Functional Analysis of the ATG8 Homologue Ao atg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

Abstract

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.Autophagy is a protein degradation system that is conserved in eukaryotic cells and used to recycle macromolecules and aid cell survival under nutritional starvation conditions (12, 13). When autophagy is induced, bulk cytoplasm and/or organelles are sequestered within double-membrane vesicles termed autophagosomes. The outer membranes of the autophagosomes then fuse to the vacuolar/lysosomal membrane and deliver single-membrane vesicles, called autophagic bodies, into the lumina of the vacuoles/lysosomes. The subsequent breakdown of the vesicle membranes allows the degradation of the contents of the autophagic bodies by vacuolar hydrolases. ATG8 is an autophagy-related gene found in Saccharomyces cerevisiae that plays an important role in the formation of autophagosomes (9). Atg8 is localized in the membranes of preautophagosomal structures (PAS), autophagosomes, and autophagic bodies and has therefore been used as a marker of these organelles (33).In addition to helping cells to survive starvation, autophagy is involved in stress-induced differentiation and development. In S. cerevisiae diploid cells, atg mutations block starvationinduced sporulation (34). In Dictyostelium discoideum, starvation, overcrowding, and high temperature induce the formation of fruiting bodies and atg mutations block these multicellular developmental processes (25). Additionally, in Caenorhabditis elegans, atg mutations result in abnormal dauer development, which is also induced by starvation, overcrowding, high temperature and so on (17). Recent studies have suggested that autophagy might participate in diseases such as cancer, liver disease, muscula...

Autophagy-assisted glycogen catabolism regulates asexual differentiation inMagnaporthe oryzae

“…6,7,26,27 Apart from an essential role in sexual sporulation in yeast, 45 autophagy has recently been implicated in conidiation and conidial germination in Aspergillus and Magnaporthe. [26][27][28] Our results add to these findings and suggest a functional role for Silver-stained profile of the SDS-PAGE gel depicting total proteins extracted from conidiating mycelial cultures from wild type (WT), atg8Δ or the atg8Δ complemented strain. Proteins differentially regulated and identified by mass spectrometry have been listed together with their predicted molecular masses.…”

“…26 Conidiation defects upon loss of autophagy have been reported recently in Magnaporthe 27 and Aspergillus spps. 28,29 Autophagy was initially defined as a nonselective, bulk degradation system within cells, until the first evidence for specific sequestration of the smooth ER by autophagy was reported. 30 Recent reports on glycogen autophagy, the breakdown of intracellular glycogen reserves within autophagic vacuoles, also showed that autophagy does possess some selectivity.…”

Linkage of autophagy to fungal development, lipid storage and virulence inMetarhizium robertsii

“…2 Deletion of AoATG8 in Aspergillus oryzae leads to the impairment of conidiation and aerial hyphal growth. 15 Disruption of ATG8 in the plant pathogen Ustilago maydis abolishes autophagosome accumulation in the vacuoles and impairs fungal growth and virulence. 16 Besides ATG8, serial gene deletions of other ATG genes in M. orzyae leads to loss of virulence in 16 of 22 null mutants, due to impairment of appressorium differentiation/maturation and conidial programmed cell death.…”

MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae

“…The conidiation was also affected by deletion of the following genes such as funga1 hydrophobin gene MHP1 (Kim et al, 2005), PAK kinase genes CHM1 and MST20 (Li et al, 2004), G-beta subunit gene MGB1 (Nishimura et al, 2003), a blue light receptor gene MgWC-1 (Lee et al, 2006), and a type III integral transmembrane protein encoding gene MTP1 . Although the mechanism of conidiation reduction in ∆moflp1 mutants remains unclear, it was reported that conidium formation relies on the endogenous sources for nutrient supply through autophagy in Aspergillus oryzae or M. oryzae (Kikuma et al, 2006;Liu et al, 2007). Therefore, further study is still needed to confirm the relationships between MoFLP1 gene and other known signal pathways such as autophagy.…”