In search for a role Of NADPH-oxidase generated reactive oxygen species In Aspergillus niger

Mark Arenthorst, Min Jin Kwon, Cees van den Hondel, Arthur Ram

Author address: 

Institute of Biology, Leiden, Netherlands

Abstract: 

RacA encodes a small GTPase that belongs to the sub-family of Rho-like GTPases. Analysis of the phenotype of a RacA deletion strain revealed an effect on polarized growth as well as a strong conidiation defect. To identify proteins that interact with RacA, a yeast two hybrid screen was performed using an activated form of RacA (RacAG12V) protein as bait. This resulted in the identification of two RacAG12V interacting proteins. One of the two RacA interacting protein, shows a strong interaction with both RacAG12V and RacA, and is highly homologous to the mammalian p67phox protein and was named NoxR. In mammalian cells, p67phox is part of the NADPH oxidase complex which is regulated by Rac1 and is active during phagocytosis by producing reactive oxygen species (ROS). To study the possible role of RacA in activation of the NADPH-complex during conidiation, knock out strains of noxR and noxA (the catalytic subunit of the NADPH-complex) were made and phenotypically analyzed. Both knock out strains of noxR and noxA show a slightly reduced conidiation phenotype, whereas the D racA strain shows a severe reduction in conidiation. Apparently, the formation of ROS via the NADPH-complex is not necessary for a-sexual conidiation in A. niger. To examine a possible role for ROS generation in polarized cells growth, the germination and further growth of the noxA and noxR mutants was examined in detail. No difference in germination and no morphological differences were observed in these strains when compared to a wild type strain. We also showed (see also poster by Min Jin Kwon et al) that RacA and CftA are synthetically lethal which suggests an overlapping function of the two GTPases. To study the possible importance of RacA-NoxR mediated ROS production in a D cftA background, double knockouts of cftA with either noxR or noxA were constructed. Both double mutants do not show any additional phenotype compared to the D cftA strain. This indicates that ROS production via the NADPH oxidase complex is dispensable for polarized growth in A. niger, even in a D cftA strain
2008

abstract No: 

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Full conference title: 

9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
    • ECFG 9th (2008)