Cloning, Expression Of An Elicitin Gene From Phytophthora Parasitica And Plant Defense Activated By The Elicitin

Elicitins, proteinaceous elicitors produced by species of two oomycetes genera, Phytophthora and Pythium, induce hypersensitive reaction (HR) and systemic acquired resistance (SAR) in tobacco. They can be classified into basic elicitins produced by species such as Phytophthora crytogea and acidic elicitin produced by species such as P. parasitica. P. parasitica var. nicotianae, the causal agent of tobacco black shank disease, produces an α-elicitin, parasiticein. This study was aimed to determine mechanisms in some respects, by which elicitins activate plant defense.We have firstly cloned and characterized parA1 homologies from 4 isolates of P. parasitica isolated from tobacco and locust in China. Amplification products were got by polymerase chain reaction (PCR) of DNA from the 4 isolates, using primers specific to a parA1 reported previously. Their nucleotide sequences are 354 bp in size and identical one to another and to the parA1 gene previously cloned. Therefore, we have successfully cloned the parA1 gene from the Chinese isolates of P. parasitica. These homologies were cloned into the vector pET30a (+) and transformed them into Escherichia coli BL21. Elicitin proteins produced by the recombinant bacteria contained 118 amino acids. The proteins induced HR in tobacco, were thermostable and sensitive to protease K. Therefore, we have established a recombinant E. coli that is capable of producing the elicitin. This is the first report of cloning of elicitin genes from locust strain of the pathogen.Then, experiments to determine whether the elicitin produced by recombinant E. coli can induce resistance to pathogen were performed. Application of elicitin at 30 nM resulted in increased resistance to tobacco mosaic virus (TMV), as compared with the treatment sprayed with water; Induced resistance was strongest at 4-5 days and lasted over 20 days after inoculation. Moreover, tobacco and tomato displayed enhanced resistance tofungal, bacterial, and viral pathogens after treatment with elicitin, pathogens tested include Alternaria longipes, Phytophthora parasitica, Pseudomonas solanacearum and TMV.To investigate molecular signals and whether they have effects on the occurrence of the HR in tobacco leaves treated with elicitin, investigations of SA accumulation,Ca2+ channel response and H2O2 accumulation were carried out. The in vivo SA in tobacco treated and nontreated with the elicitin was tested based on determination by high-performance liquid chromatography (HPLC). Concentration of SA in leaf tissues treated with the elicitin increased by 1.65-2.88 fold compared with the control in 6 days, SA concentration showed two peaks, the first was at 1st day and the second one at 4th day after treatment. In an attempt to determine whether Ca2+ plays a role in elicitin-induced HR, a Ca2+channel inhibitor, La3+was used in injecting the tobacco leaves. The results that La3+ inhibited expression of HIN1, an HR-associated gene, indicated that the HR induction is dependent on Ca2+ channel. Meanwhile, Catalyase (CAT) activity in tobacco leaves tested conspicuously decreased. Consistently, H2O2 concentration increased in elicitin-treated leaves to a limited level, which could not be thought as an "oxygen burst" characterized by many-fold increase in active oxygen species. Based on these data, SA, Ca2+ channel, and H2O2 seems to affect the formation of the HR induced by the elicitin.To determine whether the alterations of the molecular markers mentioned above affect the HR, we assayed elicitin and water-treated tobacco for expressions of HIN1 and PR-1b genes and changes of phenylalanine ammonia lyase (PAL) activity. HIN1 was expressed in tobacco leaves infiltrated with elicitin; the optimal level of HIN1 gene expression was found by 30 minutes after treatment. PR-1b gene started to express in plants at 24h after treatment with elicitin. Meanwhile, PAL activity increased after treatment with elicitin. To illustrate whether the elicitin induces microscopic hypersensitive response (micro-HR), tobacco leaves sprayed...