Isolation and molecular characterization of a phytoplasma-induced cDNA clone encoding a lipoamide dehydrogenase from Sesamum alatum implicated in disease resistance

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With a long-term objective of making cultivated variety Sesamum indicum tolerant to phyllody disease, phytoplasma specific cDNA clone was isolated by a differential display from S. alatum which is tolerant to phyllody disease. For this, total RNAs were isolated from the leaves of S. indicum , S. alatum and S. alatum on which S. indicum having phyllody had been grafted and differential display was performed. Comparison of cDNA products obtained at various stages of grafting allowed picking fifteen putative cDNA clones. To confirm that which of these cDNA fragments are truly unique to the target tissues, the differential display experiment was repeated and reverse northern was performed. Four cDNA clones were finally selected, reamplified and subsequently cloned in pMOS vector. One of the cDNA clones also showed hybridization signal in S. indicum which was affected with phyllody but not in the healthy S. indicum . This cDNA clone was not only constitutive and defense related but also phytoplasma specific. This partial cDNA clone (AP13 + C1) obtained after the differential display was used as a probe for isolation of the full-length cDNA library from S. alatum . By colony hybridization, a homogenous cDNA clone of 1.2 kb was isolated. Nucleotide sequence translation and their BLAST search revealed 99.82% homology to putative lipoamide dehydrogenase. According to the created phylogenetic tree, it was inferred that the Poaceae ( Oryza sativa & Oryza brachyantha ) were grouped with Pedaliaceae ( S. alatum ) and formed monophyletic clade. Further, its physicochemical analysis revealed that the protein was somewhat acidic, hydrophobic, thermostable and stable. Genetic manipulation of this gene may thus pave the way to make cultivated sesamum phyllody resistant.

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