Recent studies have implicated miR-21 in the development of cancer resistance

P in epidermal cells, corresponding bright field image, and overlay of GFP signal onto bright field image. doi: 10.1371/journal.pone.0083963.g005 synergistically regulated by ethylene and JA were also unaffected at the transcript level. Discussion Thaumatin-like proteins have been isolated and characterized from different plants and tissues. They are classified under the PR-5 proteins and are shown to be involved effectively in alleviating both biotic and abiotic stress tolerance. In this study, a pathogen induced, full length 726 bp cDNA encoding a thaumatin-like protein from wild peanut, Arachis diogoi was amplified and cloned using partial AdDR-11 template as reference sequence that was identified as one of the upregulated genes in the wild peanut in pathogen challenge. For our study, the gene was named as AdTLP. Sequence analysis revealed that AdTLP encodes a predicted 19774075 protein of 241 amino acids which exhibited a 21 amino acid long N-terminal signal peptide with 25.01 kDa molecular weight and 4.71 theoretical pI. Phylogeneitc analysis revealed that the leguminous MtTLP and GmTLP showed closest similarity to AdTLP with 68% and 66% respectively. The induction of TLP upon pathogen treatment was reported in several plant-microbe interactions. The transcript level of AdTLP got upregulated during Phaeseoropsis personata treatment and reached to the highest level after 72 hpi possibly showing its activity during the later stages of infection. The stress hormones involved in the signalling of biotic and abiotic stress responses like SA, JA and ABA had a positive induction effect on the AdTLP transcript levels suggesting a possible role of AdTLP in different stress responses with a significant cross talk. The recombinant TLP proteins like TaPR5-GFP and CkTLP-GFP were mainly identified as extracellular proteins during transient expression. Other TLPs like RlemTLP and CsTL1, despite being predicted as extracellular, were found predominantly localized to both periphery of plasma TG-101348 price membrane and cytoplasm and involved in anti-fungal activities as well. The localization analysis of recombinant AdTLPGFP protein also showed extracellular localization and the observation was consistent with the prediction of secretory signal peptide of 21 amino acids. However, the AdTLP-GFP protein expression in subcellular structures, possibly ER regions, needs further investigation for confirmation. The presence of AdTLP-GFP protein in the nuclear boundaries could be due to the extension of ER from plasma membrane to nucleus or due to the overexpression of GFP fusion protein under 35S promoter. The antifungal activity of the recombinant AdTLP that was purified after induction in the E.coli prokaryotic system was checked using spore germination and plate assays with different filamentous fungal pathogens that attack various crop plants. During spore germination assay, various 9 A Thaumatin like Protein Gene of Wild Peanut doi: 10.1371/journal.pone.0083963.g006 concentrations of protein were used and it was observed that 5g/ mL of recombinant protein was sufficient to inhibit spore germination completely in the case of F. oxysporum, F. solani and B. cinerea for which the calculated IC50 values were less than 1g/ mL. These values were significantly low compared to a legume TLPs as 3986806 well as osmotins and TLPs from other plants. Apart from growth inhibitory activity, the AdTLP also induced morphogenic changes like hyperbranching in the mycelium of fungal pathogen, B.cinerea