Antifungal properties of a thaumatin-like protein from watermelon

Abstract

Fusarium wilt is a causal disease that threatens watermelon production. In this work, we report the identification and antifungal activity of a thaumatin-like protein (ClTLP27) that was identified from a Fusarium oxysporum (F. oxysporum)-responsive proteomic analysis during watermelon and F. oxysporum interaction. A two-dimensional gel electrophoresis approach was used to compare changes in the leaf proteome profiles of rootstock-grafted watermelon upon F. oxysporum inoculation, and the abundance of a thaumatin-like protein was found to be differentially accumulated. This thaumatin-like protein gene was further cloned from watermelon and named ClTLP27 (accession no. MF445020). ClTLP27 contains 807 nucleotides and encodes a putative polypeptide of 268 amino acids with a calculated molecular mass of 28.93 kDa and a theoretical pI of 7.65. Sequence alignment showed that ClTLP27 contains the conserved motif with 16 cysteines. Phylogenetic analysis indicated that ClTLP27 belongs to the thaumatin-like protein cluster, and is closely related to the Cucumis TLP gene with a sequence identity of 90%. Real-time PCR analysis revealed that ClTLP27 was expressed in all tissues examined, with the highest levels of expression occurring in the roots. Expression profiles of ClTLP27 following F. oxysporum inoculation revealed that the transcript level of ClTLP27 varied in susceptible and resistant seedlings. ClTLP27 was further cloned into the PET28a(+) vector to obtain a bacterially expressed recombinant His-ClTLP27 protein. Antifungal activity analysis showed that the His-ClTLP27 protein significantly inhibited the mycelial growth of F. oxysporum f.sp. niveum race 1, Fusarium solani f.sp. cucurbitae race 1, F. oxysporum f.sp. melonis, Fusarium verticillioides and Didymella bryoniae. This work implies that ClTLP27 could be used as botanical fungicide or as a potential gene in the engineering of disease-resistant watermelon.