| Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen which attacks more than 75 plant families comprising of about 450 plant species. This destructive pathogen causes serious disease in agronomically important crop species. The pathogen produces a toxin, oxalic acid, which is a pathogenic factor. Up to now, few genetic sources of resistance to the pathogen are available to researchers and the molecular basis of plant defense to this pathogen is poorly understood. Therefore, it is of significance that cloning resistance gene and studying molecular mechanisms of host resistance to S. sclerotiorum.This study is divided into two parts. In the first part, an oxalic acid insensitive mutant named 275-7 was screened from a T-DNA insertion mutant library (Chemical-inducible Activation Tagging mutant library) of Arabidopsis thaliana (about 1000 lines). In the second part, using the two PCD-related mutants, the preliminary molecular mechanisms of cell death and ROS-mediated resistance to S. sclerotiorum were studied. The main results were as follows.With an optimal concentration of oxalic acid (3mmol/L) determined by a gradient concentration test in wide-type A. thaliana an oxalic acid- insensitive mutant named 275-7 was screened from the T-DNA insertion mutant library (about 1000 lines). In vivo leaf inoculation test was undertaken in order to assess the resistance to S. sclerotiorum of mutant 275-7. The result indicated that mutant 275-7 exhibited a higher resistance to S. sclerotiorum than wild type Arabidopsis (P<0.05). To further investigate the molecular basis of the 275-7 phenotype, we examined the accumulation of ROS in both wild type and 275-7 leaves. The result showed that ROS accumulation in 275-7 is more than wild type plants. Using real-time quantitative PCR, we examined the expressions of defense response-related genes PDF1.2 (JA-responsive marker gene) and PR1 (SA-responsive marker gene) in 275-7 and in wild type Arabidopsis. The results indicated that expression of PDF1.2 in 275-7 were significantly up-regulated (12 fold) in comparison with wild type plants,but PR1 expression level was not significantly different, which suggested that the enhanced resistance to S. sclerotiorum in the mutant 275-7 might be mediated by the JA signal pathway.The autononous PCD (spc1-1) and no PCD(fbr11-1) mutant and wild type plants were studied on roles of ROS and programmed cell death in molecular mechanisms of resistance to S. sclerotiorum in Arabidopsis. In vivo leaf inoculation test was undertaken in order to assess the resistance to S. sclerotiorum of mutants fbr11-1 and spc1-1. The result indicated that the mutant fbr11-1 exhibited a higher resistance to S. sclerotiorum than wild type Arabidopsis (P<0.05) and the mutant spc1-1 exhibited a lower resistance to S. sclerotiorum than wild type (P<0.05) . Further we examined the accumulation of ROS in both wild type and mutant leaves. The results indicated that the accumulation of hydrogen peroxide and hyperoxide in the fbr11-1 was less than wild type and in the spc1-1 was more than wild type. Cell death was detected in the mutant and wild type by Evans Blue staining,it was found that there was deeper staining in the spc1-1 than wild type and weaker in the fbr11-1 than wild type. Using real-time quantitative PCR, we examined the expressions of PDF1.2 and PR1 in the mutants and in wild type. The results indicated that expressions of PDF1.2 and PR1 in spc1-1 were significantly up-regulated in comparison with wild type plants and expression of PDF1.2 in fbr11-1 were significantly up-regulated,but PR1 expression level was down- regulated.These results indicated that spc1-1 plants with PCD and more active oxygen accumulation of is more susceptible to Sclerotinia disease, accompanied with activation of the salicylic acid and jasmonic acid pathways while fbr11-1 plants with no PCD and less active oxygen accumulation is more resistance to Sclerotinia disease, accompanied just with activation of the jasmonic acid pathway. |
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