Isolation and characterization of polygalacturonase genes (pecA and pecB) from Aspergillus flavus

Abstract

Two genes, pecA and pecB, encoding endopolyglacturonases were cloned from a highly aggressive strain of Aspergillus flavus. The pecA gene consisted of 1,228 bp encoding a protein of 363 amino acids with a predicted molecular mass of 37.6 kDa, interrupted by two introns of 58 and 81 bp in length. Accumulation of pecA mRNA in both pectin-or glucose-grown mycelia in the highly aggressive strain matched the activity profile of a pectinase previously identified as P2c. Transformants of a weakly aggressive strain containing a functional copy of the pecA gene produced P2c in vitro, confirming that pecA encodes P2c. The coding region of pecB was determined to be 1,217 bp in length interrupted by two introns of 65 and 54 bp in length. The predicted protein of 366 amino acids had an estimated molecular mass of 38 kDa. Transcripts of this gene accumulated in mycelia grown in medium containing pectin alone, never in mycelia grown in glucose-containing medium, for both highly and weakly aggressive strains. Thus, pecB encodes the activity previously identified as P1 or P3. pecA and pecB share a high degree of sequence identity with polygalacturonase genes from Aspergillus parasiticus and Aspergillus oryzae, further establishing the close relationships between members of the A. flavus group. Conservation of intron positions in these genes also indicates that they share a common ancestor with genes encoding endopolyglacturonases of Aspergillus niger.

pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger

“…When compared with other genes, the A. niger pga genes are highly identical in structural organization of introns and exons with those of A. tubingensis [28], A. oryzae [29], A. fla us [13] and A. parasiticus [30].…”

“…However, little is known about the role of the different PGs in these micro-organisms. Only recently, studies on pga gene expression in the phytopathogenic fungi Colletotrichum lindemuthianum [12] and A. fla us [13], and the analysis of secreted PG isoforms during the growth of S. sclerotiorum on polygalacturonic acid [14] showed differential expression of these genes and secretion of the proteins. These results suggest that, although fungal PGs often show a high degree of sequence identity, their physiological function is different.…”

Analysis of a polygalacturonase gene of Ustilago maydis and characterization of the encoded enzyme

“…A typical reaction mixture contained 500 ml of sample and 500 ml of substrate (PGA 0.5%, w/v), which was incubated for 1 h at 37°C. The temperature effect was determined by incubating the reaction assay at 28,34,37,40,42,45, and 50°C in a water bath. The pH effect was determined at 34°C with 50 mM sodium acetate buffer at pH of 4.3, 4.5, 4.7, 4.9, 5.0, 5.3, 5.6, and 6.0.…”

The Aspergilli and Their Mycotoxins: Metabolic Interactions With Plants and the Soil Biota

“…The plant cell wall, the first line of defense against microbial pathogens, is primarily made up of polysaccharides cellulose, hemicellulose, and pectin. While opportunistic fungi usually infect plants through wounds (e.g., mechanical or pest damages), pathogenic ones actively penetrate cell walls, often through the secretion of a range of polysaccharide-degrading enzymes such as pectinesterase, arabinofuranosidase, mannosidase, and galacturonidase along with amylases or proteases (Whitehead et al, 1995;Bellincampi et al, 2014;Wang et al, 2016b). In peanuts resistant to A. flavus infection, feruloyl esterase, pectinesterase, arabinofuranosidase, mannosidase, polygalacturonase, and galacturonidase fungal activities were significantly downregulated compared to the sensitive plants (Wang et al, 2016a).…”