Bernardus Johannes Clemens Cornelissen

Synergistic activity of chitinases and β-1,3-glucanases enhances fungal resistance in transgenic tomato plants

SummarySimultaneous expression of a tobacco class I chitinase and a class I β-1,3-glucanase gene in tomato resulted in increased fungal resistance, whereas transgenic tomato plants expressing either one of these genes were not protected against fungal infection. After infection with Fusarium oxysporum f.sp. lycopersici, a 36% to 58% reduction in disease severity was observed in resistant tomato lines. Two transgenic lines largely recovered from the initial infection by the time wild-type tomato plants had died.The overall results are consistent with the observation that class I chitinases and class I β-1,3-glucanases synergistically inhibit the growth of fungi in vitro and provide the first experimental support to the hypothesis that such synergy can contribute to enhanced fungal resistance in planta.

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.

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco.

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.