Alejandro Fuentes

Single-chain antibody fragments specific to the hepatitis B surface antigen, produced in recombinant tobacco cell cultures.

An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-histidine N-terminal tag was produced in cultured transgenic tobacco cells. Western blot and antigen-specific chromatography showed high levels of biologically active scFv in the culture supernatant (1 mg l−1) and in cells (5 mg kg−1). A simple one-step scFv purification was developed using immobilized metal ion affinity chromatography.

Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation

A transient gene‐expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules. The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way. Conditions such as pre‐culture of tobacco cells and the co‐cultivation period were identified as determinants to achieve high expression levels. Under established conditions the activity strength of CaMV (cauliflower mosaic virus) 35 S and ToMoTV (tomato mottle taino virus) AL1 promoters were compared. A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western‐blot analysis. A monoclonal antibody against anti‐(hepatitis B virus surface antigen) was produced in such quantities as to allow testing of biological activity and preliminary characterization.

Field Trial and Molecular Characterization of RNAi-Transgenic Tomato Plants That Exhibit Resistance to Tomato Yellow Leaf Curl Geminivirus.

RNA interference (RNAi) is a widely used approach to generate virus-resistant transgenic crops. However, issues of agricultural importance like the long-term durability of RNAi-mediated resistance under field conditions and the potential side effects provoked in the plant by the stable RNAi expression remain poorly investigated. Here, we performed field trials and molecular characterization studies of two homozygous transgenic tomato lines, with different selection markers, expressing an intron-hairpin RNA cognate to the Tomato yellow leaf curl virus (TYLCV) C1 gene. The tested F6 and F4 progenies of the respective kanamycin- and basta-resistant plants exhibited unchanged field resistance to TYLCV and stably expressed the transgene-derived short interfering RNA (siRNAs) to represent 6 to 8% of the total plant small RNAs. This value outnumbered the average percentage of viral siRNAs in the nontransformed plants exposed to TYLCV-infested whiteflies. As a result of the RNAi transgene expression, a common set of up- and downregulated genes was revealed in the transcriptome profile of the plants selected from either of the two transgenic events. A previously unidentified geminivirus causing no symptoms of viral disease was detected in some of the transgenic plants. The novel virus acquired V1 and V2 genes from TYLCV and C1, C2, C3, and C4 genes from a distantly related geminivirus and, thereby, it could evade the repressive sequence-specific action of transgene-derived siRNAs. Our findings shed light on the mechanisms of siRNA-directed antiviral silencing in transgenic plants and highlight the applicability limitations of this technology as it may alter the transcriptional pattern of nontarget genes.

Macroptilium yellow mosaic virus, a New Begomovirus Infecting Macroptilium lathyroides in Cuba

Macroptilium lathyroides (L) is a weed that is widely distributed in Cuba. Frequently, leaves show bright yellow mosaic symptoms, which suggest the incidence of a viral disease. Since begomovirus occurrence in Macroptilium lathyroides has been previously reported in other islands of the Caribbean (1,3), symptomatic plants from three distant places in Cuba (Havana, Villa Clara, and Camaguey), were collected and tested for the presence of begomoviruses. Plant DNA extracts were analyzed by Southern blot hybridization and polymerase chain reaction with two sets of degenerate primers (2). The presence of a bipartite begomovirus was evident through strong hybridization signals obtained with the DNA-A and DNA-B of Taino tomato mottle virus as probes at low stringency. Furthermore, 1.4-kb and 1.2-kb PCR amplified fragments were obtained with DNA-A degenerate primers, PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722, respectively. Both PCR fragments from the samples from the three locations were cloned, and restriction fragment length polymorphism analysis of the 1.4-kb fragments were performed using PstI, EcoRI, HincII, XbaI and BglII. Restriction fragment patterns were the same for the three clones. The DNA-A sequence (GenBank Accession No. AJ344452) of the isolate from Villa Clara was compared with sequences available for other geminiviruses using CLUSTAL program. For the coat protein (CP) gene, the comparisons had the highest percentage of identity with various strains of Bean golden yellow mosaic virus (BGYMV, GenBank Accession Nos. AF173555, M91604, and L01635) (85 to 87% and 93 to 94%, nucleotide and amino acid sequences, respectively). For Rep gene (1,044 nt), the best percentages of identities were with BGYMV (81 to 82% and 80 to 82% nucleotide and amino acid sequences, respectively), Tomato leaf crumple virus (GenBank Accession No. AF101476) (78 and 81%, nucleotide and amino acid sequences, respectively), and Sida golden mosaic virus from Florida (GenBank Accession No. AF049336) (78 and 79%, nucleotide and amino acid sequences, respectively). Finally, the comparative analysis of the intergenic region (i.e. the common region plus the CP gene promoter) had the highest identity with BGYMV (56 to 55%) and Tomato severe rugose virus (GenBank Accession No. AY029750) (49%). Interestingly, this virus has in this region the three G-box elements that are characteristic of BGYMV but it differs in the Rep protein-binding iterative motif that is GGTGA instead of GGAGA, for BGYMV. These data indicate that this virus is a new begomovirus and the name of Macroptilium yellow mosaic virus (MaYMV) is proposed. References: (1) A. M. Idris et al. Plant Dis. 83:1071, 1999. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) M. E. Roye et al. Plant Dis. 81:1251, 1997.

Cuban Isolate of Bean golden yellow mosaic virus is a Member of the Mesoamerican BGYMV Group

In Cuba, the emergence of bean golden mosaic was associated with high populations of Bemisia tabaci in common bean (Phaseolus vulgaris L.) plantings in the 1970s (1). During the last two decades, the disease has caused significant economic losses, forcing some growers to abandon bean production. In Holguín, one of the main bean producing provinces of the country, about 2,000 ha of beans were abandoned in 1991 due to the high incidence of this whitefly-transmitted virus. At that time, yield losses associated with this disease reached 90 to 100% in farmers fields. In spite of various control measures, the disease affected 33, 28, and 6.5% of the total area planted in Cuba to common bean in 1990, 1992, and 1996, respectively. For this investigation, common bean leaves showing systemic yellowing symptoms were collected in fields located in the provinces of Havana, Matanzas, and Holguín during 1998-1999. Sap and total DNA leaf extracts were used to inoculate healthy bean plants by manual and biolistic procedures, respectively. Characteristic yellowing symptoms were more efficiently reproduced using a particle gun device than by manual inoculation (18/20 plants and 5/20 plants, respectively, for a Holguín virus isolate). DNA extracts were further analyzed by polymerase chain reaction using two degenerate primer sets: PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (2). Fragments of approximately 1.4 and 1.2 kb were amplified and cloned. Restriction fragment length polymorphism analysis of the cloned 1.4-kb fragments was performed with BglII, HincII, SalI, EcoRI, PstI, and XbaI, indicating that selected isolates from the three Cuban provinces shared identical restriction patterns. The nucleotide sequence obtained from two clones of a virus isolate from Holguín, was compared to sequences available for other begomoviruses using BLAST. The Cuban isolate shared up to 94% nt sequence identity with various strains of Bean golden yellow mosaic virus (BGYMV) in the first 250 nt of the rep gene. For the common region (CR), scores were 93% for BGYMV-GA (Guatemala), 92% for BGYMV-MX (southern Mexico) and BGYMV-PR (Puerto Rico), and 91% for BGYMV-DR (Dominican Republic). The iterative sequence ATGGAG was identified in the CR of the Cuban BGYMV isolate, as reported for other BGYMV isolates. Finally, the Cuban begomovirus, hereafter referred to as BGYMV-CU, shared nt and aa sequence identities of 94 and 100%, respectively, with the coat protein gene of BGYMV-MX. We conclude that the begomovirus isolated from mosaic-affected common bean plants in the province of Holguín is a member of the Mesoamerican BGYMV group (3). References: (1) N. Blanco and C. Bencomo. Cienc. Agric. 2:39, 1978. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) Morales and Anderson, Arch. Virol. 146:415, 2001.

Production of Plantibodies in Nicotiana Plants

Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from the isolation of the monoclonal antibody genes to the biochemical and biological characterization of the plant-expressed monoclonal antibody are described.

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%.