Investigation Of Mechanism Of LePerl Anti Tomato Yellow Leaf Curl Virus Disease By Eugenol

Tomato yellow leaf curl virus (TYLCV) disease has become one of the serious diseases for tomato cultivation. The application of traditional chemical antiviral agent is limited because of the low efficiency and residues contamination. Our lab finds that eugenol is a naturally plant-derived antiviral agent. Pot study suggests that the control effect of eugenol on TYLCV infection is about75.8%, which is much better than chemical antiviral agent moroxydine hydrochloride. Treatment with eugenol alleviates disease symptoms of TYLCV infection by remarkably inhibiting the expression of TYLCV pathogenic genes (e.g. CP, Rep, REn) in tomato plant. In order to clarify the mechanism of eugenol-induced tomato resistance against TYLCV, this study clones the tomato R gene LePerl and investigates the regulation of eugenol on the expression of LePerl.First, the regulation of LePerl expression has been investigated. Salicylic acid (SA) and nitric oxide (NO) are vital signaling molecules in plant immunity. RT-PCR detection suggests that exogenous application of SA and NO donor sodium nitroprusside (SNP) can induce the expression of LePerl. Treatment with eugenol is able to induce the generation of endogenous NO in both root and stem of tomato plant by using NO-specific fluorescent probe DAF-FM DA. These results suggest that endogenous NO mediates eugenol-induced LePerl expression in tomato plant. The previous study in our lab indicated that eugenol could induce the biosynthesis of SA in host plant. Therefore, it can be suggested that eugenol induces LePerl expression by stimulating endogenous SA biosynthesis in plants. Due to the fact that SA is able to stimulate the generation of endogenous NO, NO acts as downstream of SA in plant immunity. Therefore, NO could be an importance messenger molecule operating downstream of SA through a linear signaling pathway during eugenol-induced expression of LePerl in tomato plant. In addition, other plant hormones (e.g. JA and GA) can also induce LePerl expression in tomato plant, suggesting that LePerl is a common downstream target in multiple plant hormone-mediated immunity pathways. However, whether and how eugenol regulates LePerl expression through other plant hormones remain to be further investigated.Second, cloning, structure analysis and functional prediction of LePerl. The full cDNA sequence of LePerl with length of1862bp containing the integrated ORF has been obtained by segment cloning, sequencing, and assembling. LePerl gene contains14exons. Blast analysis suggests that LePerl is highly conserved in plant kingdom. LePerl is predicted to be a membrane protein by the analysis of hydrophobicity and trans-membrane as well as the prediction of cellular location. The analysis of conserved domain combined with homologous modeling of the3D structure of LePerl protein suggests that LePerl is a permease with the ability of transporting nucleoside and ascorbate acid. There are several protein modification sites (e.g. Protein kinase C phosphorylation, N-glycosylation, Casein kinase II phosphorylation, and N-myristoylation) in LePerl, suggesting that LePerl may be involved in kinase-mediated signal transduction. However, the detailed role of LePerl and its downstream cassette in tomato plant against TYLCV infection needs to be further studied by using transgenic approach.