Plants have developed various defense mechanisms through evolution. The low water content of the seed and the hard seed coat provide effective physical barriers to defend plants against phytopathogens and herbivorous animals. Another array of defense mechanisms includes the production of antimicrobial phytoalexins, pathogenesisrelated proteins, and a large group of cysteine-rich proteins/peptides such as lipid transfer proteins, plant defensins, hevein, knottin-type peptides, four-cysteine-type peptides, 2S albumins and thionins. Plant defensins have been reported to play important roles in protecting plants from pathogens. We recently isolated a plant defensin named VrD1 from bruchid resistant mungbean seeds. In addition to antimicrobial activity, VrD1 also exhibits bruchid resistant activity. We study the structure and function of mungbean defensin as to pave the way to elucidate the role of defensin in mungbean defense mechanism.
Some small cysteine-rich proteins, including plant defensins, have been shown to play an important role in protecting plants from pathogens. However, little is known about the role such proteins might play in protecting legume seeds from insect pests. A cDNA encoding a small cysteine-rich protein designated as VrD1 was isolated from a bruchid resistant mungbean Vigna radiata VC6089A. VrD1 encodes a protein of 73 amino acids, containing a 27 amino acid signal peptide and 8 cysteines. VrD1 protein was obtained by overexpression of signal peptide deleted VrD1 in an IMPACT (inteinmediated purification with an affinity chitinbinding tag) expression system. The purified VrD1-TSP(TSP stands for truncated signal peptide) was identified by its molecular weight (5,944) and N-terminal amino acid sequencing. The amino acid sequence of VrD1 is similar to some plant defensins. Artificial mungbean seeds containing 0.8% VrD1-TSP killed larvae of the bruchid Callosobruchus chinensis at first instar stage. A VrD1-TSP level as low as 0.06% was sufficient to delay larval development. VrD1-TSP at 3.42 £gM completely arrested growth of insect cells (Spodoptera frugiperda, Sf21) in culture. VrD1-TSP is also a potent inhibitor of protein synthesis and arrests growth of some fungi and bacteria.
Based on the amino acid sequence similarity and biological activities, it is suggested that VrD1 is a member of the plant defensins. VrD1 is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C. chinensis.
To isolate VrD1 protein in a large quantity for its characterization, VrD1 cDNA was expressed in Pichia pastoris and the recombinant defensin (rVrD1) was purified. The recombinant VrD1 was shown to inhibit the growth of fungi such as Fusarium oxysporum and Rhizoctonia solani, and development of bruchid larva. The protein also inhibits in vitro protein systhesis. These biological activities are similar to that of the bacterially expressed defensin VrD1. Functional expression of VrD1 in Pichia pastoris provides a highly feasible system to study the structure-function relationship of of VrD1 using the mutagenesis approach.
Shyur, L. F., Wen, T. N., and Chen, C. S. 1992. cDNA cloning and gene expression of the major prolamins of rice. Plant Mol. Biol. 20: 323-326.
Chen, H. J., Yang, C. Y., Jane, W. N., and Chen, C. S. 1999. Increase of pigments, plastoglobuli and the mRNA of a 23 kDa polypeptide of PSII oxygenevolving complex in a transgenic tobacco cell line RP3/2. J. Plant Physiol. 155: 584-590.
Yang, C. Y., Chen, H. J., Chen, Y. F., Chen, M. H., and Chen, C. S. 2000. Seed-specific expression of a rice prolamin PR3/GUS chimeric gene in Nicotina tabacum cv. Wisconsin 38. J. Plant Physiol. 156: 100-150.
Chen, H. J., Yang, C. Y., Chen, Y. C., and Chen, C. S. 2000. Expression of rice prolamin RP3 promoter exhibits a positive association with cellular pigment contents in transgenic tobacco suspension cultures. Bot. Bull. Acad. Sinica 41: 93-98.
Chen, G. H., Tang, S. J., Chen, C. S., and Jiang, S. T. 2000. Overexpression of the soluble from of chicken cystatin in Escherichia coli and its purification. J. Agr. Food Chem. 48: 2602-2607.
Su, P. H., Yu, S. M., and Chen, C. S. 2001. Spatial and temporal expression of a rice prolamin gene RP5 promoter in transgenic tobacco and rice. J. Plant Physiol. 158: 247-254.
Li, S. C., Han, J. W., Chen, K. C., and Chen, C. S. 2001. Purification and characterization of isoforms of £]-galactosidases in mung bean seedlings. Phytochemisty 57: 349-359.
Li, S. C., Chung, M. C., and Chen, C. S. 2001. Cloning and characterization of a DEAD box RNA helicase from the viable seedlings of aged mung bean. Plant Mol. Biol. 47: 761-770.
Chen, K. C., Lin, C. Y., Kuan, C. C., Chung, M. C., Sung, H. Y., and Chen, C. S. 2002. Cloning and characterization of a cDNA encoding an antimicrobial protein from mung bean seeds. Bot. Bull. Acad. Sinica 43: 251-159.
Chen, K. C., Lin, C. Y., Kuan, C. C., Sung, H. Y., and Chen, C. S. 2002. A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. J. Agr. Food Chem. 50: 7258-7263.
Su, P. H., Yu, S. M. and Chen, C. S. 2004. Expression analyses of a rice 10 kDa sulfur-rich prolamin gene. Bot. Bull. Acad. Sinica. 45: 101-109.
Chen, J. J., Chen, G. H., Hsu, H. C. Li, S. S. and Chen, C. S. 2004. Cloning and functional expression of a mungbean defensin VrD1 in Pichia pastris. J. Agric. Food Chem. 52: 2256-2261.
Chen, G. H., Hsu, M. P., Tan, C. H., Sung, H. Y., Fan, M. J., Chen, H. M., and Chen, C. S. 2005. Cloning and characterization of a plant defensin VrD1 from azuki bean. J. Agr. Food Chem. 53: 982-988.
Lin, C., Chen, C. S., and Horng, S. B. 2005. Characterization of resistance to Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in a mungbean variety VC6089A and its resistanceassociated protein VrD1. J. Econ. Entomol. 98: 1369-1373.
