| Research Abstract |
Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) are the best-characterized positive-sense single-stranded RNA viruses. In this project, the signal transduction pathways for the resistance to CMV and TMV were studied. CMV-Arabidopsis system: A. thaliana ecotype C24 is resistant to a yellow strain of CMV [CMV(Y)]. This resistance to CMV(Y) was conferred by a single dominant gene, RCY1, which is a member of RPP8/HRT resistance gene family. Our analysis of Arabidopsis mutants deficient in SA, JA and ethylene signaling pathways indicated that an SA-and ethylene-dependent signaling pathway played an important role in the expression of the high level of resistance to CMV(Y) conferred by the RCY1 gene, However, the combined impairment of SA and ethylene signaling in the nahG etrl RCY1 double mutant did not completely abolish the RCY1-conferred resistance to CMV(Y). We therefore proposed that in addition to SA and ethylene signaling, a novel uncharacterized signaling pathway was requ
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ired for the full resistance to CMV(Y) conferred by the RCY1 gene. Moreover, cross-talk between the SA and JA signaling pathways is an important modulator of RCY1-conferred resistance to CMV(Y). The analysis of double Arabidopsis mutants indicated that a CO/1-dependent mechanism negatively modulated the RCY1-mediated activation of SA signaling pathway and resistance to CMV(Y). The gene-for-gene resistance to Turnip mosaic virus (TCV) conferred by HRT of ecotype Di-17 was dependent on the SA-mediated signaling pathway, but not on the JA and ethylene signaling pathways. Although RCY1 in ecotype C24 was an ortholog of HRT in the ecotype Di-17, the extent of the requirement for SA signaling in conferring resistance to CMV(Y) and TCV was different between RCY1-and HRT-containing plants. The difference in the requirement of defense mechanisms targeting these two viruses is further exemplified by studies with the Arabidopsis ssi2 mutant. While the ssi2 mutant exhibited enhanced resistance to CMV(Y), it did not confer enhanced resistance to TCV. In summary our results suggest that the Arabidopsis RCYI and HRT genes evolved to recruit different combinations of signal transduction pathways for conferring resistance to two different viruses. TMV-Tobacco system: The, signaling pathway for acquired resistance to pathogen infection has been examined using a synchronous HR cell-death-inducing system in TMV-infected tobacco plants carrying the temperature-sensitive N resistance gene, as well as the wound-signaling pathway which cross-talks with HR-signaling. We studied the function of genes whose expression were induced or repressed 3 h after HR triggering. WRK: A receptor-like protein kinase gene was induced rapidly after HR triggering and wounding. WRK was constitutively expressed at a low level, and induced by a simple temperature transfer from 30'C to 20'C independent of the N gene. In transgenic tobacco with suppressed WRK expression, wound-induced phenomena such as activation of defense-related MAPKS (WIPK and SIPK), JA accumulation, and expression of wound-inducible genes such as PI-II were inhibited, suggesting involvement of WRK at least in wound-signaling. HSP90 : In transgenic tobacco plants with suppressed HSP90 gene expression, the level of HR-induced electrolytic leakage, an indicator of cell death, was considerably inhibited with attenuated resistance to TMV. Treatment with geldanamycin, a specific inhibitor of Hsp90, reduced the ion leakage and wound-induced activation of WIPK and SIPK, indicating the involvement of HSP90 in HR-signaling. CaM (calmodulin) : We isolated 13 tobacco genes encoding three plant-specific types of CaMs, some of which were induced by HR-triggering or wounding. The amount of each type CaM dynamically changed after HR or wounding, Tobacco CaMs activates target enzymes specifically in a Ca^<2+>dependent and type-specific manner. As a new CaM-binding protein, a MAPK phosphatase (MKP1) was isolated. The gene product inactivated SIPK in vitro. In MKP1 overexpressors, wound-induced activation of WIPK and SIPK was inhibited, suggesting Ca^2/CaM signal functions upstream in MAPK-cascade at least in wound-signaling. WAF-1: WIPK is activated enzymatically in response to wounding and HR by TMV infection. (11E,13E)-labda-11,13-diene-8a,15-diol, designated WAF-1 was identified as WIPK-activating substance from TMV-infected tobacco leaves. WAF-1, a novel diterpene, functions as an endogenous signal mediating defense responses of tobacco plants to TMV infection and wounding. Less
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