1.2.3
SIGNAL TRANSDUCTION IN THE HYPERSENSITIVE RESPONSE OF CITRUS LIMON AGAINST ALTERNARIA ALTERNATA

L PEREZ, P CASTANEDA, X ORTEGA and R POLANCO

University of Chile, Santiago, Chile

Background and objectives
The hypersensitive response (HR) is one of the most effective defence mechanisms of plants to prevent pathogen infection disease development. An efficient signal transduction system is essential for induction of the HR in plants. We have studied the HR in a model using Citrus limon seedlings and Alternaria alternata. This HR includes the activation of the phenylpropanoid pathway for the biosynthesis of umbelliferone and scoparone [1], and for lignin deposition at the sites of fungal interaction with the plant. In addition, pathogenesis-related proteins such as chitinases and glucanases are also synthesized de novo. Calcium ions and calcium channels are involved in the early steps of signal transduction [2]; nevertheless, other signalling components are unknown in this system. Therefore, we propose to study if some components of signal transduction described in animal systems, such as G protein, phospholipase C and phosphoinositides (Ip3), protein kinases and calmodulin (CaM), are involved in signal transduction for the development of the HR in lemon seedlings against A. alternata.

Materials and methods
The participation of CaM, G protein, phosphoinositides (Ip3) and protein kinases was studied, using either inhibitors or activators of these systems. Trifluoperazine (TFP) and W-7 (antagonists of CaM); cholera toxin (G protein activator); neomicin (phospholipase C, PLC, inhibitor); lithium ions (inhibitors of phosphoinositide-degrading phosphatases); phorbol esters and staurosporine (activator and inhibitor of protein kinase C and PKC, respectively); dihydroxycinnamate methyl ester (DHCM) and lavendustin A (inhibitors of protein tyrosine kinases and TPK) were tested on lemon seedlings inoculated with fungal conidia. Seedlings were pre-treated with each of the compounds mentioned above and inoculated with conidia of A. alternata, and 4 and 46 h post-inoculation phenylalanine ammonia-lyase (PAL) activity and phytoalexins, respectively, were measured as markers of the HR.

Results and conclusions
PAL induction as well as phytoalexin biosynthesis were prevented by (i) TFP and W-7; (ii) neomicin and long-term lithium ion treatment; (iii) staurosporine; and (iv) DHCM and lavendustin A, suggesting the participation of CaM, PLC and Ip3, PKC, and PTKs, respectively, in the signal transduction pathway for the development of HR. In addition, PAL induction and phytoalexin synthesis were observed after treatment with (i) lithium ions for short periods and (ii) phorbol esters, suggesting the participation of Ip3 and PKC, respectively, thus supporting the above results.

We conclude that in the development of the HR in lemon seedlings inoculated with A. alternata, calmodulin, phospholipase C, phosphoinositides, and PKC- and PTK-like protein kinases are participating in signal transduction, along with the calcium ions and calcium channels previously described [2].

Supported by grants from FONDECYT 1940441 and 1970532.

References
1. Pérez LM, Pavani M, Quaas A, Roco A, 1994. Fitopatologia 29, 94-101.
2. Castañeda P, Pérez LM, 1996. Phytochemistry 42, 595-598.