Screening Of CaM Interacting Protein In Process Of Wheat Leaves Invaded By Leaf Rust Fungi

The increase of intracellular Ca²⁺ transient was triggered by early signs of plant defense responses. Calmodulin (CaM), a ubiquitous Ca²⁺ sensor protein minter is sensitive and can respond to the instantaneous rise of Ca²⁺, Ca²⁺/CaM is one of the most intensive and wide investigations in the plant cell signal transduction. CaM binds its downstream Calmodulin binding protein (CaMBP) regulate cell physiological function. Identification and analysis CaMBP could make a further understanding of Ca²⁺/CaM.Using wheat-leaf rust interaction system as investigated subject, it was showed in our preliminary work of the changing of extracellular Ca²⁺ and CaM concentration, blocking or activating the Ca²⁺ signaling. Ca²⁺ subcellular localization, rising of cytoplast Ca²⁺ in different wheat varieties in elicitor stimulate protoplasts of mesophyll cell showed that Ca²⁺ is involved in the signal transduction of wheat leaf rust (Puccinia triticina) infection, and rising of cytoplast Ca²⁺ may induced by the Ca²⁺ influx. The western blotting analysis and Real-time quantitative RT-PCR results showed that at 4h after inoculation the expression of CaM was increased in the incompatible combination compared with the control, and reached the maximum at 8h after inoculation. These results suggest that in the early stages of wheat-leaf rust interaction, CaM is induced to express to participate in the signal transduction in resistant the infection of leaf rust.To have an insight into the function of CaM and lay a foundation for investigating the signal pathway as well as the role of CaM gene in t resistant the infection of leaf rust, yeast two-hybrid system was used to screen proteins interacting with CaM. The TaCaM2-3 and CaM4-1 of wheat (Triticum aestivum L.) was cloned into PGBKT7 vector firstly. Then we screened the yeast two hybrid cDNA library of wheat leaf which invaded by leaf rust. Eventally 8 putative positive clones were obtained. One of them is high homology with rice, which we called TaCAMTA-Like temporary. The bimolecular fluorescence complementation ( BiFC) conformed that TaCAMTA-Like can interact CaM in the tobacco cell, and the interaction happened in the cytoplasm and also in the nucleus. The analysis of TaCAMTA-Like amino acid sequence revealed conserved regions: IQ motifs, known as calmodulin-binding sites. In electrophoresis mobility shift assay (EMSA), peptides containing IQ motifs can bind CaM in the presence of Ca²⁺. On this basis, RT-PCR was performed to further investigate relative expression of the TaCAMTA-Like in the different time after wheat inoculated leaf rust, in order to discuss the response of defending pathogen infection. The result showed that, the relative expression of TaCAMTA-Like in incompatible combination is higher than CK, and remained a high level during 4-48 h, after 72 h the level of relative expression declined to the normal level; in compatible combination the expression of TaCAMTA-Like had no change compared to CK. These results showed that resistance of wheat to leaf rust infection of wheat during the CaM signal transduction pathway laid a foundation of TaCAMTA-Like gene function research.