Transgenic plants: an emerging approach to pest control.

Insect pests are a major cause of damage to the world's commercially important agricultural crops. Current strategies aimed at reducing crop losses rely primarily on chemical pesticides. Alternatively transgenic crops with intrinsic pest resistance offer a promising alternative and continue to be developed. The first generation of insect-resistant transgenic plants are based on insecticidal proteins from Bacillus thuringiensis (Bt). A second generation of insect-resistant plants under development include both Bt and non-Bt proteins with novel modes of action and different spectra of activity against insect pests.

The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects.

The Vip3A protein is a member of a newly discovered class of vegetative insecticidal proteins with activity against a broad spectrum of lepidopteran insects. Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the black cutworm (Agrotis ipsilon) and fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelium cells resulting in larval death at concentrations above 40 ng/cm2. The European corn borer (Ostrinia nubilalis), a nonsusceptible insect, does not develop any pathology upon ingesting Vip3A. While proteolytic processing of the Vip3A protein by midgut fluids obtained from susceptible and nonsusceptible insects is comparable, in vivo immunolocalization studies show that Vip3a binding is restricted to gut cells of susceptible insects. Therefore, the insect host range for Vip3A seems to be determined by its ability to bind gut cells. These results indicate that midgut epithelium cells of susceptible insects are the primary target for the Vip3A insecticidal protein and that their subsequent lysis is the primary mechanism of lethality. Disruption of gut cells appears to be the strategy adopted by the most effective insecticidal proteins.

Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.

A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from Bacillus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in approximately 15% of Bacillus strains analyzed. The sequence of the vip3A(b) gene, a homologue of vip3A(a) isolated from B. thuringiensis strain AB424 is also reported. Vip3A(a) and (b) proteins confer upon Escherichia coli insecticidal activity against the lepidopteran insect larvae mentioned above. The sequence of the gene predicts a 791-amino acid (88.5 kDa) protein that contains no homology with known proteins. Vip3A insecticidal proteins are secreted without N-terminal processing. Unlike the B. thuringiensis 5-endotoxins, whose expression is restricted to sporulation, Vip3A insecticidal proteins are expressed in the vegetative stage of growth starting at mid-log phase as well as during sporulation. Vip3A represents a novel class of proteins insecticidal to lepidopteran insect larvae.

Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase.

A CryV-type protein (CGCryV) has been isolated from supernatant fluids of Bacillus thuringiensis AB88 cultures. Previous reports have suggested the cryptic nature of the cryV-type genes on the basis of the absence of CryV-type proteins in parasporal crystals. The CryV-type protein reported here is expressed early in stationary phase, and evidence indicates that it is an exported protein. Analysis of the deduced protein sequence from this gene reveals the presence of an N-terminal domain that likely acts as a signal peptide. The CGCryV protein is the first reported case of a delta-endotoxin being a secreted protein, which may influence the biological relevance of these proteins.

Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase.

A calcium-dependent calmodulin-independent protein kinase (CDPK) has been cloned from maize (Zea mays). The sequence predicts a 550-amino acid (predicted molecular mass is 60 kDa) protein with two major functional domains: an N-terminal catalytic domain highly homologous to protein kinases and a C-terminal domain resembling calmodulins. Northern analysis shows that the expression of the maize CDPK gene is pollen specific and that its transcription is restricted to late stages of pollen development. Western blots reveal a major abundance of CDPK protein at the stage of pollen germination. In vitro germination and pollen tube growth are impaired upon addition of a calmodulin antagonist (calmidazolium), CDPK inhibitors (W-7), and antisense oligonucleotides directed against CDPK mRNA. These observations indicate that the function of the pollen-specific maize CDPK protein is required for germination and pollen tube growth.

Plant activating sequences: positively charged peptides are functional as transcriptional activation domains.

Plant sequences that act as transcriptional activation domains in yeast as well as in plants have been isolated by genetic selection in yeast. The selection was based on the reconstitution of a functional GAL4 transcriptional activator. Since the peptides show no homology with reported activation domains, they represent a new class of activating sequences. The sequence P1, which is 10 amino acids long, is the shortest functional activation domain reported. A cDNA that encodes the P14 class (peptides P14-P18) activating sequence have been cloned. The protein exhibits strong homology (higher than 50% amino acid identity) with the BBC1-related sequences, a highly conserved family of basic proteins containing nuclear localization signals. The P14 and P15 peptides are the most effective plant activating sequences. The P14 and P15 peptides are highly hydrophilic, positively charged and mostly unstructured. These properties are at odds with the ones usually found in known activation domains.

Floral development and expression of floral homeotic genes are influenced by cytokinins.

Tobacco plants that are somatic mosaics for the expression of a cytokinin-synthesizing gene have viviparous leaves. Epiphyllous buds can be either vegetative or floral. Floral adventitious buds can be either normal or abnormal. Abnormalities of floral development correlate with: (i) a local activation of the cytokinin-synthesizing gene, (ii) a drastic increase in floral cytokinin content, and (iii) a decrease in the steady-state levels of mRNA homologous of the homeotic genes DEFA, GLO and PLENA of Antirrhinum majus. Thus, these data show in planta that cytokinins, a class of phytohormones, are able to alter the development of floral organs and to decrease the expression of three homeotic floral genes.

NTGLO: a tobacco homologue of the GLOBOSA floral homeotic gene of Antirrhinum majus: cDNA sequence and expression pattern.

We report the cloning and DNA sequence of a cDNA from Nicotiana tabacum, NTGLO, as well as the pattern of expression of the NTGLO gene in wild-type tobacco plants. The NTGLO cDNA encodes a protein of 209 amino acids, which shows 73% identity with the GLO protein encoded by the GLO gene of Antirrhinum majus, a homeotic gene involved in the genetic control of flower development. Northern blot analysis shows that the NTGLO gene is expressed mainly in floral organs and, within the flower, expression is restricted to petals and stamens. The NTGLO gene most probably represents a true homologue of the GLO gene because: i) the MADS boxes, of the two genes are highly homologous (56 out of 58 amino acids are identical): ii) at the carboxy-terminal a block of 19 amino acids is perfectly conserved between the NTGLO and GLO proteins and iii) their expression patterns in floral organs are identical.

Anther-specific expression of the rolB gene of Agrobacterium rhizogenes increases IAA content in anthers and alters anther development and whole flower growth.

The promoter activity of a 2.2-kb DNA fragment from the 5' flanking sequence of the tap1 gene of snapdragon has been characterised in tobacco with the ß-glucuronidase reporter gene. The tap1 promoter conferred to the reporter gene an expression that was limited to the early stages of anther development. Expression of the rolB gene of Agrobacterium rhizogenes under the control of the tap1 promoter impaired the development of tobacco flowers. Tobacco plants transgenic for the tap1-rolB chimeric gene showed altered anthers and a reduction in whole flower growth. Such growth alterations were correlated with an increase in free IAA content and a decrease in gibberellin activity present in anthers. Since the rolB gene codes for an indole ß-glucosidase (Estruch et al. 1991), we interpret the phenotypic alterations to be a consequence of the increased content and activity of auxin in anthers. The perturbation of anther development would in turn affect gibberellin production and content, which is reflected in a reduced elongation of flowers.

The protein encoded by the rolB plant oncogene hydrolyses indole glucosides.

The rolB gene of Agrobacterium rhizogenes, whose expression stimulates the formation of roots by transformed plant tissues and other growth alterations in transgenic plants, codes for a beta-glucosidase able to hydrolyse indole-beta-glucosides. Indeed, we show that extracts of bacteria and/or plant tissue expressing the rolB protein hydrolyse indoxyl-beta-glucoside (plant indican). Because of the structural similarity between indoxyl-beta-glucoside and indole-3-acetyl-beta-glucoside (IAA-beta-glucoside), we propose that the physiological and developmental alterations in transgenic plants expressing the rolB gene could be the result of an increased intracellular auxin activity caused by the release of active auxins from inactive beta-glucosides. Thus two of the oncogenes carried by the T-DNA of the plant pathogen Agrobacterium rhizogenes (rolB and rolC) perturb plant growth and development by coding for beta-glucosidases with distinct specificities. Whereas the rolC beta-glucosidase releases cytokinins from their glucoside conjugates, the rolB encoded protein hydrolyses indole-beta-glucosides. The combined action of these two genes therefore is expected to modulate the intracellular concentration of two of the main growth factors active in plants.

Viviparous leaves produced by somatic activation of an inactive cytokinin-synthesizing gene.

Tobacco plants that are somatic mosaics for expression of a cytokinin-synthesizing gene have viviparous leaves. Such a formation of shoots in an abnormal position represents a significant deviation from the usual organization of the plant body where a central axis produces shoots only in the axils of lateral leaf appendages and according to a precise phyllotactic pattern. This report links vivipary to the expression of a gene whose product is involved in the synthesis of the phytohormone cytokinin.

The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates.

The rolC gene of Agrobacterium rhizogenes, which drastically affects growth and development of transgenic plants, codes for a cytokinin-beta-glucosidase. Indeed, rolC protein expressed in Escherichia coli as a fusion protein hydrolyses cytokinin glucosides, thus liberating free cytokinins. Furthermore, beta-glucosidase activity present in E. coli extracts expressing the rolC protein was inhibited by affinity-purified antibodies specific for the rolC protein. Finally, rolC proteins expressed in transgenic plants were shown to be responsible for cytokinin-beta-glucosidase activity. Morphological and phytohormonal analysis, performed on transgenic plants that are somatic mosaics for the expression of the rolC gene, extend and confirm our interpretation that the developmental, physiological and morphological alterations caused by rolC expression in transgenic plants are primarily due to a modification of the cytokinin balance. These observations shed new light on the control of growth and differentiation in plants by growth factors.

Cytosolic localization in transgenic plants of the rolC peptide from Agrobacterium rhizogenes.

The rolC gene of Agrobacterium rhizogenes codes for a peptide with an apparent molecular weight of approximately 20 kDa. Immunolocalization of the rolC peptide, in leaves of transgenic plants which are genetic mosaics for the expression of the rolC gene, is restricted to the phenotypically altered sectors. Subcellular fractionation of homogenates from 35S-rolC transgenic leaves shows the cytosolic localization of the rolC peptide.

Gibberellic acid stimulates acid invertase secretion in pea ovary protoplasts.

Protoplasts purified from mesocarp of nonpollinated pea (Pisum sativum L.) ovaries released acid invertase to the incubation medium. The association of the acid invertase with microsomal fractions, and the sensitivity to energy-metabolism inhibitors and to tunicamycin, indicated the secretory nature of the release process. In the presence of GA3 (10 microM), the protoplasts increased their invertase secretion at about 60 min, this effect being counteracted by tunicamycin but not by cycloheximide. Subcellular fractionation of GA3-treated protoplasts showed that higher invertase secretion was the result of a promotion of invertase transfer from endoplasmic reticulum (ER) to Golgi apparatus.

Sucrose Loading in Isolated Veins of Pisum sativum: Regulation by Abscisic Acid, Gibberellic Acid, and Cell Turgor.

Enzymatically isolated vein networks from mature pea (Pisum sativum L. cv Alaska) leaves were employed to investigate the properties of sucrose loading and the effect of phytohormones and cell turgor on this process. The sucrose uptake showed two components: a saturable and a first-order kinetics system. The high affinity system (K(m), 3.3 millimolar) was located at the plasmalemma (p-chloromercuriphenylsulfonic acid and orthovanadate sensitivity). Further characterization of this system, including pH dependence and effects of energy metabolism inhibitors, supported the H(+)-sugar symport concept for sucrose loading. Within a physiological range (0.1-100 micromolar) and after 90 min, abscisic acid (ABA) inhibited and gibberellic acid (GA(3)) promoted 1 millimolar sucrose uptake. These responses were partially (ABA) or totally (GA(3)) turgor-dependent. In experiments of combined hormonal treatments, ABA counteracted the GA(3) positive effects on sucrose uptake. The abolishment of these responses by p-chloromercuriphenylsulfonic acid and experiments on proton flux suggest that both factors (cell turgor and hormones) are modulating the H(+) ATPase plasmalemma activity. The results are discussed in terms of their physiological relevance.