Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ALTERED DEFENSE RESPONSE TO FUSARIUM OXYSPORUM IN TOMATO (LYCOPERSICON ESCULENTUM, MILL.) LINES TRANSFORMED WITH ROL GENES OF AGROBACTERIUM RHIZOGENES
BETTINI P. *, SANTANGELO E.**, MICHELOTTI S.*, MARVASI M.*, BINDI D.*, MOSCONI P.**, CRINÒ P.***, BUIATTI M.*
*) Dipartimento di Biologia animale e Genetica “Leo Pardi”, Università degli Studi di Firenze
**) Consorzio Agrital Ricerche, Maccarese (Roma)
***) ENEA C.R. Casaccia, UTS Biotecnologie, Protezione della Salute Umana e degli Ecosistemi, Roma
tomato, rol genes, plant-pathogen interaction, Fusarium oxysporum, phytohormones
The role of phytohormones in plant defense response to pathogens has been clearly established, even if evidence concerning their effect is still controversial. In fact, both an inhibition and an increase in host resistance to fungi, bacteria and viruses have been reported after treatment with growth regulators or transformation with genes involved in auxin and cytokinin biosynthesis (Martinez Noel et al. 2001, Plant Physiol. Biochem. 39: 815-823; Clarke et al. 1999, Plant Physiol. 120: 547-552; Beckman and Ingram 1994, Physiol. Mol. Plant Pathol. 45: 229-246; Sitbon F et al. 1999, Plant Science 141: 165-173). The aim of the present study is to analyse the effect of the modification of endogenous hormone equilibria on the horizontal defense response of plants. For this purpose, Lycopersicon esculentum cv. Tondino, kindly provided by Petoseed Italia, has been transformed with Agrobacterium rhizogenes rolA, B, C, D genes, known to be involved in the phytohormone metabolic network. The transgenic plants and the corresponding controls (regenerated, not transformed) have been self-fertilized and the resulting segregating progenies have been subjected to PCR and RT-PCR molecular analyses in order to evaluate the presence and the expression of transgenes, respectively. Cosegregation of an aberrant phenotype with presence of the transgene was demonstrated for the rolA progeny. This confirmed that the observed modifications were due to the introgression of the foreign gene and not to variation induced during the in vitro regeneration process. The same progenies have been infected in vivo with the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici race 0, in order to assess if the introgression of the rol genes led to an increased tolerance/resistance to pathogens. Under controlled conditions, 15-day old plantlets (20/genotype) were infected by immersion of the rooting apparatus into a fungal suspension (1.5x106 conidia/ml), while 5-10 uninfected plants for each genotype were used as a positive control. Symptoms were evaluated 15, 27 and 40 days after inoculation. Preliminary data revealed that rolA transformants reacted better than rolD, the response of the latter ones being not significantly different from their untransformed control. In particular, the best response to the fungus has been recorded for the clone rolA23, harbouring only one copy of the transgene. Molecular analyses, carried out on a sample of 21 rolA23 plants surviving the infection, has shown a ratio of 6 transgenic : 1 not transgenic plants, this being higher than the ratio expected on the basis of a single gene segregation. This finding seems to support the hypothesis that the highest level of Fusarium resistance observed could be due to the presence of the foreign gene. The expression of PR-1 gene, considered to be a marker of Systemic Acquired Resistance (SAR), will also be analysed quantitatively by real-time PCR in a sample of infected and symptomless plants, in order to investigate the possible role of this resistance mechanism in the tolerance of the rolA plants.
Infection experiments on the progenies of rolB and rolC transformants are currently underway.
Research activity carried out in the frame of the PNR Biotecnologie Avanzate II, tema 4, coordinated by Consorzio Agrital Ricerche (Chairman: Prof. Gian Tommaso Scarascia Mugnozza).