Characterization of a pathogen induced thaumatin-like protein gene AdTLP from Arachis diogoi, a wild peanut.

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

Effect of recombinant AdTLP protein on the growth of <i>Rhizoctonia</i><i>solani</i>.

<p>C, 1, 2 & 3 represents control, 10µg/ mL, 25µg/ mL, and 50µg/ mL, respectively. Photographs were taken after 24 and 36 hrs of fungal growth (A and B).</p

<i>Rhizoctonia</i><i>solani</i> wilt bioassay with T₂ transgenic and the non-transformed control.

<p>Fungal resistance was checked in control and transgenic plants using phytopathogenic fungus <i>R. solani</i>. Control plants were seriously affected whereas high expression line appeared completely healthy. Photographs were taken after 8 (A) and 10 days (B) post inoculation of fungus.</p

Condition of root, root and shoot junction and complete plant after 10 days of post inoculation of fungus.

<p>Two plants were taken from each line. Wild type plants became completely wilted. Infection symptoms also appeared in the roots of the low expression line plants whereas high expression line plants were completely healthy.</p

Seedlings assay with 200mM NaCl.

<p>Seedlings were transferred on 200mM NaCl medium for 14 days (A). Seedlings on recovery medium (B). Seedlings condition after 10 days of recovery period of line 7 (C), line 4 (D) and the wild type (E). </p

The nucleotide, deduced amino acid sequence and gene structure of <i>AdTLP</i>.

<p>Deduced amino acid sequence of the protein is shown under the nucleic acid sequence (A). Nucleotides are numbered. (*) indicates a stop codon. A 21 amino acid N-terminal signal peptide is underlined. The gene structure of <i>AdTLP</i> is shown in Figure (B). Exon is represented by closed box and dark lines represent 5′ and 3′ UTRs. Their respective lengths were given in bp.</p

Semi-quantitative RT-PCR analysis of transgenic plants.

<p>Transcript levels of <i>AdTLP</i> were checked in T₀ (A) and T₂ (B) transgenic plants. Line 7 is high expression line and line 4 represents low expression line. Actin served as control to demonstrate equal loading.</p

SDS-PAGE analysis of purified protein.

<p>12% SDS-PAGE analysis showing the purified protein. (M) Protein marker, (P) purified recombinant AdTLP protein.</p

Seedlings assay with 300 mM Nacl.

<p>Seedlings were transferred on to 300mM NaCl medium for14 days (A). Seedlings on recovery medium (B). Seedlings condition after 10 days of recovery period of line 7 (C), line 4 (D) and the wild type (E).</p

Seedlings assay with 2% H₂O₂.

<p>Seedlings were transferred on 2% H₂O₂ medium for 12 days (A). Seedlings on recovery medium (B). Seedlings condition after 10 days of recovery period of line 7 (C), line 4 (D) and wild type (E).</p