Evidence For Involvment Of Calcium-dependent Protein Kinase And Adenylyl Cyclase In Verticillium Toxin Induced Signal Transduction In Arabidopsis Thaliana

In plants, intracellular Ca²⁺ levels are modulated in response to various signals including abiotic and biotic stresses, and specific calcium signatures can be transduced into downstream effects by different calcium sensors. CDPKs are unique calcium sensors in higher plants and are likely to be crucial mediators of diverse calcium signaling cascades induced by abiotic and biotic stress signals. However the precise biological function of most CDPKs remain elusive.Verticillium wilt caused by toxins of Verticillium dahlia is a serious threat to many important crops. The dose-dependent inhibition of Arabidopsis seed germination and seedling growth by the toxins was observed, and the inoculation of leaves by the toxins induced significant local necrosis. Twenty-six homozygous T-DNA insertion lines of Arabidopsis CDPK-CRK genes were screened in the toxin-containing medium. Two independent mutant lines (cpk18A and cpk18B) for CPK18 showed significant increased sensitivity to the toxins. Complementation of CPK18 mutation reduced the sensitivity to the toxins and resulted in similar phenotype compared to wild type plants in the presence of toxins, whereas overexpression of CPK18 resulted in increased toxin tolerance. The sensitivity of various plants (wild type, transgenic lines and mutants) was external Ca²⁺-dependent. Northern blot analysis showed toxin-induced accumulation of PR1 transcripts was significantly delayed in cpk18A or cpk18B mutants and accelerated in the CPK18-overexpressed lines. The results suggests that CPK18 may involve in toxin-induced signaling and may act upstream to PR proteins.The previous studies have provided evidence that cAMP may activate signaling cascades in plant hypersensitive reactions in response to pathogen infection. Our previous work has demonstrated that endogenous cAMP in plant cells may play roles in regulation of SA synthesis in response to Verticillium toxins. To further obtain direct evidence for involvement of endogenous cAMP in signal transduction of Arabidopsis plants in response to the toxins, the functions of genes encoding adenylyl cyclase (AC) were evaluated. The AC189 and AC388 T-DNA insertion mutants showed significant increased sensitivity to the toxins. The endogenous cAMP contents in both mutants were significantly lower than that in wild type plants. The results indicate that the increased sensitivity of the mutants to the toxins may result from the decreased cAMP contents. The full-length cDNA of AC189 was cloned and AC189-overexpressed lines showed increased tolerance to the toxins compared to wild type plants. It is concluded that endogenous cAMP is an important upstream regulator for plant responses to fungal elicitors.