Session V: Local and systemic resistance
The molecular biology of extreme resistance against potato virus X
Rx-mediated resistance against potato virus X (PVX) in potato involves a surveillance system detects a virus-encoded protein and a response that has broad spectrum antiviral activity. The Rx protein is a component of the surveillance system that recognizes the PVX coat protein. However, as we have been unable to demonstrate a direct interaction of Rx and the coat protein, it seems likely that the surveillance mechanism is a complex of several proteins. The antiviral response has a primary phase resulting in suppression of virus accumulation and a secondary phase resulting in death of the infected cell. This second phase is activated when the primary phase is too late or too weak to prevent continued virus accumulation.
To investigate the signaling components involved in Rx and other resistance responses we are using a system based on virus induced gene silencing. I will describe the basis of this system and its application in different examples of disease resistance system.
Analysis of the jasmonate signal pathway in Arabidopsis
Jasmonates (JAs), which include jasmonic acid and its cyclopentanone derivatives, are widely distributed throughout the plant kingdom. They are synthesised by the octadecanoic pathway from linolenic acid in undamaged tissues and apparently by a different pathway in wounded tissues. JAs are formed when plants are wounded, and have strikingly diverse effects: they inhibit root growth, promote fruit ripening, senescence, pollen development, tuber formation, tendril coiling, and defence against insect pests and pathogens. They are associated with altered gene expression at the level of transcription, RNA processing, and translation. However, the molecular basis of the JA signal pathway and its regulation by the wounding is only starting to be elucidated.
We have started to dissect the JA signal pathway through the isolation and characterisation of Arabidopsis mutants. Of the three mutant classes isolated one, named coil, exhibits no JA-induced responses. We isolated the COI1 gene by map-based cloning and showed that it encodes an F-Box protein. We have used yeast two-hybrid screens to show that COI1 links the JA response to the ubiquination pathway. Six COI1 ASSOCIATED PROTEIN (CAP) genes were isolated. CAP1, CAP2, and CAP3 are homologues of SKP1, which forms an essential component of the ubiquitination complex. CAP4, CAP5 and CAP6 are candidate repressor protein targets for COI1-mediated ubiquitination. Further evidence for the function of the CAP proteins in the JA pathway was by co-immunoprecipitation and by reverse genetics. The results indicate that selective proteolysis is required for the JA perception-response pathway in Arabidopsis. Other mutants with altered JA responses define negative regulators. These mutants were and were isolated in a screen for constitutive expression of the JA-responsive promoter from the vegetative storage protein (VSP) gene fused to the luciferase structural gene. They formed two distinct types: the CONSTITUTIVE EXPRESSION OF VSP1 (cevl) mutant had constitutive expression of JA-induced and ethylene-induced responses, and was generally resistant to pests and diseases. cev2-cev8 had JA-induced overexpression of JA responses. Characterisation of these three types of mutant has enabled us to predict a model for the JA signal pathway, and its interaction with other signal pathways in Arabidopsis during the wound response.
Variable interactions between salicylic acid and jasmonic acid signalling
pathways in Arabidopsis thaliana and tobacco
Salicylic acid (SA), is a key signal in the establishment of systemic acquired resistance whilst jasmonic acid (JA) is associated with wound-activated events. A well established model suggests that SA and JA signalling is mutually exclusive with SA suppressing JA synthesis at a cyclo-oxygenase step in a manner which is analogous to the reduction of prostaglandin levels by analgesics. We have reported however, the simultaneous accumulation of SA and JA in developing hypersensitive response (HR) lesions (Kenton et al., 1999) and significantly JA levels were not elevated within the HR occurring in salicylate hydroxylase expressing tobacco plants were SA levels are reduced. Reciprocally dosing Arabidopsis with various concentrations of SA, JA and the JA precursor of linolenic acid revealed that the SA-mediated suppression of JA-associated expression of PDF1.2 and thionin only occurred at >250M whilst at lower levels expressed was in fact augmented. Addition of low concentrations of JA was also observed to increase SA-induced PR1a-expression. Furthermore, addition of higher levels of SA or JA resulted in cell-death, an effect that could be suppressed by the apoplastic application of catalase. Significantly, the H2O2-responsive transgene, AoPR1-GUS also exhibited SA/JA synergistic effects in tobacco and Arabidopsis and associated increases in apoplastic H2O2 levels were measured in planta. These data suggests that SA/JA effects are concentration dependent with lower levels probably augmenting the oxidative burst during the hypersensitive response.
Reference: Kenton et al., (1999) MPMI 12:74-78.