Marianne Beatrix Sela-Buurlage

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.

A Novel Pathogen- and Wound-Inducible Tobacco (Nicotiana tabacum) Protein with Antifungal Activity

A novel pathogen- and wound-inducible antifungal protein of 20 kD was purified from tobacco (Nicotiana tabacum) Samsun NN leaves inoculated with tobacco mosaic virus (TMV). The protein, designated CBP20, was purified by chitin-affinity chromatography and gel filtration. In vitro assays demonstrated that CBP20 exhibits antifungal activity toward Trichoderma viride and Fusarium solani by causing lysis of the germ tubes and/or growth inhibition. In addition it was shown that CBP20 acts synergistically with a tobacco class I chitinase against F. solani and with a tobacco class I [beta]-1,3-glucanase against F. solani and Alternaria radicina. Analysis of the protein and corresponding cDNAs revealed that CBP20 contains an N-terminal chitin-binding domain that is present also in the class I chitinases of tobacco, the putative wound-induced (WIN) proteins of potato, WIN1 and WIN2, and several plant lectins. The C-terminal domain of CBP20 showed high identity with tobacco pathogenesis-related (PR) proteins, PR-4a and PR-4b, tomato PR-P2, and potato WIN1 and WIN2. CBP20 is synthesized as a prepro-protein, which is processed into the mature protein by the removal of an N-terminal signal peptide and a C-terminal propeptide, most likely involved in the vacuolar targeting of the protein. The intracellular localization of CBP20 and its induction upon TMV infection and wounding indicate that CBP20 is the first class I PR-4 type protein purified.

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and β-1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.

In Vitro Anti-Microbial Activities of Defense Proteins and Biotechnology

The phenomenon of induced resistance in plants is accompanied by the induction of the synthesis of a large number of proteins. Here we demonstrate that among these proteins the intracellular class I chitinases, s-1,3-glucanases and AP24 exhibit antifungal activity in vitro. In contrast, the class II isoforms of these induced proteins show limited or no antifungal activity. Furthermore, we show here that in transgenic plants expressing modified forms of either a class I chitinase gene, a class I s-1,3-glucanase gene or an AP24 gene, these proteins are targeted extracellularly. The secreted proteins have retained their antifungal activity in vitro.