Purification and Characterization of a CkTLP Protein from Cynanchum komarovii Seeds that Confers Antifungal Activity

Abstract

Background Cynanchum komarovii Al Iljinski is a desert plant that has been used as analgesic, anthelminthic and antidiarrheal, but also as a herbal medicine to treat cholecystitis in people. We have found that the protein extractions from C. komarovii seeds have strong antifungal activity. There is strong interest to develop protein medication and antifungal pesticides from C. komarovii for pharmacological or other uses.Methodology/Principal FindingsAn antifungal protein with sequence homology to thaumatin-like proteins (TLPs) was isolated from C. komarovii seeds and named CkTLP. The three-dimensional structure prediction of CkTLP indicated the protein has an acid cleft and a hydrophobic patch. The protein showed antifungal activity against fungal growth of Verticillium dahliae, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea and Valsa mali. The full-length cDNA was cloned by RT-PCR and RACE-PCR according to the partial protein sequences obtained by nanoESI-MS/MS. The real-time PCR showed the transcription level of CkTLP had a significant increase under the stress of abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), NaCl and drought, which indicates that CkTLP may play an important role in response to abiotic stresses. Histochemical staining showed GUS activity in almost the whole plant, especially in cotyledons, trichomes and vascular tissues of primary root and inflorescences. The CkTLP protein was located in the extracellular space/cell wall by CkTLP::GFP fusion protein in transgenic Arabidopsis. Furthermore, over-expression of CkTLP significantly enhanced the resistance of Arabidopsis against V. dahliae.Conclusions/SignificanceThe results suggest that the CkTLP is a good candidate protein or gene for contributing to the development of disease-resistant crops.

Purification, characterization of a CkChn134 protein from Cynanchum komarovii seeds and synergistic effect with CkTLP against Verticillium dahliae

“…12,28 Although, a little known the synergistic mechanism of chitinase and thaumatin-like genes, they degrade the structural components of fungal cell or include alterations in fungal cell membrane integrity to inhibit the fungal cell growth. [29][30][31][32] The CkTLP protein from C. komarovii was located in the extracellular space/cell wall 13 and the CkChn134 had same distribution in plant, it showed the two proteins accumulated in the extracellular space/cell wall in plant to inhibit the invasion and spread of fungal pathogens, which may potentially be a significant reason for synergistic protective interaction of the two proteins in transgenic cotton against V. dahliae.…”

“…Previous work in our laboratory had shown the enhance tolerance to V. dahliae by overexpression of CkTLP in Arabidopsis. 13 In this work, it is the first-time report the development of V. dahliae-resistant transgenic Arabidopsis with the expression of CkChn134 and coexpression of CkChn134 and CkTLP. We found transgenic plants expressed constitutively CkChn134 and CkTLP, which showed more significantly enhanced resistance to V. dahliae than overexpression CkChn134 and CkTLP, respectively (Fig.…”

“…Antifungal activity of the purified protein against the test pathogenic fungus V. dahliae was displayed in Figure 2. To further examine the combination of CkChn134 and CkTLP from C. komarovii seeds, 13 the IC 50 values of these fungi were tested. When the two proteins were used in combination, the IC 50 values lower than the proteins were used individually (Table I).…”

“…The antifungal activity assay in vitro and determination of IC 50 were performed as previously described. 13 The conidial suspensions of V. dahliae were prepared from a culture grown for 5 days at 25 C in Czapek liquid medium (NaNO 3 2 g, K 2 HPO 4 1 g, MgSO 4 .7H 2 O 0.5 g, KCl 0.5 g, FeSO 4 0.01 g, sucrose 30 g, distilled water 1 L, and pH 7.2). All fungi were used for the detection of antifungal proteins in vivo.…”

“…So far, a novel thaumatinlike protein (TLP) was isolated from C. komarovii seeds, which displayed strong antifungal activity and resistance to V. dahliae in transgenic Arabidopsis. 13 To screening more antimicrobial proteins, we continue to choose this plant to develop more valuable proteins to control the verticillium wilt of cotton. Cotton losses due to this disease-causing organism are $50% in production in China and bring huge economic losses every year.…”