Transformation of Potato (Solanum tuberosumcv.Savalan) by Chitinase and β-1,3-Glucanase Genes of Myco-Parasitic Fungi Towards Improving Resistance to Rhizoctonia solani AG-3

Document Type: Research Paper

Authors

National Institute for Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran

Abstract

Potato (Solanum tuberosum L.) an agro-economically important food crop in the world, is sensitive to many fungal pathogens including Rhizoctonia solani (AG-3), the causal agent of stem and root rot diseases. Chitinase and glucanase are cell wall degrading enzymes which have been shown to have high antifungal activity against a wide range of phytopathogenic fungi. In the present study, plasmid pBIKE3 harboring a double-gene cassette containing the chitinase (chit42) and b-1,3-glucanase (bgn13.1) genes was constructed. In this construct, the chit42 gene is located between the CaMV 35S promoter and nos terminator derived from pBI121, while the bgn13.1 gene is downstream of a modified CaMV 35S promoter, followed by the nos terminator both of which were derived from the pRTL plasmid. Micro-tubers of potato plants (the Savalan cultivar) were transformed with the pBIKE3 construct via the Agrobacterium delivery system. Integration of these two genes into the potato genome and their expression at the transcriptional level was confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). The radial diffusion assay showed that the heterologous expressed chitinase and glucanase enzymes demonstrated antifungal activity on R. solani (AG-3).

Keywords


Potato (Solanum tuberosum L.) is one of the major crops of many countries like Iran with an annual global production of approximately 300 million tones (Banerjee et al., 2006). Because of its high nutritional quality and ease of production, the potato is a suitable candidate crop for genetic engineering-related improvement projects. Like many other crops, the production of this crop is challenged by fungal pathogens.
Fungal diseases are rated as one of the most important factors contributing to yield losses in many economical crops including the potato. The mycoparasitic process in Trichoderma involves various fungal cell wall degrading enzymes, including hydrolytic enzymes (Chet et al., 1998). Among these enzymes, chitinases and b-1,3-glucanases are attractive molecules because they have activity against a wide range of fungi (Walsh et al., 2000; Lorito, 1998; Stone and Clarke, 1993). Chitinases and b-1,3-glucanases from Trichoderma sp. have been shown to have strong antifungal activities when used individually and highly synergistic in combination (Lorito et al., 1998). Chitinases also confer broad resistance to other biotic and abiotic stresses, such as bacterial pathogens, salinity and heavy metals (Dana et al., 2006).
In this work the chitinase gene (chit42) from Trichoderma atroviride and the b-1,3-glucanase gene (bgn13.1) from Trichoderma virens are used to prepare a double gene construct containing these two genes. Potato plants are transformed by this construct via Agrobacterium-mediated transformation method. The putative transgenic plants are analyzed by PCR and RT-PCR, and tested for their antifungal activity on Rhizoctonia solani, the causal agent of potato root and stem rot.
Materials and methods

Enzymes and chemicals: All chemicals, culture media, plant growth regulators and antibiotics were purchased from Merck (USA) and Sigma (USA) at the highest purity available, unless stated otherwise. Restriction enzymes and other DNA-modifying enzymes were obtained from Roche (Switzerland) and Fermentas (CA).

Plant material: An economically important potato (S. tuberosum) of the Savalan cultivar, which was released by the Seed and Plant Improvement Institute, Karaj, Iran, was used as the receptor.

Microorganisms and growth conditions:  R. solani, (AG-3), the causal agent of potato root and stem rot was propagated on potato dextrose agar (PDA) and subcultured as needed. Escherichia coli DH5a (Cinnagen, Iran) was used in all molecular experiments and Agrobacterium tumefaciens LBA4404 was used for the plant transformation procedure. Bacteria were grown in Luria-Bertani (LB) medium at appropriate temperatures (37ºC  for E. coli and 28ºC for A. tumefaciens) with shaking (150 rpm).

General procedures: Plasmid DNA preparation and electrophoresis of DNA fragments were performed by routine procedures (Sambrook and Russell, 2001). Enzymatic

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