Miguel A. Gómez-Lim

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants.

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.

Expression of the Newcastle disease virus fusion protein in transgenic maize and immunological studies

Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins, including antibodies, antigens and hormones. Here, we report the expression of the fusion (F) gene of the Newcastle disease virus (NDV) in transgenic maize plants. The expression of the transgene, driven by the maize ubiquitin promoter, caused accumulation of the F protein in maize kernels. The presence of the transgene was verified by Southern and western blots. Feeding chickens with kernels containing the F protein induced the production of antibodies, which conferred protection against a viral challenge. This protection was comparable to that conferred by a commercial vaccine. Possible uses of this plant-based F protein as a potential mucosal vaccine are discussed.

Alternative oxidase from mango (Mangifera indica, L.) is differentially regulated during fruit ripening

Alternative oxidase is a respiratory-chain component of higher plants and fungi that catalyzes cyanide-resistant oxygen consumption. The activity of a alternative oxidase has been detected during ripening in several climacteric fruit including mango (Mangifera indica L.). Synthetic oligonucleotides, corresponding to conserved regions of the Sauromatum guttatum and Arabidopsis thaliana nucleotide sequences, were used as primers for polymerase chain reaction to amplify genomic DNA extracted from mango leaves. The 623-bp fragment was found to encode an open reading frame of 207 amino acids showing high identity to the S. guttatum enzyme. Using this fragment to screen a ripe mango mesocarp cDNA library, one full-length cDNA clone, designated pAOMI.1, was obtained that contained an open reading frame encoding a polypeptide of 318 amino acids. The predicted amino-acid sequence exhibited 62, 64 and 68% identity to the S. guttatum, soybean, and A. thaliana enzymes respectively, indicating that this cDNA encodes a mango homologue of the alternative oxidase. Gel blot hybridization showed that pAOMI.1 is likely to be encoded by a single-copy gene. The 1.6 kb-transcript was induced during mango fruit ripening although the transcript was clearly detectable in unripe and developing fruit. Antibodies raised against the S. guttatum enzyme recognized three bands of ≈27, ≈33 and ≈36 kDa from mitochondrial mango proteins. Two of the bands were detectable before ripening and increase in ripe fruit, the other band (27 kDa) was barely present in unripe fruit but accumulated during ripening. The clone pAOMI.1 was able to complement an Escherichia coli hemA mutant deficient in cytochrome-mediated aerobic respiration. This is the first report on the analysis of alternative oxidase at the molecular level during the ripening of a climacteric fruit.

Isolation and characterization of a gene involved in ethylene biosynthesis from Arabidopsis thaliana

The ethylene forming enzyme (EFE) is a key factor in ethylene biosynthesis. To understand better the regulation of ethylene biosynthesis in vegetative tissues, we set out to isolate and characterize a complementary DNA (cDNA) encoding the EFE from Arabidopsis thaliana. An A. thaliana cDNA library was screened with pTOM 13, a tomato cDNA coding for the EFE. A cDNA clone (pEAT1) was isolated. The cDNA is 1200 nucleotides (nt) in length and predicts a protein of M(r) 36,663. The insert includes the complete open reading frame of 972 bp and shows strong homology with several reported sequences, both at the nt and amino acid level. In whole seedlings, expression of pEAT1 was enhanced by wounding, ethrel, Fe2+, and 1-amino-cyclopropane-carboxylic acid (ACC) treatments. In contrast, heat shock had no effect on the expression.

Genetic transformation of perennial tropical fruits

SummaryGenetic transformation provides the means for modifying single horticultural traits in perennial plant cultivars without altering their phenotype. This capability is particularly valuable for perennial plants and tree species in which development of new cultivars is often hampered by their long generation time, high levels of heterozygosity, nucellar embryony, etc. Most of these conditions apply to many tropical and subtropical fruit crops. Targeting specific gene traits is predicated upon the ability to regenerate elite selections of what are generally trees from cell and tissue cultures. The integrity of the clone would thereby remain unchanged except for the altered trait. This review provides an overview of the genetic transformation of perennial tropical and subtropical fruit crops, i.e., citrus (Citrus spp.), banana and plantain (Musa groups AAA, AAB, ABB, etc.), mango (Mangifera indica L.), pineapple (Ananas comosus L.), avocado (Persea americana Mill.), passion fruit (Passiflora edulis L.), longan (Dimocarpus longan Lour.), and litchi (Litchi chinensis Sonn.).

Physical methods for genetic plant transformation.

Production of transgenic plants is a routine process for many crop species. Transgenes are introduced into plants to confer novel traits such as improved nutritional qualities, tolerance to pollutants, resistance to pathogens and for studies of plant metabolism. Nowadays, it is possible to insert genes from plants evolutionary distant from the host plant, as well as from fungi, viruses, bacteria and even animals. Genetic transformation requires penetration of the transgene through the plant cell wall, facilitated by biological or physical methods. The objective of this article is to review the state of the art of the physical methods used for genetic plant transformation and to describe the basic physics behind them.

A novel and highly efficient method for genetic transformation of fungi employing shock waves.

Genetic transformation of filamentous fungi is an essential tool in many areas such as biotechnology, medicine, phytopathology and genetics. However, available protocols to transform fungi are inefficient, laborious and have low reproducibility. We report the use of underwater shock waves as a novel method to transform filamentous fungi. An experimental piezoelectric shock wave generator was designed to expose fungal conidia to heterologous DNA. The device was successfully tested in Aspergillus niger, Fusarium oxysporum, Trichoderma reesei and Phanerochaete chrysosporium. The transformation frequency per number of conidia was between two and four orders of magnitude higher in comparison to previously published methods. For example, the frequency of transformation in A. niger was improved up to 5400-fold as compared with Agrobacterium protocols. Transformation was verified by expression of the green fluorescent protein, PCR and Southern blot. Our method offers new possibilities for fast, easy and efficient genetic manipulation of diverse fungal species.

Induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein

The introduction of exogenous genes into plants permits the development of a new generation of biological products, i.e., edible vaccines. Cereals, especially maize, have been the systems of choice for the expression of antigenic proteins because the proteins can be expressed at high levels in the kernel and stored for prolonged periods without excessive deterioration. The utilization of plant-derived antigens for oral delivery provides an alternative strategy for the control of pathogens in animals compared to the current vaccine administration methods, such as injection. However, there is some doubt about the efficacy of these types of vaccines in polygastric animals due to the features of their digestive system. Here, we report the efficacy of an edible vaccine against rabies evaluated in sheep. Kernels containing different doses of G protein (0.5, 1, 1.5 and 2mg) were given in a single dose by the oral route. Cumulative survival was better in groups that received 2mg of G protein and for the positive control (inactivated rabies vaccine); this observation was supported by the presence of neutralizing antibodies. Animals in the control group died after challenge. The degree of protection achieved for 2mg of G protein was comparable to that conferred by a commercial vaccine. In conclusion, this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model.

The abundant 31-kilodalton banana pulp protein is homologous to class-III acidic chitinases

We have identified and characterized the abundant protein from the pulp of banana fruit (Musa acuminata cv. Grand Nain), and have isolated a cDNA clone encoding this protein. Comparison of the amino terminal sequence of the purified 31 kDa protein (P31) suggests that it is related to plant chitinases. Western analyses utilizing rabbit anti-P31 antiserum demonstrate that this protein is pulp-specific in banana. A full-length cDNA clone homologous to class III acidic chitinase genes has been isolated from a pulp cDNA library by differential screening. The identity of this clone as encoding P31 was verified by comparisons between the amino-terminal peptide sequence and the cDNA sequence and cross-hybridization of the translation product of the cDNA clone with P31 antiserum. Northern and western blot analyses of RNA and protein isolated from banana pulp at different stages of ripening indicate that the cDNA and protein are expressed at high levels in the pulp of unripe fruit, and that their abundance decreases as the fruit ripens. Based on its expression pattern and deduced amino acid sequence and composition, we hypothesize that the physiological role of P31 is not for plant protection, but as a storage protein in banana pulp.

Peroxisomal thiolase mRNA is induced during mango fruit ripening

Fruit ripening is a complex, developmentally regulated process. A series of genes have been isolated from various ripening fruits encoding enzymes mainly involved in ethylene and cell wall metabolism. In order to aid our understanding of the molecular basis of this process in a tropical fruit, a cDNA library was prepared from ripe mango (Mangifera indica L. cv. Manila). By differential screening with RNA poly(A)+ from unripe and ripe mesocarp a number of cDNAs expressing only in ripe fruit have been isolated. This paper reports the characterization of one such cDNA (pTHMF 1) from M. indica which codes for a protein highly homologous to cucumber, rat and human peroxisomal thiolase (EC 2.3.1.16), the catalyst for the last step in the β-oxidation pathway.The cDNA for the peroxisomal mango thiolase is 1305 bp in length and codes for a protein of 432 amino acids with a predicted molecular mass of 45 532 Da. Mango thiolase is highly homologous to cucumber thiolase (80%), the only other plant thiolase whose cloning has been reported, and to rat and human thiolases (55% and 55% respectively).It is shown by northern analysis that during fruit ripening THMF 1 is up-regulated. A similar pattern of expression was detected in tomato fruit. Wounding and pathogen infection do not appear to affect THMF 1 expression. The possible involvement of thiolase in fatty acid metabolism during fruit ripening will be discussed. To our knowledge this is the first report cloning of a plant gene involved in fatty acid metabolism showing an induction during fruit ripening.