Isolation and characterization of an exopolygalacturonase from Fusarium oxysporum f.sp. cubense race 1 and race 4

Abstract

BackgroundFusarium wilt is an economically devastating disease that affects banana production. Although Cavendish banana cultivars are resistant to Fusarium oxysporum f.sp. cubense race 1 (FOC1) and maitain banana production after Gros Michel was destructed by race 1, a new race race 4 (FOC4) was found to infect Cavendish.ResultsAn exopolygalacturonase (PGC2) was isolated and purified from the supernatant of the plant pathogen Fusarium oxysporum f.sp. cubense race 4 (FOC4). PGC2 had an apparent Mr of 63 kDa by SDS-PAGE and 51.7 kDa by mass spectrometry. The enzyme was N-glycosylated. PGC2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. To obtain adequate amounts of protein for functional studies between the PGC2 proteins of two races of the pathogen, pgc2 genes encoding PGC2 from race 4 (FOC4) and race 1 (FOC1), both 1395 bp in length and encoding 465 amino acids with a predicted amino-terminal signal sequence of 18 residues, were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC2 products, r-FOC1-PGC2 and r-FOC4-PGC2, were expressed and purified as active extracellular proteins. Optimal PGC2 activity was observed at 50°C and pH 5. The Km and Vmax values of purified r-FOC1-PGC2 were 0.43 mg.mL-1 and 94.34 units mg protein-1 min-1, respectively. The Km and Vmax values of purified r-FOC4-PGC2 were 0.48 mg.mL-1 and 95.24 units mg protein-1 min-1, respectively. Both recombinant PGC2 proteins could induce tissue maceration and necrosis in banana plants.ConclusionsCollectively, these results suggest that PGC2 is the first exoPG reported from the pathogen FOC, and we have shown that fully functional PGC2 can be produced in the P. pastoris expression system.

Isolation and Heterologous Expression of a Polygalacturonase Produced by Fusarium oxysporum f. sp. cubense Race 1 and 4

“…The sample application on SDS-PAGE showing one single protein band indicated that PGC3 was purified to homogeneity and had a molecular weight of 45 kDa according to the markers ( Figure 1 ), which was different from PGC2 (63 kDa) purified previously [ 14 ]. It is obvious that there is more than one PG in FOC4 like other pathogenic fungus.…”

“…Both purified recombinant PGC3 from both FOC1 and FOC4 exhibited optimum activity at pH 4.5 ( Figure 4 a), and the highest activity at 50 °C ( Figure 4 b). Similarly, the optimum pH and temperature of PGC2 from FOC4 were 5 and 50 °C, respectively [ 14 ], and the pH value and temperature optima for the PG activity from PG2 of Fusarium oxysporum f. sp. lycopersici (FOL) were 5 and 55 °C [ 17 ].…”

Development and evaluation of polyclonal antibodies for detection of Pythium aphanidermatum and Fusarium oxysporum in ginger

“…Protein banding pattern of fungal isolates allowed identification of multiple protein bands as shown in Figure 1. The molecular weights of the P. aphanidermatum proteins ranged from approximately 15 to 95 kDa (Anusuya & Sathiyabama, 2014;Ray et al, 2016) and of F. oxysporum proteins from 14 to 116 kDa (Bhuvanendra et al, 2010;Dong & Wang, 2011). Analysis of fungal proteins showed several specific protein bands which were absent in healthy ginger rhizomes.…”

Proteomics of Fusarium oxysporum Race 1 and Race 4 Reveals Enzymes Involved in Carbohydrate Metabolism and Ion Transport That Might Play Important Roles in Banana Fusarium Wilt

“…The virulence proteins that have been characterized from F1 and F4 are involved in signal transduction, transcription, defense system, and colonization [2] . They include G-proteins [7] , mitogen-activated protein kinase [22] , signal transcription factor [13] , [23] , glucosidase [23] , polygalacturonase [24] , and mitochondrial carrier protein [25] . Proteomics studies in Fusarium graminearum [26] , [27] and F. oxysporum [28] , [29] identified 174 cell wall proteins and 19 proteins carrying predicted signal peptides in F. oxysporum , including glucose-regulated protein, acid phosphatase, cell wall glucanosyltransferase, glucanase, secreted aspartic protease, and vacuolar protease [28] .…”