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Different isoforms of chitinases and @-1,3-glucanases of tobacco (Nicotiana fabacum cv Samsun NN) were tested for their antifungal activities. l h e class I, vacuolar chitinase and @-1,3-gIucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In addition, we observed that the class I chitinase and @-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 @-1,3-glucanase. The class II @-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 @-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their Cterminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and @-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.

We determined whether T‐DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPTII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T‐DNA, one carrying a 5′ deleted NPTII gene and the other a NPTII gene with a 3′ deletion. A restored NPTII gene was found in 1‐4% of the protoplasts that had been cotransformed with both T‐DNAs. Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T‐DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T‐DNA insertion carrying a defective NPTII gene with a 3′ deletion was transformed via Agrobacterium with a T‐DNA containing a defective NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T‐DNA introduced via A. tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.

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A nove1 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 CBPZO, was purified by chitin-affinity chromatography and gel filtration. In vitro assays demonstrated that CBPZO 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 CBPZO acts synergistically with a tobacco class I chitinase against F. solani and with a tobacco class I &1,3-glucanase against F. solani and Alternaria radicina. Analysis of the protein and corresponding cDNAs revealed that CBPZO 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, W l N l and WINZ, and severa1 plant lectins. The C-terminal domain of CBPZO showed high identity with tobacco pathogenesis-related (PR) proteins, PR-4a and PR-4b, tomato PR-P2, and potato W l N l and WIN2. CBPZO is synthesized as a preproprotein, 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 CBPZO and its induction upon TMV infection and wounding indicate that CBPZO is the first class I PR-4 type protein purified.