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 report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.

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.

Cytoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C-terminus of these CTL epitopes is predominantly generated by the proteasome.Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus, and a clinically important epitope from melanoma-protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing and nardilysin contributed to both C-terminal and N-terminal CTL epitope generation. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

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Link to publication Citation for published version (APA):Jongedijk, E., Tigelaar, H., Roekel, J. S. C., Bres-Vloemans, S. A., Dekker, I., van den Elzen, P. J. M., ... Melchers, L. S. (1995). Synergistic activity of chitinases and beta-1,3-glucanases enhances fungal resistance in transgenic tomato plants. EUPHYTICA, 85,[173][174][175][176][177][178][179][180]

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