Prediction, isolation, overexpression and antifungal activity analysis of Medicago truncatula var. truncatula putative thaumatin like proteins (TLP-1,-2,-3,-4 and-5)

Pathogenesis-related proteins (PR-proteins) are induced in response to environmental stresses such as osmotic and drought stress, wounding, microbial infections and treatment with specific plant hormones and elicitors. These proteins are classified into several groups (PR-1 through PR-17) based on their amino acid sequence and biochemical functions. The present study focuses on prediction, isolation, over-expression and analysis of the antifungal activities of the thaumatin-like proteins (i.e. PR-5) in the model legume M. truncatula var. truncatula. Analysis of M. truncatula genome sequence, available freely on the NCBI website, indicated the presence of at least 15 PR-5 Open Reading Frames (ORFs), 5 of them (dubbed TLP-1, -2, -3, -4 and -5) were selected for this study. Using gene-specific primers, the genomic coding sequences were isolated, sequenced and all confirmed to match with those reported in the database. All the fragments were, then, cloned in Escherichia coli isolate BL21 (DE3), using pET-21c(+) plasmids for subsequent overexpression (overexpression). All 5 genes were expressed as inclusion bodies (IBs) with masses, estimated by SDS PAGE, corresponding to the theoretical values. As expected, none of the protein IBs had no detectable effect on the phytopathogenic fungi Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium solani, Verticillium sp. and Phytophtora spp. However, when the in vitro refolded IB preparations were applied, all displayed comparable strong antifungal activities against the tested fungi. The current study is the first report of overexpression and evaluation of antifungal activities of PR-5 family of proteins from M. truncatula Var. truncatula, and provides experimental evidence that all investigated proteins have the potential for enhancing resistance against some important fungal pathogens.

PDF

___

Acharya K, Pal AK, Gulati A, Kumar S, Singh AK et al. (2013). Overexpression of Camellia sinensis thaumatin-like protein, CsTLP in potato confers enhanced resistance to Macrophomina phaseolina and Phytophthora infestans infection. Molecular Biotechnology 54 (2): 609-622. doi: 10.1007/s12033-012- 9603-y.
Anand A, Lei Z, Sumner L, Mysore K, Arakane Y et al. (2004). Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal. Molecular Plant Microbe Interaction 17 (12): 1306-1317.
Anzlovar S, Dermastia M (2003). The comparative analysis of osmotins and osmotin-like PR-5 proteins. Plant Biology 5:116- 124.
Boccardo NA, Segretin ME, Hernandez I, Mirkin FG, Chacón O et al. (2019). Expression of pathogenesis-related proteins in transplastomic tobacco plants confers resistance to filamentous pathogens under field trials. Scientific Reports 9 (1): 2791
Bryngelsson T, Green B (1989). Characterization of a pathogenesisrelated, thaumatin-like protein isolated from barley challenged with an incompatible race of mildew. Physiological and Molecular Plant Pathology 35:45-52.
Frendo P, Didierjean J, Passelegue E, Burkard G (1992). Abiotic stresses induce a thaumatin-like protein in maize: cDNA isolation and sequence analysis. Plant Science 85:61-69.
Frendo P, Didierjean L, Passelegue E, Burkard G (1992). Abiotic stresses induce a thaumatin-like protein in maize; cDNA isolation and sequence analysis. Plant Science 85 (1): 61-69. doi: 10.1016/0168-9452(92)90094-3.
Ghosh R, Chakrabarti C (2008). Crystal structure analysis of NP24-I: a thaumatin-like protein. Planta 228 (5): 883-890.
Grenier J, Potvin C, Trudel J, Asselin A (1999). Some thaumatin-like proteins hydrolyse polymeric β-1, 3-glucans. Plant Journal 19: 473-480.
Grenier J, Potvin C, Asselin A (1993). Barley pathogenesis‐related proteins with fungal cell wall lytic activity inhibit the growth of yeasts. Plant Physiology 103: 1277-1283.
Hohnjec N, Vieweg FM, Puhler A, Becker A, Kuster H (2005). Overlaps in the Transcriptional Profiles of Medicago truncatula Roots Inoculated with Two Different Glomus Fungi Provide Insights into the Genetic Program Activated during Arbuscular Mycorrhiza. Plant Physiology 137 (4): 1283-1301.
Hu X, Reddy A (1997). Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein. Plant Molecular Biology 34 (6): 949-959.
Karen LM, Diane B, Chengsong L, Cheryl HA Edwards (2003). Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins. Protein Science 12 (9): 2073-2080.
Kitajima S, Sato F (1999). Plant pathogenesis-related proteins: molecular mechanisms of gene expression and protein function. Journal of Biochemistry 125: 1-8.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018). MEGA X: Molecular Evolutionary Genetics 44 Analysis across Computing Platforms. Molecular Biology and Evolution 35 (6): 1547-1549. doi: 45 10.1093/molbev/msy096.
Misra RC, Sandee P, Kamthan M, Kumar S, Ghosh S (2016). A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis. Scientific Reports 6: 25340.
Moosavi-Movahedi AA, Sabourya AA, Hosseinkhanic S, LohrasbiNejadd A, Habibi M et al. (2016). Protein Stability, Folding, Disaggregation and Etiology of Conformational Malfunctions. Biomacromolecular Journal 2 (1): 8-20.
Munis MF, Tu L, Deng F, Tan J, Xu L et al. (2010). A thaumatin-like protein gene involved in cotton fiber secondary cell wall development enhances resistance against Verticilliumdahliae and other stresses in transgenic tobacco. Biochemical and Biophysical Research Communication 393: 38-44. doi: 10.1016/j.bbrc.2010.01.069.
Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15 (3): 473-497. doi: 10.1111/j.1399-3054.1962.tb08052.x.
Murray MG Thompson WF (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8 (19): 4321–4325. doi: 10.1093/nar/8.19.4321.
Piggott N, Ekramoddoullah AKM, Liu JJ, Yu X (2004). Gene cloning of thaumatin-like (PR-5) protein of western white pine (Pinus monticola D. Don) and expression studies of members of the PR-5 group. Physiological and Molecular Plant Pathology 64: 1-8.
Rebmann G, Mauch F, Dudler R (1991). Sequence of wheat cDNA encoding a pathogen-induced thaumatin-like protein. Plant Molecular Biology 17: 283-285.
Roberts WK, Selitrennikoff CP (1990). Zeamatin, an antifungal protein with membrane-permeabilizing activity. Microbiology 136: 1771-1778.
Selitrennikoff CP (2001). Antifungal proteins. Applied and Environmental Microbiology 67: 2883-2894.
Singh NK, Kumar KRR, Kumar D, Shukla P, Kirti PB (2013). Characterization of a pathogen induced thaumatin-like protein gene AdTLP from Arachis diogoi, a wild peanut. Plos One 8(12): 83963. doi: 10.1371/journal.pone.0083963.
Skadsen RW, Sathish P, Kaeppler HF (2000). Expression of thaumatinlike permatin PR-5 genes switches from the ovary wall to the aleurone in developing barley and oat seeds. Plant Science 156: 11-22.
Van der Wel H, Loeve K (1972). Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. European Journal of Biochemistry 31: 221-225. doi:10.1111/j.1432-1033.1972. tb02522.x.
Van Loon LC, Rep M, Pieterse CMJ (2006) Significance of Inducible Defense-related Proteins in Infected Plants. Annual Review of Phytopathology 44: 135-162.
Velazhahan R, Muthukrishnan S (2003). Transgenic tobacco plants constitutively overexpressing a rice thaumatin-like protein (pr5) show enhanced resistance to Alternaria alternata . Biologia Plantarum 47: 347-354.
VelazhahanR, Datta SK, Muthukrishnan S (1999). The PR-5 family: thaumatin-like proteins. In Pathogenesis-Related Proteins in Plants. In: S.K. Datta, S. Muthukrishnan (editors). Boca Raton, FL, NY: CRC Press, pp. 107-129.
Zareie R, Melanson DL, Murphy PJ (2002). Isolation of fungal cell wall degrading proteins from barley (Hordeum vulgare L.) leaves infected with Rhynchosporium secalis. Molecular Plant Microbe Interaction 15: 1031-1039.
Zhang J, Du X, Wang Q, Chen X, Lv D et al. (2010). Expression of pathogenesis related genes in response to salicylic acid, methyl jasmonate and 1-aminocyclopropane-1-carboxylic acid in Malus hupehensis (Pamp.) Rehd. BMC Research Notes 3: 208. doi: 10.1186/1756-0500-3-208.
Zhang J, Wang F, Liang F, Zhang Y, Ma L et al. (2018). Functional analysis of a pathogenesis-related thaumatin-like protein gene TaLr35PR5 from wheat induced by leaf rust fungus. BMC Plant Biology 18: 76. doi: 10.1186/s12870-018-1297-2.