A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger

Abstract

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes.

Updating genome annotation for the microbial cell factoryAspergillus nigerusing gene co-expression networks

“…In this study, we performed a transcriptomic meta-analysis to generate a high-quality gene coexpression network, and used this to predict biological processes for 9,579 (~65%) of all A. niger Depicted are 23 predicted transcription factors in the anafp sub-network which either positively (19) or negatively (4) correlate with the expression of the anafp gene. Two of these (velC and stuA) were selected for in vivo verification by establishing velC and stuA deletion strains in an anafp::mluc reporter strain published earlier (strain PK2.9) (24). (B, C) Analysis of anafp promoter activity in a strain deleted for velC (strain BBA17.9) and for stuA (strain BBA13.2), respectively, which was compared to the progenitor strain PK2.9 abbreviated as 'wt'.…”

“…Ribosomally synthesized antifungal peptides of the AFP family are promising molecules for use in medical or agricultural applications to combat humanand plant-pathogenic fungi (40). We and others could show that expression of their cognate genes are under tight temporal and spatial regulation in their native hosts and precedes asexual sporulation (24,(41)(42)(43).…”

“…The gene encoding AnAFP (An07g01320), is co-expressed with 986 genes (ρ ≥ |0.5|; 605 positively correlated / 381 negatively correlated (24)). GO enrichment analyses of positively correlated sub-networks uncovered that anafp gene expression parallels with fungal secondary metabolism, carbon limitation and autophagy (24). In total, 23 predicted transcription factors are co-expressed at a stringent level among which were the transcription factors VelC (An04g07320) and StuA (An05g00480; Fig.…”

“…We normalized and interrogated gene expression across 155 published transcriptomic analyses for the A. niger laboratory wildtype strain N402 (ATCC 64974) and its descendants, comprising of 283 Affymetrix microarray experiments in total (24). Experimental parameters include a diverse range of cultivation conditions (agar plate, bioreactor, shake flask), developmental and morphological stages (germination, mycelial growth, sporulation), deletion and disruption mutants, stress conditions (antifungals, secretion stress, pH), different carbon and nitrogen sources, starvation, and co-cultivation with bacteria.…”

When green and red mycology meet: Impressions from an interdisciplinary forum on virulence mechanisms of phyto- and human-pathogenic fungi

“…The session was closed by Sascha Jung from Berlin, Germany, proposing a novel biologic role of the A. niger antifungal protein AnAFP. 47 All contributions, together with the ones that had not been nominated for oral presentation, were also on display as posters throughout the entire event to support their scientific debate. This was further promoted by a dedicated lunch break poster session sponsored by BIOMED CENTRAL and the open access journal Fungal Biology and Biotechnology.…”