and radical production in head-kidney leukocytes compared with the control group. In addition, oral administration of CS-WT significantly increased the expression levels of pro-inflammatory cytokines. These findings support the use of the corn smut as a biofactory and oral delivery vehicle of novel immunostimulants, such as CTB, for fish aquaculture.

Plants have developed mechanisms that allow them to tolerate different abiotic stresses. Among these mechanisms, the accumulation of specific proteins such as dehydrins (DHNs) and aquaporins (AQPs) can protect other proteins from damage during dehydration and may allow the control of water loss, respectively. Although both types of proteins are involved in plant protection against dehydration stress, a direct interaction between them has not been explored. A previous screen to identify potential OpsDHN1 protein interactions revealed an aquaporin as a possible candidate. Here, we used the Bimolecular Fluorescence Complementation (BiFC) approach to investigate the direct interaction of the cactus OpsDHN1 protein with the Arabidopsis plasma membrane PIP family aquaporin AtPIP2B (At2G37170). Since AtPIP2B is a membrane protein and OpsDHN1 is a cytosolic protein that may be peripherally associated with membranes, we propose that OpsDHN1/AtPIP2B interaction takes place at cellular membranes. Furthermore, we also demonstrate the interaction of AtPIP2B with the three Arabidopsis dehydrins COR47 (AT1G20440), ERD10 (At1g20450), and RAB18 (At5g66400).

Ustilago maydis is a dimorphic fungus that has emerged as a model organism for the study of fungal phytopathogenicity and RNA biology. In a previous study, we isolated the U. maydis UmRrm75 gene. The deletion of the UmRrm75 gene affected morphogenesis and pathogenicity. UmRrm75 gene encodes a protein containing three RNA recognition motifs. Here we determined that UmRrm75 has chaperone activity in Escherichia coli using the transcription anti-termination assay. Subsequently, we analyzed the growth of ΔUmRrm75 mutants at 15 °C and 37 °C, observing that mutant strains had reduced growth in comparison to parental strains. UmRrm75 gene expression was induced under these non-optimal temperatures. ΔUmRrm75 mutant colonies displayed a dark-brown color at 28 °C, which was confirmed to be melanin based on spectroscopic analysis and spectrometric data. Furthermore, ΔUmRrm75 mutant strains showed the presence of peroxisomes, and increased H2O2 levels, even at 28 °C. The ΔUmRrm75 mutant strains displayed a higher expression of redox-sensor UmYap1 gene and increased catalase activity than the parental strains. Our data show that deletion of the UmRrm75 gene results in higher levels of H2O2, increased melanin content, and abiotic stress sensitivity.

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; México Fil: Jiménez Bremont, Juan Francisco. Instituto Potosino de Investigación Científica y Tecnológica; México Fil: Becerra Flora, Alicia. Instituto Potosino de Investigación Científica y Tecnológica; México Fil: Juárez Montiel, Margarita. Instituto Potosino de Investigación Científica y Tecnológica; México Fil: 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); Argentina Fil: 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); Argentina Fil: García de la Cruz, Ramón Fernando. Universidad Autónoma de San Luis Potosí. Facultad de Ciencias Químicas; México Fil: Rodríguez Kessler, Margarita. Universidad Autónoma de San Luis Potosí. Facultad de Ciencias; México

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.

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.

Polyamines are low molecular weight aliphatic compounds involved in various biochemical, cellular and physiological processes in all organisms. In plants, genes involved in polyamine biosynthesis and catabolism are regulated at transcriptional, translational, and posttranslational level. In this research, we focused on the characterization of a PEST sequence (rich in proline, glutamic acid, serine, and threonine) of the maize spermine synthase 1 (ZmSPMS1). To this aim, 123 bp encoding 40 amino acids of the C-terminal region of the ZmSPMS1 enzyme containing the PEST sequence were fused to the GUS reporter gene. This fusion was evaluated in Arabidopsis thaliana transgenic lines and onion monolayers transient expression system. The ZmSPMS1 PEST sequence leads to specific degradation of the GUS reporter protein. It is suggested that the 26S proteasome may be involved in GUS::PEST fusion degradation in both onion and Arabidopsis. The PEST sequences appear to be present in plant spermine synthases, mainly in monocots.

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.

Polyamines are new plant growth regulators that participate in various physiological processes modulating cell division and differentiation, stimulating secondary metabolite production and in stress responsiveness. In the present study, we evaluated the effect of polyamine application on CYCB1-GUS reporter line in Arabidopsis, in order to monitor changes in cell division. We observed that polyamines modulate the expression of CYCB1-GUS, most likely in an amine-specific manner. In particular, spermidine and spermine induced significant increases in CYCB1-GUS expression in shoot apex and root meristems. According of this view, mainly the higher polyamines stimulate the lateral root formation in Arabidopsis. Furthermore, the application of d-arginine and methylglyoxal bis-(guanylhydrazone) polyamine inhibitors drastically reduced Arabidopsis CYCB1-GUS root growth and plant fresh weight, as well as CYCB1-GUS expression. Another key point on this study was to analyze the effect of polyamines on CYCB1-GUS expression under salt stress. Salt stress treatments repressed CYCB1-GUS expression in a concentration dependent manner; this negative effect was ameliorated by polyamine application, in particular by spermidine and spermine, even at 125 mM NaCl, allowing the maintenance of CYCB1-GUS levels under salt stress. This work is one more contribution on the role of polyamines in cell cycle modulation and abiotic stress protection.

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.

Proline accumulation in two different bean (Phaseolus vulgaris L.) cultivars, one drought-sensitive (Canario 60) and one drought-resistant (Pinto Villa) was investigated. Both tolerated salt concentrations up to 150 mM NaCl, but the sensitive Canario 60 did not survive at 400 mM NaCl. In response to salt stress, both cvs. accumulated proline in all the analyzed tissues, the lowest contents were detected in roots. Pinto Villa accumulated higher proline concentrations than Canario 60 only at 400 mM NaCl. The addition of polyamines or ornithine increased proline content in plant tissues without stress, while they decreased it under salt stress.

The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field. In this study, Ustilago maydis, 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.

Little is known about the genetic basis and molecular mechanisms regulating female gametogenesis in flowering plants. In many species sexuality is replaced by apomixis, a method of asexual reproduction that circumvents female meiosis and fertilization, and culminates in the formation of clonal seeds. Using a new generation of transposon based insertional mutagenesis strategies and their resulting molecular tools, we are investigating how female meiotically derived cells (megaspores) acquire their identity. We are also determining their function and interactions, and attempting the induction of apomixis initiation in the ovule of Arabidopsis. This basic knowledge will contribute to establish the transfer of apomixis into sexual crops, a major challenge faced by plant biotechnology. The introduction of apomixis as a reproductive alternative could represent a unique opportunity to simplify breeding schemes and genetically perpetuate any desired heterozygous genotype, including hybrids.

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. Fil: Paz, Rosalia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan. Centro de Investigaciones de la Geosfera y Biosfera. Universidad Nacional de San Juan. Facultad de Ciencias Exactas Físicas y Naturales. Centro de Investigaciones de la Geosfera y Biosfera; Argentina Fil: Rocco, Ruben Anibal. 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); Argentina Fil: Bermont Jiménez, Juan Francisco. Instituto Potosino de Investigación Científica y Tecnológica; México Fil: Rodríguez y Domínguez Kessler, Margarita. Universidad Autónoma de San Luis Potosí.; México Fil: Becerra Flora, Alicia. Instituto Potosino de Investigación Científica y Tecnológica; México Fil: Menendez, Ana Bernardina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Ruiz, Oscar Adolfo. 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); Argentina

Plant samples from important horticultural areas in Mexico and the southern United States were collected during several seasons and analyzed for the presence of geminiviruses by a combination of agarose gel electrophoresis, molecular hybridization, and polymerase chain reaction amplification techniques. A general detection strategy confirmed the presence of geminiviruses in all horticultural areas of Mexico in pepper, tomato, tomatillo (Physalis ixocarpa), cucurbits, and tobacco. Specific detection procedures showed that pepper huasteco virus is widely distributed in Mexico; it was found in pepper and tomato samples in both coastal areas, as well as in central Mexico. It was also found in pepper samples from the Rio Grande Valley in southern Texas. Pepper jalapeno virus (PJV) and chino del tomate virus (CdTV) showed a more restricted distribution, although, in all cases, the viruses appeared to become more widely distributed over time. Partial DNA sequences of PJV and CdTV were also obtained. Comparative sequence analysis showed that PJV and the previously described Texas pepper geminivirus are probably strains of the same virus. The name pepper jalapeno virus is, thus, withdrawn to avoid further confusion. Similarly, CdTV showed a very high level of sequence identity with the recently described tomato leaf crumple virus (TLCrV), also suggesting that they both are strains of the same virus.