Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae.

Microbial secretory phospholipases A(2) (sPLA(2)s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA(2)s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA(2)s. Two sPLA(2)s differ in pH optimum, Ca(2+) requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA(2) overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA(2) overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either DeltasplaA or DeltasplaB mutants, hyphal growth of DeltasplaB, but not that of DeltasplaA, displayed increased sensitivity to H(2)O(2) treatment. These data indicate that two A. oryzae sPLA(2) enzymes display distinct, presumably non-redundant, physiological functions.

Light represses conidiation in koji mold Aspergillus oryzae.

In the filamentous fungus Aspergillus oryzae, there has been no report on photoreaction. Here we investigated the effect of light in A. oryzae and found that conidiation was repressed by white light. This reaction is contrary to that of other Aspergilli, which show abundant conidiation under light. Moreover, red light also caused reduced conidiation. Genome sequencing of A. oryzae indicated the existence of homologs of some light-related genes in other filamentous fungi. To approach the molecular mechanism of this photoresponse, the effect of red light on the expression level of several genes putatively responsible for conidiation or photoperception, i.e., brlA, a gene known to be required for conidiation, AofphA, the putative homolog of the A. nidulans phytochrome gene fphA, and AoveA, the putative homolog of the negative regulator gene in conidiation in A. nidulans, was examined. These three genes showed no significant response to red light at the transcriptional level. The results indicate that A. oryzae perceives and responds to red light in a manner independent of the transcriptional regulation of these genes.

Visualization of the endocytic pathway in the filamentous fungus Aspergillus oryzae using an EGFP-fused plasma membrane protein.

Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner.