RESUMO
Proteins of the aegerolysin family have a high abundance in Fungi. Due to their specific binding to membrane lipids, and their membrane-permeabilization potential in concert with protein partner(s) belonging to a membrane-attack-complex/perforin (MACPF) superfamily, they were proposed as useful tools in different biotechnological and biomedical applications. In this work, we performed functional studies on expression of the genes encoding aegerolysin and MACPF-like proteins in Aspergillus niger. Our results suggest the sporulation process being crucial for strong induction of the expression of all these genes. However, deletion of either of the aegerolysin genes did not influence the growth, development, sporulation efficiency and phenotype of the mutants, indicating that aegerolysins are not key factors in the sporulation process. In all our expression studies we noticed a strong correlation in the expression of one aegerolysin and MACPF-like gene. Aegerolysins were confirmed to be secreted from the fungus. We also showed the specific interaction of a recombinant A. niger aegerolysin with an invertebrate-specific membrane sphingolipid. Moreover, using this protein labelled with mCherry we successfully stained insect cells membranes containing this particular sphingolipid. Our combined results suggest, that aegerolysins in this species, and probably also in other aspergilli, could be involved in defence against predators.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Hemolisinas/metabolismo , Perforina/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/genética , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica/genética , Proteínas Hemolisinas/fisiologia , Proteínas de Membrana/metabolismo , Perforina/genética , Esfingolipídeos/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
A TaqMan(®) one-step reverse transcription real-time PCR (RT-qPCR) assay was developed for the specific detection and relative quantitation of Grapevine fanleaf virus (GFLV), the causal agent of grapevine fanleaf degeneration disease. The assay was targeted to a conservative region located in the 2A(HP) gene of the GFLV RNA2 molecule. The assay specificity was evaluated on GFLV isolates from a wide range of geographical regions and on other viruses infecting grapevines. The sensitivity of the developed assay for GFLV detection was approximately 1000-fold higher than the sensitivity of the conventional ELISA. Concentrations as low as 10 genome copies of GFLV per reaction were reliably detected using RT-qPCR. The new method offers a fast, reliable, specific and sensitive identification test for GFLV that is easily applicable for high-throughput diagnosis of GFLV in different types of grapevine material, including dormant phloem scrapings. The quantitative nature of the assay was evaluated by monitoring the seasonal variation of the amount of GFLV present in the plant phloem.
