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Int. J. Mol. Sci. 2015, 16(4), 7595-7607; doi:10.3390/ijms16047595

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

1
Department of Plant Protection, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
2
Laboratory of Physiological Plant Pathology, South China Agricultural University, Guangzhou 510642, China
*
Author to whom correspondence should be addressed.
Academic Editor: Jan Schirawski
Received: 8 December 2014 / Revised: 4 March 2015 / Accepted: 23 March 2015 / Published: 3 April 2015
(This article belongs to the Special Issue Plant Microbe Interaction)
View Full-Text   |   Download PDF [1679 KB, uploaded 3 April 2015]   |  

Abstract

Fusarium wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The Km and Vmax values of PGC3 from FOC4 were determined to be 0.70 mg·mL−1 and 101.01 Units·mg·protein−1·min−1, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3–7 and >50% activity in 10–50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris. View Full-Text
Keywords: polygalacturonase; Fusarium oxysporum f. sp. cubense; Fusarium wilt; banana polygalacturonase; Fusarium oxysporum f. sp. cubense; Fusarium wilt; banana
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Dong, Z.; Wang, Z. Isolation and Heterologous Expression of a Polygalacturonase Produced by Fusarium oxysporum f. sp. cubense Race 1 and 4. Int. J. Mol. Sci. 2015, 16, 7595-7607.

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