| Summary: | 博士 === 國立東華大學 === 生命科學系 === 101 === Plant interactions with microbial bio-control agents are used as experimental models to understand resistance related molecular adaptations of plants. In a hydroponic three-way interaction study, a novel Trichoderma harzianum ETS 323 mediated mechanism was found to induce resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets. The R. solani challenge on leaves initiate an increase in lipoxygenase activity and associated hypersensitive tissue damage with characteristic “programmed cell death” that facilitate the infection. However, B. oleracea plantlets whose roots were briefly (6 h) colonized by T. harzianum ETS 323 developed resistance to R. solani infection through a significant reduction of the host hypersensitive tissue damage. The resistance developed in the distal leaf tissue was associated with the expression of a H2O2 inducible glutathione S-transferase (BoGST) which scavenges cytotoxic reactive electrophiles, and of a deoxycytidine deaminase (BoDCD), which modulates the host molecular expression and potentially neutralizes the DNA adducts and maintains DNA integrity. The cDNAs of BoGST and BoDCD were cloned and sequenced; their expressions were verified by RT- PCR analysis and were found to be transcriptionally activated during the three-way interaction. To determine the in vitro and in vivo biochemical and functional properties of BoDCD the enzyme was cloned into an E. coli expression system and their potential to neutralize the toxic analogs of 2′-deoxycytidine was examined. BoDCD transformant E. coli cells were found to be resistant to 2′-deoxycytidine analogs in all the concentrations tested. The BoDCD enzyme was also over-expressed as a histidine-tagged protein and purified using Nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 KDa as visualied by SDS-PAGE. The substrate specificity and other kinetic properties determined with its possible natural substrates and with cytosine β-D-arabinofuranoside, a deoxycytidine analog, show that BoDCD is found to be potentially more active in neutralizing 2′-deoxycytidine adducts than deaminating 2′-deoxycytinde to 2′-deoxyuridine in nuclei acids or metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5 respectively. The Km and Vmax values of BoDCD for their natural substrate 2′deoxycytidine were 91.3 µM and 1.475 mM respectively and with the respective values of 63 µM and 2.072 mM the enzyme were found to be relatively more active with cytosine β-D-arabinofuranoside. Moreover, cytosine β-D-arabinofuranoside showed better kcat/Km values of 0.697 µM s-1 than 2′-deoxycytidine (0.342 µM s-1) and cytidine (0.034 µM s-1). The BoDCD can effectively bind to analogs modified at C2 position than their substrates of established metabolism and therefore BoDCD can efficiently play a major role in neutralizing cytotoxic dC analogs and establish T. harzianum ETS 323 induced resistance in B. oleracea. BoDCD is the first ever resistance related DCD in plants to be reported and characterized.
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