Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis.

Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.

A Phaseolus vulgaris lipoxygenase gene expressed in nodules and in Rhizobium tropici inoculated roots.

A genomic clone encoding a common bean lipoxygenase (PvLOX5) was isolated from a Phaseolus vulgaris library. Reverse transcription-polymerase chain reaction analysis revealed that PvLOX5 is expressed during nodule development and in Rhizobium tropici inoculated roots. There was no detectable expression of PvLOX5 in non-inoculated roots, healthy leaves, leaves after Pseudomonas syringae pv. tabaci infection, floral buds or dry seeds.

Analysis of lipoxygenase mRNA accumulation in the common bean (Phaseolus vulgaris L.) during development and under stress conditions.

Plant lipoxygenases (LOX, EC 1.13.11.12) have been involved in processes such as stress responses and development. The levels of these enzymes and their corresponding mRNAs are modulated during these processes as well as by different effectors such as jasmonic acid (JA), its methyl ester (MeJA) or abscisic acid (ABA). A new lipoxygenase (LOX) cDNA clone, PvLOX2, was isolated from a Phaseolus vulgaris nodule library and used to study the LOX mRNA accumulation pattern in some developmental stages and in plants subjected to hormone and stress treatments. In nodules, LOX mRNA reaches a maximum level around day 14 to 16 after Rhizobium tropici inoculation, as compared with LOX mRNA present in uninoculated and inoculated roots at the same days. LOX antigen is detected in the nodule parenchyma and in the uninfected cells. During germination, bean LOX transcripts start to accumulate 48 h after imbibition, remains at the same level until 72 h after imbibition and then declines. In hypocotyl, LOX mRNA is abundant in the growing region and almost absent in the mature region. After water stress or ABA treatment, this mRNA increases in the mature region and decreases in the growing region. In bean seedlings, LOX mRNA is accumulated in response to some types of stresses such as cold and desiccation. Wounding, MeJA or ABA treatment of mature leaves also induces LOX mRNA accumulation. These results indicate that in common bean plants LOX is required during development and stress conditions.

An allosteric hammerhead ribozyme.

We have constructed an RNA molecule containing a hammerhead ribozyme that is under allosteric control. In the inactive state, the RNA enzyme is unable to cleave a suitable substrate. The formation of the active state of the ribozyme is triggered by a specific interaction with a DNA oligonucleotide effector that is complementary to a single-stranded loop in the RNA enzyme molecule. Other DNA or RNA molecules containing unrelated nucleotide sequences do not function as allosteric effectors. This work demonstrates the feasibility of designing RNA enzymes that are specifically activated in response to an artificially designed molecular recognition event. Such enzymes may have practical applications.

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L.

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N(2)-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.