Cloning And Functional Analysis Of Trehalase Gene From Puccinia Striiformis F.sp. Tritici

Puccinia striiformis f. sp. tritici（Pst） is an obligate plant pathogenic fungi, preferring cool and cold climate for growth and development. Pst has strong adaptability to low temperature, and wheat stripe rust caused by Pst is a kind of major diseases in wheat. However, up to now, there was no study on adaptation mechanism of Pst to low temperature. Trehalose hydrolase（Trehalase）, the key enzyme in microbial trehalose hydrolysis pathway, can not only be involved in stress response of microorganism, but also participate in the process of microbial growth and development. Trehalose hydrolase genes including PstNTH1 and PstNTH2 were cloned from Pst in this study. The gene expression pattern of trehalose hydrolase genes under low temperature stress and the influence on Pst virulence was analyzed in order to further understand adaptation mechanism of the wheat stripe rust to low temperature. The main results are as following:1. Trehalose hydrolase genes including PstNTH1 and PstNTH2 were cloned from Chinese yellow rust 32（CYR32）, both of them containing conserved domain of trehalose hydrolase family. PstNTH1 shares 99% amino acid sequence identity with hypothetical protein PSTG₀4138 of P. striiformis f. sp. tritici PST-78 and 84% with alpha, alpha-trehalase of P. graminis f. sp. tritici CRL 75-36-700-3. PstNTH2 shares 99% amino acid sequence identity with trehalase of PST-78 and 64% with hypothetical protein PGTG₁2486 of CRL 75-36-700-3.2. The relative expression levels of PstNTH1 and PstNTH2 gene were confirmed through real-time fluorescent quantitative PCR during Pst infection. Results showed that the relative expression level of PstNTH2 at 6 h after inoculation was significantly increased suggesting that PstNTH2 might participate in the germination process of Pst urediniospores. Under low temperature stress（0℃ and 5℃）, the expression levels of PstNTH1 and PstNTH2 were significant increased, which indicated that PstNTH1 and PstNTH2 might be involved in participating in cold stress response and pathogenic process.3. The expressioin of PstNTH1 and PstNTH2 genes was silenced using BSMV-mediated HIGS（host-induced gene silencing, HIGS） technology. Results showed that no effect on sporulation of Pst was found when these genes were silenced. However, the colony area of Pst in PstNTH2-silenced plants was increased which indicated that PstNTH2 might be involved in the expansion procedure of the hyphae.