Testing the efficacy of RNA interference constructs in Aspergillus fumigatus.
We recently developed a silencing vector in Aspergillus fumigatus which carries a hygromycin resistance marker and a transcriptional unit for hairpin RNA expression under the control of the inducible glucoamylase promoter (pGla) (Mouyna et al. in FEMS Microbiol Lett 237:317-324, 2004). We showed previously that this vector can be used for the RNA interference application of two genes ALB1 and FKS1 of which reduced mRNA levels occurred for both, with phenotypic consequences resembling disruptions of genes involved in melanin (ALB1) and beta(1-3)glucan biosynthesis (FKS1). We reported here the silencing of KRE6 and CRH1, two other genes putatively involved in cell wall biosynthesis using a similar construction under the control of the constitutive promoter glyceraldehyde-3-phosphate dehydrogenase (pgpdA). Silencing of the expression of these two genes was obtained. Further analysis of the transformants showed however that (1) a 100% loss of expression was never achieved for all genes tested (2) the vector used for RNAi is lost or modified over successive transfers resulting in an inhibition of the silencing. These disadvantages of RNAi indicate that classical gene disruption by gene replacement remains the most efficient method for a molecular analysis of gene function in A. fumigatus.
Henry
Christine
C
Institut Pasteur, Unité des Aspergillus, 25 rue du Docteur Roux, 75724, Paris Cedex 15, France. imouyna@pasteur.fr
Mouyna
Isabelle
I
Latgé
Jean-Paul
JP
D001232:Aspergillus fumigatus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D005964:Glucosyltransferases / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D014170:Transformation, Genetic
D017931:DNA Primers; D004271:DNA, Fungal; C034070:1,6-beta-glucan synthetase; D005964:Glucosyltransferases; D006026:Glycoside Hydrolases