Differential Roles of the ChiB Chitinase in Autolysis and Cell Death of Aspergillus nidulans

Abstract

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (⌬flbA) mutant autolyzed completely after a 3-day incubation, the ⌬flbA ⌬chiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the ⌬flbA-induced autolysis. However, importantly, both ⌬flbA and ⌬flbA ⌬chiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the ⌬flbA and ⌬flbA ⌬chiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, ␤-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.

Nitric Oxide and ProteinS-Nitrosylation Are Integral to Hydrogen Peroxide-Induced Leaf Cell Death in Rice

“…Thus, S-nitrosylation of both GAPDH and TRX in noe1 but not in wild-type plants parallels the development of cell death in animal systems. Ascorbate peroxidase (Vacca et al, 2004), glutaredoxin (Saeed et al, 2010), acyl carrier protein (Feng et al, 2009), phosphomannomutase (Hoeberichts et al, 2008), Fru-bisP aldolase isozyme (Yao et al, 2004), triose phosphate isomerase (Gnerer et al, 2006), pyruvate kinase (Steták et al, 2007), acidic 27-kD endochitinase (Shin et al, 2009), and lactate/malate dehydrogenase (Matthews et al, 2004) have all previously been reported to be involved in programmed cellular execution (Table I; Supplemental Table S4). However, the molecular mechanisms associated with the functions of these proteins in cell death remain to be established.…”

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls

“…They are thought to be associated with the degradation of empty hyphae supporting fungal growth under carbon starvation conditions [51,52]. In particular, NagA have been suggested to mediate the breakdown of chito-oligomers [45,53]. PrtA role was inferred since deletion of the encoding gene has significantly decreased proteolysis during autolysis [51].…”

NsdD Is a Key Repressor of Asexual Development inAspergillus nidulans

“…However, mRNA accumulation of aflR (encoding an activating TF) and stcU was much reduced in the DnsdD mutant compared to WT. These results indicate that NsdD negatively affects ST production acting downstream of FluG, FlbB, FlbA, and RgsA, likely at a posttranscriptional level.The deletion of flbA causes accelerated and enhanced cell death and autolysis (Lee and Adams 1994b;Shin et al 2009). As DnsdD partially restored conidiation and suppressed autolysis of the DflbA mutant in solid culture, we further quantified its effects on suppressing cell death and hyphal disintegration in the DflbA mutant in liquid culture.…”

“…The supernatant was placed into the 96-well plates excluding mycelial aggregates for analysis of absorbance at A 570 and A 600 . The percentage of AB reduction was detected by Synergy HT (BIO-TEK), using KC4 v3.1 software, and was calculated by a formula, (117,216 3 A 570 of sample -80,586 3 A 600 of sample)/(155,677 3 A 600 of media -14,652 3 A 570 of media) 3 100 (Shin et al 2009). After getting the sample for the AB reduction, the remaining cultures were collected by filtering to determine the mycelial mass.…”

“…Then, 0.5 ml of mycelial aggregates was aliquoted into 1 ml of fresh liquid medium containing 150 ml of the AB reagent. The samples were incubated for 6 hr at 37°in the dark as described in Shin et al (2009). The supernatant was placed into the 96-well plates excluding mycelial aggregates for analysis of absorbance at A 570 and A 600 .…”