Knock down of chitosanase expression in phytopathogenic fungus Fusarium solani and its effect on pathogenicity
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Abstract
Chitosanases are lytic enzymes involved in the degradation of chitosan, a component of fungal cell walls. The phytopathogenic fungus Fusarium solani produces an extracellular chitosanase, CSN1, the role of which in the physiology and virulence of the fungus remains to be expounded. Here, we studied the expression of the CSN1 gene through gene silencing and examined its effect on fungal pathogenicity. A vector construct encoding a hairpin RNA (hpRNA) of CSN1 was constructed and introduced into the F. solani 0114 strain. The results revealed that majority of the transformants exhibited a significant reduction in chitosanase activity compared with the wild-type strain. Further, transformants with silenced CSN1 exhibited no change in mycelial growth and spore formation. However, pea pod and seedling bioassays indicated that transformants with silenced CSN1 were more virulent compared with the wild-type strain, and in sharp contrast to strains in which overexpression of the CSN1 gene resulted in virulence reduction. Although the mechanism remains unclear, our findings did suggest that F. solani chitosanase has a negative effect on fungal pathogenicity.
Keywords
Chitosanase Fusarium solani RNA silencing PathogenicityNotes
Acknowledgment
The authors express their gratitude to Dr. Roberta Greenwood for editing the manuscript.
References
- Bandyopadhyay R, Mwangi M, Aigbe SO, Leslie JF (2006) Fusarium species from the cassava root rot complex in west Africa. Phytopathology 96:673–679CrossRefPubMedGoogle Scholar
- Caracuel Z, Roncero MI, Espeso EA, González-Verdejo CI, García-Maceira FI, Di Pietro A (2003) The pH signalling transcription factor PacC controls virulence in the plant pathogen Fusarium oxysporum. Mol Microbiol 48:765–779CrossRefPubMedGoogle Scholar
- de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842CrossRefPubMedGoogle Scholar
- dos Reis MC, Pelegrinelli Fungaro MH, Delgado Duarte RT, Furlaneto L, Furlaneto MC (2004) Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana. J Microbiol Methods 58:197–202CrossRefPubMedGoogle Scholar
- Fukamizo T, Brzezinsk R (1997) Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function. Biochem Cell Biol 75:687–696CrossRefPubMedGoogle Scholar
- Gao XD, Katsumoto T, Onodera K (1995) Purification and characterization of chitin deacetylase from Absidia coerulea. J Biochem 117:257–263CrossRefPubMedGoogle Scholar
- Hadwiger LA, Beckman JM (1980) Chitosan as a component of pea-Fusarium solani interactions. Plant Physiol 66:205–211CrossRefPubMedGoogle Scholar
- Hadwiger LA, Beckman JM, Adams MJ (1981) Localization of fungal components in the pea-Fusarium interaction detected immunochemically with anti-chitosan and anti-fungal cell wall antiseta. Plant Physiol 67:170–175CrossRefPubMedGoogle Scholar
- Kikot GE, Hours RA, Alconada TM (2008) Contribution of cell wall degrading enzymes to pathogenesis of Fusarium graminearum: a review. J Basic Microbiol 48:1–11CrossRefGoogle Scholar
- Lee JR, Park SC, Kim MH, Jung JH, Shin MR, Lee DH, Cheon MG, Park Y, Hahm KS, Lee SY (2007) Antifungal activity of rice Pex5p, a receptor for peroxisomal matrix proteins. Biochem Biophys Res Commun 359:941–947CrossRefPubMedGoogle Scholar
- Li Y, Sommerfeld M, Chen F, Hu Q (2008) Consumption of oxygen by astaxanthin biosynthesis: a protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae). J Plant Physiol 165:1783–1797CrossRefPubMedGoogle Scholar
- Liu H, Bao X (2009) Overexpression of the chitosanase gene in Fusarium solani via Agrobacterium tumefaciens-mediated transformation. Curr Microbiol 58:279–282CrossRefPubMedGoogle Scholar
- Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
- Mahr SER, Stevenson WR, Sequeira L (1986) Control of bottom rot of head lettuce with iprodione. Plant Dis 70:506–509CrossRefGoogle Scholar
- Maiti IB, Kolattukudy PE (1979) Prevention of fungal infection of plants by specific inhibition of cutinase. Science 205:507–508CrossRefPubMedGoogle Scholar
- McDonald T, Brown D, Keller NP, Hammond TM (2005) RNA silencing of mycotoxin production in Aspergillus and Fusarium species. Mol Plant Microbe Interact 18:539–545CrossRefPubMedGoogle Scholar
- Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91:73–80CrossRefGoogle Scholar
- Nakayashiki H, Nguyen QB (2008) RNA interference: roles in fungal biology. Curr Opin Microbiol 11:494–502CrossRefPubMedGoogle Scholar
- Osusky M, Zhou G, Osuska L, Hancock RE, Kay WW, Misra S (2000) Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nat Biotechnol 18:1162–1168CrossRefPubMedGoogle Scholar
- Oyarzun PJ, Dijst G, Zoon FC, Maas PW (1997) Comparison of soil receptivity to Thielaviopsis basicola, Aphanomyces euteiches, and Fusarium solani f sp. pisi causing root rot in pea. Phytopathology 87:534–541CrossRefPubMedGoogle Scholar
- Prapagdee B, Kotchadat K, Kumsopa A, Visarathanonth N (2007) The role of chitosan in protection of soybean from sudden death syndrome caused by Fusarium solani f sp. glycines. Bioresour Technol 98:1353–1358CrossRefPubMedGoogle Scholar
- Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343–3353CrossRefPubMedGoogle Scholar
- Schmidhauser TJ, Lauter FR (1990) Cloning, sequence, and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa. Mol Cell Biol 10:5064–5070PubMedGoogle Scholar
- Shimosaka M, Nogawa M, Ohno Y, Okazaki M (1993) Chitosanase from the plant pathogenic fungus, Fusarium solani f. sp. Phaseoli—purification and some properties. Biosci Biotec Biochem 57:231–235CrossRefGoogle Scholar
- Shimosaka M, Sato K, Nishiwaki N, Miyazawa T, Okazaki M (2005) Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis. J Biosci Bioeng 100:545–550CrossRefPubMedGoogle Scholar
- Sriwilaijaroen N, Boonma S, Attasart P, Pothikasikorn J, Panyim S, Noonpakdee W (2009) Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA directed against malaria histone deacetylase. Biochem Biophys Res Commun 381:144–147CrossRefPubMedGoogle Scholar
- Sweigard JA, Orbach MJ, Valent B, Chumley FG (1990) A miniprep procedure for isolating genomic DNA from Magnaporthe grisea. Fungal Genet Newsl 37:108–111Google Scholar
- Yonni F, Moreira MT, Fasoli H, Grandi L (2004) Simple and easy method for the determination of fungal growth and decolourative capacity in solid media. Int Biodeterrio Biodegrad 54:283–287CrossRefGoogle Scholar
- Zeng R, Liao Q, Feng J, Li D, Chen J (2007) Synergy between cucumber mosaic virus and zucchini yellow mosaic virus on Cucurbitaceae hosts tested by real-time reverse transcription-polymerase chain reaction. Acta Biochim Biophys Sin (Shanghai) 39:431–437CrossRefGoogle Scholar