Carlos Borroto

Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants ☆

The application of bioengineering to plants for production of biological products for human and animal use has expanded in recent years. The reasons for this expansion are several and include advances in the technology for novel production systems and the need for very large quantities of therapeutic proteins. The process of growing pharmaceutical proteins in plants, extracting, and purifying is a hard task considering the lack of available information concerning these topics. In this work, a recombinant murine monoclonal antibody specific for the hepatitis B surface antigen, expressed in stably transformed transgenic Nicotiana tabacum plants, was purified by means of a recombinant protein A Streamline chromatography as the main purification step. The antibody expression level varied with the age of the plants and the number of harvests from 40 to 15microg/ml and the maximum process yield was about 25mg of plantibody/kg of biomass. Protein A Streamline chromatography was successfully used in the purification process yielding a recovery of about 60% and a plantibody SDS-PAGE purity of over 90% but unexpectedly, previous clarification steps could not be totally avoided. The amino acid sequence recognized by this affinity purified plantibody was similar to its murine counterpart verifying the potentiality of plants to replace animals or bioreactors for large-scale production of this monoclonal antibody.

NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high‐level pathogen resistance under greenhouse and field conditions

Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions.

Novel gene isolated from Caligus rogercresseyi: A promising target for vaccine development against sea lice

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chiles salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccination-challenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.

EIL2 Transcription Factor and Glutathione Synthetase Are Required for Defense of Tobacco Against Tobacco Blue Mold

In order to identify tobacco (Nicotiana megalosiphon) genes involved in broad-spectrum resistance to tobacco blue mold (Peronospora hyoscyami f. sp. tabacina), suppression subtractive hybridization was used to generate cDNA from transcripts that are differentially expressed during an incompatible interaction. After differential screening by membrane-based hybridization, clones corresponding to 182 differentially expressed genes were selected, sequenced, and analyzed. The cDNA collection comprised a broad repertoire of genes associated with various processes. Northern blot analysis of a subset of these genes confirmed the differential expression patterns between the compatible and incompatible interaction. Subsequent virus-induced gene silencing (VIGS) of four genes that were found to be differentially induced was pursued. While VIGS of a lipid transfer protein gene or a glutamate decarboxylase gene in Nicotiana megalosiphon did not affect blue mold resistance, silencing of an EIL2 transcription factor gene and a glutathione synthetase gene was found to compromise the resistance of Nicotiana megalosiphon to P. hyoscyami f. sp. tabacina. Potentially, these genes can be used to engineer resistance in blue mold-susceptible tobacco cultivars.

'Candidatus Liberibacter asiaticus', Causal Agent of Citrus Huanglongbing, Is Reduced by Treatment with Brassinosteroids.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

Molecular characterization of tobacco leaf rugose virus, a new begomovirus infecting tobacco in Cuba.

Tobacco (Nicotiana tabacum L.) is the main raw material for the cigar industry and one of the most important crops in Cuba comprising 49,654 ha. During the past 20 years, foliar rugosity and stunting symptoms have been observed in several tobacco producing areas. These symptoms were correlated with the presence of typical geminivirus nuclear inclusions and the transmission of the causal agent by whiteflies (Bemisia tabaci Genn) (1). To identify the suspect geminivirus, diseased leaf samples were collected in Havana province in 2000 and 2001. Sap extracts or leaf pieces were used to inoculate healthy tomato and tobacco plants by mechanical and graft inoculation procedures. Characteristic symptoms were reproduced in tobacco plants only by grafting (8 to 10 plants). DNA extracts from symptomatic plants were analyzed by Southern blot and polymerase chain reaction. The presence of a bipartite begomovirus was supported by the observation of hybridization signals (1.6 kb to 3 kb) at low stringency to probes derived from DNA-A and DNA-B of Taino tomato mottle virus. Furthermore, typical begomovirus amplicons of approximately 1.4 kb and 1.2 kb were amplified using the primer sets PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (2), respectively. Amplicons were cloned, and their nucleotide sequences (nt) obtained from two clones each. Sequence for component A was assembled, and some fragments were compared with those for other begomoviruses using CLUSTAL W. For the CP gene (756 nt) (GenBank Accession No. AJ488768), the comparison revealed the highest percentages of nt identity with Sida golden mosaic virus from Florida (SiGMV-F, GenBank Accession No. AF049336) (86%), Tomato mottle virus (GenBank Accession No. L14460) (83.5%), and the yellow vein strain of Sida golden mosaic virus from Honduras (GenBank Accession No. Y11099) (83.3%). In addition, the percentages of nt identity obtained using the core region (a 540-nt fragment located between positions 147 and 687) of the CP gene from the tobacco virus were calculated. The best scores were as follows: SiGMV-F, 87.8%; Jatropha mosaic virus (JMV) from Puerto Rico (GenBank Accession No. AF058025), 86.9%; and Tomato rugose mosaic virus (GenBank Accession No. AF291705), 86.3%. Finally, comparisons of the common region (CR, 144 nt) revealed the highest values with JMV from Jamaica (JMV-JM) DNA-A and DNA-B (GenBank Accession Nos. AF324410 and AF324411; 89% and 91.1%, respectively). Interestingly, the CR analysis revealed the presence of the Ori-associated iterative motif GGGGT, which is the same in the CR of JMV-JM. Although the data suggest that the tobacco begomovirus is related to the JMV-JM isolate, it is a new species, and the name of Tobacco leaf rugose virus (TbLRV) is proposed. References: (1) S. Quintero and J. Santiesteban, Agrotec. Cuba 11(1), 1979. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry.

A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors.

Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.

Expression of a Nicotiana tabacum pathogen-induced gene is involved in the susceptibility to black shank

Many host genes induced during compatible plant-pathogen interactions constitute targets of pathogen virulence factors that act to suppress host defenses. In order to identify Nicotiana tabacum L. genes for pathogen-induced proteins involved in susceptibility to the oomycete Phytophthora parasitica var. nicotianae, we used SuperSAGE technology combined with next-generation sequencing to identify transcripts that were differentially upregulated during a compatible interaction. We identified a pathogen-induced gene (NtPIP) that was rapidly induced only during the compatible interaction. Virus-induced gene silencing of NtPIP reduced the susceptibility of N. tabacum to P. parasitica var. nicotianae. Additionally, transient expression of NtPIP in the resistant species Nicotiana megalosiphon Van Heurck & Mull. Arg. compromised the resistance to P. parasitica var. nicotianae. This pathogen-induced protein is therefore a positive regulator of the susceptibility response against an oomycete pathogen in tobacco.

NmEXT Extensin Gene: a Positive Regulator of Resistance Response Against the Oomycete Phytophthora nicotianae

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae.