Pn-AMPs, the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato, Lycopersicum esculentum

Abstract

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.

Introduction

Effective and sustained control of fungal pathogens is an important issue in agricultural systems and the global losses caused by pathogens are estimated to be about 12% of the potential crop production, despite the continued release of new resistant cultivars and fungicides. Many fungi are continually becoming resistant to existing fungicides, with a few of the latter even being withdrawn from the market for environmental and safety reasons. Further, fungi often lower crop quality by producing toxins that affect human and animal health. Additional methods of disease control are therefore highly desirable.

One of the most important objectives in modern agriculture is to develop strategies for utilization of natural host plant resistance mechanisms to control diseases. As an extension of these objectives, recombinant DNA, gene transformation and molecular biological techniques provide the potential to “engineer” traits for resistance that are effective against both specific and broad spectrum of pathogens. Therefore, significant effort has been directed toward the identification of antifungal proteins and their expression in transgenic plants (Broglie et al., 1991, Collinge et al., 1993, Florack and Stiekema, 1994, Yun et al., 1997, Koo et al., 1998, Garcia-Olmedo et al., 1998, Gao et al., 2000).

Plants produce a wide number of antifungal compounds as part of pre-existing and developmentally regulated defense barriers, and also as components of the defense response induced upon pathogen infection. The best studied among them are several classes of proteins having anti-microbial properties which include chitinases (Melchers et al., 1993, Collinge et al., 1993), glucanases (Garcia-Omeldo et al., 1998), ribosome inactivating proteins (Bolognesi et al., 2002, Chen et al., 2002), defensins (Lehrer et al., 1991, Selsted et al., 1993) and a variety of glycine/cysteine-rich small molecular weight proteins (Florack and Stiekema, 1994, Terras et al., 1995, Yun et al., 1997, Epple et al., 1997). Transgenic plants constitutively expressing several of these proteins have shown enhanced resistance to a number of pathogens (Jach et al., 1995, De Bolle et al., 1996, Epple et al., 1997, Munch-Garthoff et al., 1997, Fagoaga et al., 2001, Oldach et al., 2001, Chen et al., 2002).

Hevein and hevein-like small molecular weight anti-microbial proteins (AMPs) have been reported from rubber tree latex (Broekaert et al., 1990), seeds of amaranth (Broekaert et al., 1992) and Pharbitis nil (Koo et al., 1998), fruits of elderberry (Van Damme et al., 1999), leaves of sugar beet (Nielsen et al., 1997), Arabidopsis (Potter et al., 1993) and bark of Eucommia ulmoides (Huang et al., 2002). All of these AMPs are peptides containing 40–43 amino acid residues and possess a characteristic putative cysteine/glycine rich chitin-binding domain. Hence, it has been proposed that these proteins bind to chitin in the cell wall and disrupt cell wall synthesis. However, the hevein-like anti-microbial proteins from P. nil (Pn-AMPs) exhibit considerable antifungal activity against a broad spectrum of fungi, including those that do not contain chitin in their cell walls (Koo et al., 1998).

Since Pn-AMPs have potential antifungal activities, transgenic tomato plant lines over-expressing Pn-AMP2 were obtained to examine whether its in situ expression imparts resistance to chitin-free Phytophthora capsici and/or chitin-containing Fusarium oxysporum. Our experiments showed that transgenic plants constitutively over-expressing Pn-AMP2 exhibited enhanced resistance to these two phytopathogens. These results confirm our earlier observations (Koo et al., 1998) that hevein-like Pn-AMPs have in vitro antifungal activity against fungi that do not contain chitin in their cell wall. Furthermore, we propose that Pn-AMP2 can be used to engineer plants to mediate effective and durable resistance in plants against phytopathogens.