Processing, Targeting, and Antifungal Activity of Stinging Nettle Agglutinin in Transgenic Tobacco

Abstract

UDA is a single-chain peptide found in roots and rhizomes of stinging nettle (Urtica dioica L.) (Peumans et al., 1984; Van Damme and Peumans, 1987). In most ecotypes UDA is present as a mixture of isolectins with similar chitin-binding and agglutination activities (Van Damme and Peumans, 1987; Van Damme et al., 1988). Recently, one acidic and five basic UDA isolectins were identified in the Weerselo ecotype of the stinging nettle (Does et al., 1999). In in vitro assays, UDA showed antifungal activity toward various plant pathogenic fungi containing chitin in their cell walls (Broekaert et al., 1989).

UDA consists of two Cys-rich chitin-binding domains (Beintema and Peumans, 1992). These domains are homologous to hevein, a small chitin-binding peptide of 43 amino acids from the lutoid bodies of rubber tree latex (Archer, 1960; Walujono et al., 1975). Mature UDA is processed from a precursor that comprises an N-terminal signal peptide, the two chitin-binding or hevein domains, a small hinge region, and a C-terminal chitinase domain (Lerner and Raikhel, 1992; Does et al., 1999). Genes encoding precursors to UDA isolectins contain two introns in the chitinase-encoding region located at the same positions as those in class I, II, IV, and VI chitinase genes (Does et al., 1999). Because of their homology with other plant chitinases and the presence of two hevein domains, UDA precursors are classified as class V or Chia5 chitinases (Neuhaus et al., 1996).

Many hevein domain-containing proteins are localized in vacuoles. The precursors to these proteins are synthesized on the rough ER and translocated into its lumen (Blobel, 1980; Gomord and Faye, 1996). For targeting to the vacuoles, soluble proteins that pass the Golgi apparatus require additional information (Dorel et al., 1989). Such vacuole-sorting determinants reside within the C-terminal or N-terminal propeptides of the precursor proteins or within the mature protein sequence (Chrispeels and Raikhel, 1992; Neuhaus, 1996). In precursors to vacuolar hevein domain-containing proteins, these targeting sequences have been shown to be located on the C terminus. Mature vacuolar tobacco (Nicotiana tabacum) class I chitinase contains one hevein domain fused to the catalytic domain (Mauch and Staehelin, 1989; Shinshi et al., 1990).

The targeting determinant of prochitinase is contained within a C-terminal propeptide of seven amino acids and has been shown to be necessary and sufficient for vacuolar sorting (Neuhaus et al., 1991). Without this C-terminal extension the protein is secreted (Neuhaus et al., 1991; Melchers et al., 1993). The vacuolar lectins wheat germ agglutinin, rice lectin, and barley lectin are each composed of four hevein-like domains (Mishkind et al., 1983; for review, see Raikhel and Lerner, 1991) and are all processed from precursors that contain a small C-terminal propeptide (Stinissen et al., 1984; Mansfield et al., 1988; Lerner and Raikhel, 1989; Wilkins and Raikhel, 1989). Like the chitinase propeptide, the C-terminal propeptide of the barley prolectin is required for correct vacuolar deposition of the mature lectin and is able to redirect an extracellular cucumber chitinase to the vacuoles (Bednarek et al., 1990; Bednarek and Raikhel, 1991; Neuhaus et al., 1991).

Hevein is localized in vacuole-derived lutoid bodies and, similarly to UDA, is processed from a precursor with a large C-terminal domain (Archer, 1960; Lee et al., 1991). In contrast to the UDA precursor, this C-terminal domain does not show homology to chitinases but rather to tobacco PR4-type (pathogenesis-related) proteins (Broekaert et al., 1990; Linthorst et al., 1991). A putative vacuole-targeting signal has been shown to be cleaved off from the C terminus of the PR4-like domain of the hevein precursor (Soedjanaatmadja et al., 1995). Other known C-terminal propeptides containing essential vacuole-sorting determinants are present in precursors to tobacco class I β-1,3-glucanase (Shinshi et al., 1988; Sticher et al., 1992; Melchers et al., 1993), vacuolar PR5 proteins (Singh et al., 1989; Melchers et al., 1993), Brazil nut 2S albumin (Harris et al., 1993; Saalbach et al., 1996), and kiwifruit proactinidin (Paul et al., 1995).

In the present study we show that the precursor to UDA is processed to mature-sized isolectin in transgenic tobacco and that this isolectin is localized intracellularly, most likely in the vacuoles. Removal of the C-terminal 25 amino acids of the precursor results in the secretion of UDA. Extracellularly targeted UDA is correctly processed and displays similar agglutination, chitin-binding, and antifungal activities as the same isolectin from stinging nettle. The growth-inhibiting effect of UDA on B. cinerea and T. viride appears to be temporal, and adaptation of both fungi coincides with a phase of accelerated hyphal growth. C. lindemuthianum did not adapt to UDA within 2 d after addition and showed a severely stunted and inhibited growth.

Different amounts of UDA isolectin I purified from the total extract of a ΔC25 tobacco line or from stinging nettle were added to pregerminated spores of B. cinerea, T. viride, and C. lindemuthianum. Fungal growth was monitored by inverted light microscopy. BSA was used as a control. The final concentrations of UDA and BSA in water are shown. Growth inhibition is the percentage of growth compared with the water control (no UDA or BSA added).

ACKNOWLEDGMENTS

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