RESUMEN
The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae.
Asunto(s)
Quitina/análisis , Escarabajos/metabolismo , Fabaceae/metabolismo , Intestinos/química , Proteínas de Plantas/metabolismo , Plantas Medicinales , Semillas/metabolismo , Animales , Western Blotting , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Quitina/metabolismo , Fabaceae/química , Mucosa Intestinal/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Almacenamiento de Semillas , Semillas/químicaRESUMEN
The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae
Asunto(s)
Animales , Escarabajos/metabolismo , Quitina/análisis , Fabaceae/metabolismo , Intestinos/química , Proteínas de Plantas/metabolismo , Semillas/metabolismo , Western Blotting , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Quitina/metabolismo , Fabaceae/química , Intestinos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Semillas/químicaRESUMEN
Arcelins are insecticidal proteins found in some wild accessions of the common bean, Phaseolus vulgaris. They are grouped in six allelic variants and arcelin-5 is the variant with the highest inhibitory effect on the development of Zabrotes subfasciatus larvae. Characterization of the protein and its genes resulted in the identification of three polypeptides and the isolation of two genes that encode the Arc5a and Arc5b polypeptides. Here we describe a new gene, Arc5-III. The protein it encodes has 81% amino acid identity with the derived amino acid sequences of Arc5-I and Arc5-II. The Arc5-III gene is highly expressed in developing seeds and at a much lower level in roots. Data obtained by a combination of two-dimensional gel electrophoresis, protein sequencing and MALDI-TOF mass spectrometry analysis support the conclusion that Arc5-III encodes a polypeptide present in Arc5c band. Using ion-exchange chromatography, three fractions containing arcelin-5 polypeptides were eluted by increasing the salt concentration. The three fractions contain various amounts of the three arc-5 polypeptides and inhibit the growth of Zabrotes subfasciatus larvae differentially, suggesting differences in insecticidal activity among the arcelin-5 isoforms.
Asunto(s)
Glicoproteínas/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/metabolismo , Fabaceae/genética , Glicoproteínas/biosíntesis , Glicoproteínas/farmacología , Insecticidas/farmacología , Péptidos y Proteínas de Señalización Intercelular , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/farmacología , Plantas Medicinales , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TransfecciónRESUMEN
Some wild accessions of the common bean (Phaseolus vulgaris) contain a family of proteins called arcelins, that are toxic to the larvae of certain bruchid species. Among the six allelic variants of arcelin tested so far, arcelin-5 and arcelin-1 confer the highest level of resistance against the Mexican bean weevil, Zabrotes subfasciatus. The same proteins are not toxic to the bean weevil, Acanthoscelides obtectus, which is also a serious pest of cultivated beans. Arcelins belong to the bean lectin family that includes phytohemaggutinins and alpha-amylase inhibitors. Although homologous to lectins, arcelins are themselves only very weak lectins, and their binding properties have not been clearly established. The toxic properties of arcelins may be related to their recognition of and interaction with the glycoproteins and other constituents of the membranes along the digestive tract of insects. Since arcelin-1 was shown to have growth inhibitory effects for the larvae of Z. subfasciatus but not of A. obtectus, we examined the effect of an arcelin-1 containing diet on the structure of the cells that line the intestinal tract of the larvae of these two bruchid species, and used antibodies against arcelin to examine the distribution of arcelin within the cells and tissues. Here we show that dietary arcelin-1 caused an alteration of the gut structure and the penetration of arcelin into the haemolymph in Z. subfasciatus but not in A. obtectus. These results lead us to suggest that arcelins exert their toxic effect by severely damaging the epithelial cells.