Peanut (Arachis hypogaea L) is one of the widely cultivated and leading oilseed crops of the world and its yields are greatly affected by various biotic and abiotic stresses. Arachis diogoi, a wild relative of peanut, is an important source of genes for resistance against various stresses that affect peanut. In our previous study a thaumatin-like protein gene was found to be upregulated in a differential expression reverse transcription PCR (DDRT-PCR) study using the conidial spray of the late leaf spot pathogen, Phaeoisariopsis personata. In the present study, the corresponding full length cDNA was cloned using RACE-PCR and has been designated as AdTLP. It carried an open reading frame of 726 bp potentially capable of encoding a polypeptide of 241 amino acids with 16 conserved cysteine residues. The semi-quantitative RT-PCR analysis showed that the transcript level of AdTLP increased upon treatment with the late leaf spot pathogen of peanut, P. personata and various hormone treatments indicating its involvement in both, biotic and abiotic stresses. The antifungal activity of the purified recombinant protein was checked against different fungal pathogens, which showed enhanced anti-fungal activity compared to many other reported TLP proteins. The recombinant AdTLP-GFP fusion protein was found to be predominantly localized to extracellular spaces. Transgenic tobacco plants ectopically expressing AdTLP showed enhanced resistance to fungal pathogen, Rhizoctonia solani. The seedling assays showed enhanced tolerance of AdTLP transgenic plants against salt and oxidative stress. The transcript analysis of various defense related genes highlighted constitutively higher level expression of PR1a, PI-I and PI-II genes in transgenic plants. These results suggest that the AdTLP is a good candidate gene for enhancing stress resistance in crop plants.

Protease inhibitors have been reported to confer insect resistance in transgenic plants, except for a rice protease inhibitor that conferred drought tolerance in transgenic rice plants. We have cloned a protease inhibitor of tobacco that is expressed under treatment with ABA, hydrogen peroxide, methyl jasmonate and wounding. The cDNA codes for a six-domain serine protease inhibitor with a deduced sequence of 396 amino acids. We have generated transgenic tobacco plants expressing the protease inhibitor constitutively under the 35S promoter. When analyzed in the T(2) generation, these transgenic plants exhibited tolerance to sodium chloride, variable pH and sorbitol, together with the expected resistance to the insect pests Spodoptera litura and Helicoverpa armigera. The transgenic plants showed enhanced seed germination, root length and root-shoot ratio, significantly enhanced total chlorophyll content and reduced thiobarbituric acid-reactive substances under stress. Under sodium chloride treatment, the transgenic plants have enhanced protease inhibitor activity. The transgenic plants exhibited a higher potassium content and an optimum Na+/K+ ratio. To our knowledge, this is the first report of transgenic plants with constitutive protease inhibitor expression showing tolerance to a wide range variable pH in the culture medium along with other stresses.

The wild relatives of peanut are resistant to various economically important diseases including late leaf spot (LLS) caused by Phaeoisariopsis personata, compared with the susceptible cultivated peanut (Arachis hypogaea L.). The interaction of the late leaf spot pathogen, Phaeoisariopsis personata and the highly resistant, diploid peanut wild species, Arachis diogoi was analyzed at the molecular level by differential gene expression studies. Genes up-regulated with in 48 h of pathogen challenge were isolated as partial cDNAs. Some of the isolated genes, which are shown to be involved in the first line of defense in plants, were further characterized with respect to their transcriptional regulation in response to pathogen. Among the isolated clones, two were found to encode cyclophilin like proteins. One of the two isolated partial cDNAs encoding cyclophilin like proteins was extended using 5' RACE. The full length cDNA, designated as AdCyp, was 886 bp in length and encodes a polypeptide of 172 amino acids. Southern hybridization suggests that AdCyp is possibly coded by a single gene and at least one more identical gene is present in Arachis diogoi genome. AdCyp exhibits evolutionary conservation across the kingdoms. Phylogenetic analysis showed that AdCyp belongs to the subgroup I of Group I in cyclophilins. A translational fusion of GFP-AdCyp was found to localize to both cytosol and nucleus. AdCyp transcripts were found to accumulate in response to the treatments with pathogen as well as phytohormones. Constitutive heterologous expression of AdCyp resulted in enhanced resistance to Ralstonia solanacearum and reduced susceptibility towards Phytophthora parasitica var. nicotianae in transgenic tobacco and the resistance was associated with higher transcript levels of various defense related genes.

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765-775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu(2+)/Zn(2+) superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.

No abstract