Cloning and analysis of endoglucanase genes from the phytopathogenic fungus Macrophomina phaseolina

Haiyin Wang, Purdue University

Abstract

Macrophomina phaseolina, a soil-borne deuteromycete fungus, causes a seedling blight and charcoal rot of roots and stems on over 500 species of plants (Mihail and Taylor 1995). Annual loss in worldwide soybean production, due to this fungus, exceeds one million metric tons. Endoglucanase and pectinase activities were detected in both culture and infected plants (Chan and Sackston 1970). Ultrastructural investigations of infection processes of M. phaseolina on soybean seedling root tissue suggested both mechanical pressure and cell wall-degrading enzymes were involved in the penetration of interior cell walls by infection pegs (Ammon et al. 1974). To determine the relationship between endoglucanases produced by the phytopathogenic fungus M. phaseolina and disease development, endoglucanase genes were cloned and functional expressed. A CMC-induced cDNA library from the fungus M. phaseolina was made in Escherichia coli. Colonies with endoglucanase activity were screened by a CM-Cellulose/Congo Red overlay. Two types of full-length genes, designated egl1 and egl2, with endoglucanase activity, were cloned and sequenced. Egl1 lacks the cellulose-binding domain and linker region, requires cellopentaose or larger substrates, and shares high sequence homology to egl1 from the phytopathogenic bacterium Pseudomonas solanacearum. Egl2 also lacks the cellulose-binding domain and linker region, but requires cellotetraose or larger substrates, and has high sequence homology to egl3 from the saprophytic fungus Trichoderma reesei. To provide the molecular evidence for the role that EGLI plays in disease development, single-chain antibody gene fragments (ScFv) against EGLI were cloned from the spleen cells of an immunized mouse and expressed as a fusion protein on the surfaces of recombinant phages. Two ScFv clones with weak affinity to EGLI were obtained. Ideally, transgenic plants expressing the ScFv antibody gene against EGLI of the phytopathogenic fungus M. phaseolina not only provide the evidence of the role of EGLI in disease development in infected plants, but also confer genetic resistance to M. phaseolina, since no resistant cultivars of any host plant are known.

Degree

Ph.D.

Advisors

Jones, Purdue University.

Subject Area

Plant pathology|Molecular biology|Microbiology

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