Briefly, monolayers of PC-3MMM2 cells were cultured on glass coverslips

hly susceptible cultivated peanuts. Hence, they are assumed to be an important source of genes for resistance to the stresses for peanut improvement. The leaf spots, collectively termed as `Tikka’ disease is a very serious disease affecting the cultivated peanuts. One of the pathogens responsible for Tikka disease is Phaeoisariopsis personata that causes late leaf spot; and early leaf spot is caused by Cercospora arachidicola. Wild peanut, Arachis diogoi exhibits very high levels of resistance to these and other diseases that seriously affect the cultivated peanuts. Through a differential display approach, we have earlier identified several genes that get significantly upregulated in Arachis diogoi upon treatment with Phaeoisariopsis personata. One of the upregulate genes designated as AdDR-11 in the wild peanut was identified to encode a putative thaumatin-like protein. In the present study, using the available partial cDNA sequence of AdDR-11, full length cDNA was amplified and cloned from Arachis diogoi and it was named as AdTLP. We Y-27632 dihydrochloride web analyzed its transcriptional regulation in response to various treatments and its subcellular localization by translational fusion with GFP. In vitro and in vivo antifungal activity of AdTLP protein was analyzed against different fungal pathogens, Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani. Abiotic stress assays were also carried out to observe the efficacy of AdTLP in alleviating stress caused high NaCl and H2O2 conditions. Isolated RNA was quantified and the quality was evaluated through formaldehyde gel electrophoresis. Rapid amplification of cDNA ends was performed using 5/3 RACE kit following the manufacturer’s instructions. In brief, two micrograms of total RNA was reverse transcribed with gene specific primer AdDR11-146R using Transcriptor reverse transcriptase provided in the kit. The dA-tail was added to the synthesized first strand cDNA at the 5 end using terminal transferase. The dA-tailed cDNA was used as template in subsequent PCR reactions. Gene specific primers AdDR11-117R and AdDR11-64R were designed based on the available partial sequence . It was used in combination with Oligo-dT anchor primer and PCR anchor primer in the PCR reactions respectively. RACE-PCR reactions were performed using Taq DNA polymerase. Genomic DNA served as templates for the amplification of genomic sequences. All PCR products described in the study were cloned in to pTZ57R vector and sequenced commercially for sequence confirmation. All the primer details were provided in Materials and Methods Plant materials and treatments Wild peanut and tobacco plants 7473193 were maintained in the green house. For different treatments, detached leaves of A. diogoi were utilized and the experiments were performed essentially as described earlier. In brief, 105 conidia per milliliter of the peanut late leaf spot pathogen, Phaeoisariopsis personata were used for pathogen treatment. For hormone treatments, concentrations of 500 M salicylic acid, 100 M methyl jasmonate, 100 M abscisic acid, and 250 M ethephon were utilized. Samples were collected at regular intervals, quick-frozen in liquid nitrogen, and stored at -80 C till further use. Analysis of cDNA and protein sequence The cDNA 10485587 sequence data were analyzed using BLASTn and BLASTp at NCBI website. The theoretical isoelectric point and molecular mass were computed using COMPUE pI/Mw tool. Nucleotide translations were performed using translate tool at Ex