German Title: Identifizierung von hoch regulierten Genen des hyphomyceten Pilzes, Beauveria bassiana, während der Infektion von Leptinotarsa decemlineata
The aim of this research was the identification of genes over-expressed or specifically expressed by the insect pathogenic fungus, B. bassiana during infection of the Colorado potato beetle, L. decemlineata. Two different approaches for the study of B. bassiana gene expression were used: (1) gene expression in an aritficial context of infection, with the culture of the fungus in the presence of its host cuticle as inductor of the pathogenic process, (2) gene expression in a natural context of infection, with B. bassiana infecting the larvae of the Colorado potato beetle. In both cases, the method used was the cDNA representational difference analysis (cDNA RDA). The cDNA RDA has for main advantages the need of low quantities of RNA, an enrichment of the differences between two mRNA populations, and thus, a low number of false positives. Indeed, among the difference products cloned from the two last cDNA RDAs, no false-positives were found. This confirmed the adapted culture conditions for such a study with this sensitive method and the general low-rate of false-positives using cDNA RDA (i.e. in comparison with the differential-display). Eleven genes were specifically expressed or over-expressed in the cuticle medium cultures of B. bassiana. These genes encode three putative hydrolases (choline sulfatase, serine peptidase, a/b-hydrolase enzyme), three probable transporters (ABC transporter, siderophore transporter, oligopeptide transporter), a presumed metabolic enzyme (pyruvate decarboxylase), a probable V-ATPase, two putative mucin-like proteins and a putative protein, which shares homology with an unknown ORF of the ascomycete N. crassa. Grown in the presence of its host cuticle, B. bassiana seemed to over-expressed several hydrolases probably involved in the degradation of the cuticle. B. bassiana seemed to up-regulate also the expression of genes involved in stress response (i.e., siderophore transporter, choline sulfatase), detoxification (ABC transporter) and transmembrane transport (oligopeptide transporter, V-ATPase). The two putative mucin-like proteins might play a determinant role in pathogenicity. Some putative pathogenicity determinants of B. bassiana identified during this PhD opened new perspectives in the research on the pathogenic process of hyphomycetes. A functional study of the putative siderophore transporter of B. bassiana could be first realized in S. cerevisiae, because some strains lacking the high-affinity iron uptake system are available. The probable MDR-type ABC transporter identified in B. bassiana should confer resistance to diverse drugs. Thus, the effects of several toxic compounds should be tested in the wild type and deletion/promoter mutants. A parallel Northern blot analysis, using the corresponding cDNA RDA fragment as probe, would enable the confirmation of the transcriptional up-regulation of this presumed ABC transporter by toxicants. The cDNA sequence of the two putative mucin-like proteins should be cloned. The antisera raised against these two proteins would then be used in in situ hybridisation. This experiment could verify the hypothesis of the localization of these putative mucin proteins to the germ tube tip before the penetration of the insect cuticle. Finally, a differential display approach would be probably more adapted than the cDNA RDA to identified up-regulated gene expression of B. bassiana in a natural context of infection. Such a study is indispensable to understand really the host-pathogen interactions during infection by entomopathogenic fungi This thesis marks one supplementary step in our understanding of the mechanisms of fungal pathogenesis in insect, which could further allow the production of more efficient mycoinsecticides and contribute to reductions in chemical pesticides use.
|Supervisor:||Schairer, Prof. Dr. Hans Ulrich|
|Date of thesis defense:||24 July 2003|
|Faculties / Institutes:||Service facilities > Center for Molecular Biology Heidelberg|
|Subjects:||570 Life sciences|
|Controlled Keywords:||Entomopathogene Pilze, Biologische Insektenbekämpfung, Infektion, Virulenzfaktor|