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

Abstract

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.

Figure 1. The nucleotide, deduced amino acid sequence and gene structure of AdTLP.

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 AdTLP 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.

Figure 2. Multiple sequence alignment of AdTLP with TLPs from other plant species.

The sixteen cysteine residues required for the formation of eight disulfide bridges are conserved in AdTLP also and are indicated by asterisk (*). Mt: Medicago truncatula, Gm: Gycine max, Pp: Pyrus pyrifolia, At: Arabidopsis thaliana, Nt: Nicotiana tabacum.

Figure 3. Phylogenetic analysis of AdTLP with other TLPs.

A phylogenetic tree based on genetic distance of the protein sequences was constructed using MEGA 4.0.2 software. Bootstrap values are indicated at the branches. The TLP members used for construction of the tree are listed in the GenBank database under the following accession numbers: AtTLP (AAD02499.1); BhOLP (AAD53089.1); BkTLP (CBJ55937.1); BrTLP (ABV89616.1); CjTLP (BAD90814.1); CmTLP (ADN33945.1); CtjTLP (BAI63297.1); DcTLP (AAL47574.1); FpTLP (ABB86299.1); GbTLP (ABL86687.1); GmTLP1a (XP_003535214.1); HvTLP (BAJ96850.1); LjTLP (AFK33451.1); MdTLP (AAC36740.1); MtTLP (AFK34461.1); NtTLP (BAA74546.2); OsTLP (BAD34224.1); PhpTLP (XP_001784610.1); PpTLP (BAC78212.1); PmTLP (ADB97928.1); PrpTLP (AEV57470.1); PtTLP (XP_002330973.1); RcTLP (XP_002519620.1); SbTLP (XP_002465570.1); TaTLP (AAM15877.1); ThTLP (BAJ34394.1); TpTLP (BAE71242.1); VvPR5 (XP_002277548.1); ZmTLP (NP_001142502.1); At Arabidopsis thaliana, Bh Benincasa hispida, Bk Bupleurum kaoi, Br Brassica rapa, Cj Cryptomeria japonica, Cm Cucumis melo, Ctj Citrus jambhiri, Dc Daucus carota, Fp Ficus pumila, Gb Gossypium barbadense, Gm Glycine max, Hv Hordeum vulgare, Lj Lotus japonicas, Md Malus domestica, Mt Medicago truncatula, Nt Nicotiana tabacum, Os Oryza sativa, Php Physcomitrella patens, Pp Pyrus pyrifolia, Pm Pinus monticola, Prp Prunus persica, Pt Populus trichocarp, Rc Ricinus communis, Sb Sorghum bicolor, Ta Triticum aestivum, Th Thellungiella halophile, Tp Trifolium pratense, Vv Vitis vinifera, Zm Zea mays.

Figure 4. Transcript level analysis of AdTLP in A. diogoi.

Transcript level of AdTLP in A. diogoi was analyzed using semi-quantitative RT-PCR, during P. personata (A) and various hormones treatments (B).

Figure 5. Subcellular localization of AdTLP by transient expression in tobacco leaves using agroinfiltration.

Empty vector pEGAD expressing free GFP (A, B, C). AdTLP:GFP:1300 expressing translationally fused AdTLP-GFP and cells were visualized at different planes (D-I). Expression of free GFP and AdTLP-GFP in epidermal cells (A, D, G), corresponding bright field image (B, E, H), and overlay of GFP signal onto bright field image (C, F, I).

Figure 6. SDS-PAGE analysis of purified protein.

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

Figure 7. Fungal spore germination assay in the presence of different concentrations of AdTLP protein.

A. Fusarium oxysporum B. Fusarium solani C. Botrytis cinerea.

Figure 8. Effect of recombinant AdTLP protein on the growth of Rhizoctonia solani.

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).

Figure 9. Semi-quantitative RT-PCR analysis of transgenic plants.

Transcript levels of AdTLP 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.

Figure 10. Rhizoctonia solani wilt bioassay with T₂ transgenic and the non-transformed control.

Fungal resistance was checked in control and transgenic plants using phytopathogenic fungus R. solani. 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.

Figure 11. Condition of root, root and shoot junction and complete plant after 10 days of post inoculation of fungus.

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.

Figure 12. Seedlings assay with 200mM NaCl.

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).

Figure 13. Seedlings assay with 300 mM Nacl.

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).

Figure 14. Seedlings assay with 2% H₂O₂.

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).

Figure 15. Total chlorophyll and TBARS measurement.

Total chlorophyll and TBARS were measured in seedlings after 12d of NaCl treatment (A and B) and after 10d of H₂O₂ treatment (C and D). Note the significantly increased total chlorophyll content and reduced TRABS in the transgenic seedlings after stress treatments. Experiments were repeated three times and means ±SE were plotted (P < 0.05, n = 3).

Figure 16. Transcript profile of defense responsive genes.

Semi-quantitative RT-PCR was performed for transcript profiling of defense response genes in WT and transgenic plants. PR: Pathogenesis related proteins, PI: Protease inhibitor, Lox: Lipoxygenase, AOS: allene oxide synthase, ICS: isochorismate synthase, ACS: 1-aminocyclopropane-1-carboxylic acid synthase.