Poster: Plant insect-nematode interactions

Abs # 485: Purification and biological activities of two plant defensins from mungbean seeds

Presenter:	Chen, Ching-San , chingsan@gate.sinica.edu.tw
Authors	Chen, Ching-San  (A) (C)  Hsu, Ming-Pin  (A)   Tan, Chi-Hsing  (A)   Chen, Ji-Jr  (B)   Chen, Yung-Che  (C)   Chen, Huei-Mei  (D)
Affiliations:	(A): Institute of Botany, Academia Sinica (B): Graduate Institute of Bioscience and Biotechnology, National Taiwan Ocean University (C): Graduate Institute of Agricultural Chemistry, National Taiwan University (D): Asian Vegetable Research and Development Center

Plant defensins have been shown to be major constituents of the innate immune systems of plants for nonspecific defense of the host against various plant pathogens. We have isolated two plant defensins VrD1( Vigna radiate Defensin 1) and VrD2 from an insect resistant isogenic line of mungbean Vigna radiata VC6089A. VrD1 cDNA was also cloned and its nucleotide sequence was determined. Whereas the nucleotide sequence of VrD2 cDNA was obtained from previously published sequence in the data bank. The mature VrD1 and VrD2 proteins have predicted molecular masses of 5.1 and 5.5 kDa and calculated isoelectric points of 8.43 and 8.04, respectively. The amino acid sequence homology between these two mungbean defensins is 48.8%. Artificial seeds containing 0.2% VrD1 reduced percentage emergence of bruchid (Callosobruchus chinensis) from 54% (control artificial seeds) to 21%, indicating that VrD1 is resistant against bruchid. VrD1 was shown to moderately inhibit the growth of Staphylococcus aureus and Staphylococcus epidermidis. Whereas VrD2 arrests growth of some fungi such as Fusarium oxysporum, Stemphylium solani, Pyricularia oryzae and Rhizotonia solani. VrD1 cDNA was overexpressed in Pichia pastoris. The purified recombinant VrD1 (rVrD1) was shown to inhibit growth of fungi such as F. oxysporum, P. oryze, R. solani and Trichophyton rubrum, and development of bruchid larva. The protein also inhibits in vitro protein synthesis. Functional expression of VrD1 in Pichia pastoris provides a highly feasible system to study the structure-function relationship of VrD1 using mutagenesis approach.