5.3.10
TRANSGENIC TOBACCO AND POTATO PLANTS EXPRESSING ANTIFUNGAL GENES FROM TRICHODERMA ARE RESISTANT TO SEVERAL PLANT PATHOGENIC FUNGI

F SCALA1, SL WOO1, I GARCIA2, A ZOINA1, E FILIPPONE3, JA PINTOR-TORO4, G DEL SORBO1, B ALOJ1 and M LORITO1

1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale - sezione Patologia Vegetale, Universitą degli Studi di Napoli Federico II, 80055 Portici (NA), Italy; 2Inst. Genet. et Microb., Universite Paris-Sud, 91405-Orsay Cedex, France; 3Dipartimento di Agronomia e Genetica Vegetale, Universitą degli Studi di Napoli Federico II, Portici (NA), Italy; 4Instituto de Recursos Naturales y Agrobiologķa, CSIC, Sevilla, Spain

Background and objectives
Plant-pathogen interaction involves recognition and activation of pathogenesis-related genes, including those coding for chitinolytic and glucanolytic enzymes. These can be utilized transgenically to reduce plant susceptibility to fungal pathogens, with little or no effect on plant tissues.The use of plant chitinase genes produced promising results but also contradictory effects on plant disease resistance. Chitinase genes from microbes, such as biocontrol fungi or bacteria, may represent a valid alternative to the corresponding plant genes for genetically improving plant disease resistance. Mycoparasitic fungi are great producers of cell wall-degrading enzymes (CWDEs) and the corresponding genes have never been used for this purpose. Biocontrol strains of Trichoderma spp. attack and destroy many important fungal plant pathogens and secrete a variety of CWDEs that are: able to lyse the mature chitinous wall of fungi including that of sclerotia and clamydospores; more antifungal than similar enzymes purified thus far from other sources; non-toxic to plants; and strongly synergistic in antifungal activity when combined with different CWDEs and other compounds. The objective of this work was to demonstrate that the genome of these mycoparasites is a rich and novel source of genes for the improvement of plant disease resistance.

Results and conclusions
We cloned from a biocontrol strain of T. harzianum several genes encoding powerful antifungal chitinases and glucanases active against a wide range of phytopathogenic fungi (at least 20 susceptible species identified). These genes were used to improve the antifungal activity of various plants, including tobacco, potato, tomato and petunia. Plants were transformed with genomic and cDNA copies of the fungal genes containing the original Trichoderma secretion sequence, and a high level of constitutive expression was obtained. In comparison to the controls, some tobacco transformants showed a 400-fold chitinolytic activity increase in roots and leaves. Immunogold labelling with TEM revealed an extracellular accumulation of the fungal enzymes, with very little endochitinase in the cytoplasm. Several transgenic lines were significantly more resistant to foliar and soilborne pathogenic fungi, and the Trichoderma fungicidal enzymes were also able to stimulate plant defence responses. Selected transgenic tobacco and potato inoculated with the foliar pathogens Alternaria spp. showed up to ca 80% reduction in the development of disease symptoms as compared to wild-type. In soil assays with the soilborne pathogen Rhizoctonia solani, plant mortality averaged 27% for transgenics and 60.5% for controls. Infection of tobacco leaves with a virulent strain of Botrytis cinerea also showed a marked difference between the disease susceptibility of some of the endochitinase-transgenic plant lines and controls. The sizes of the lesions caused by the pathogen were larger in control plants than in transgenic lines expressing high levels of endochitinase activity, and a few selected lines that showed high levels of resistance to A. alternata and R. solani also did not develop Botrytis disease symptoms. In contrast to results from Alternaria assays, there was no correspondence between the level of enzyme activity in the transgenics and level of resistance to R. solani. This suggests that the fungal enzyme may act in planta not only as an antifungal agent, but also as an inducer of the plant defence system during infection.