| Phytophthora capsici Leonian, is a soil-borne and facultative parasite occurredworldwide, parasites wide-ranging hosts, can attack pepper, tomato, eggplant, melon etc.which belong to solanaceous and cucurbit hosts. It can harm many organs of the plantseriously and long time. In1922, Leon Leonian firstly described a blight of peppers in NewMexico and identified the pathogen—P. capsici. The pepper blight disease seriously affectsthe yield and quality of pepper, causing enormous economic losses as well as environmentaldamage.During the long co-evolutionary process between the plant and pathogen, the host hasbeen formed effectively survival system to identify specific effector molecules, and induceddefense responses. This host response is called effector molecules trigger immune response(ETI). Crinkling and necrosis-inducing proteins (CRN) is an effector molecule-cytoplasmiceffector. CRN are transported into the host cell by the infection structure, appressoria orhaustoria, during the interaction period between pathogenic oomycete and the host, andregulate the plant immune response. In the present study, more than one CRN effector genewere successfully cloned and conducted the prokaryotic expression to obtain high purification(above95%) and high concentration (10mg/ml) protein. The purified proteins were used tocrystallize. Through the X-ray diffraction of high-quality protein crystal, the structure of theCRN effector can resolve and interpret the pathogenic mechanism, broad-host mechanism,transport mechanism and co-evolutionary mechanism at the molecular level. The main resultsare as follows:（1）Five CRN effectors were screened and cloned successfully from the genome of P.capsici, and the homology was higher aligned with the original sequence. There was a littledifferences, but the conserved sequence and the position had no change. It was supposed thatthe difference maybe result from different strains or different mating types.（2）The molecular characters of PC080was consistant with CRN effectors family: Nterminus was conserved and had a host-target signal; C terminus executing effector activitywas very divergent. It was found that the C-terminal region had tyrosine kinase activitythrough the functional domain prediction, suggesting that it may be involved in thetransduction of intracellular signal. The information of the flexible region analysis, secondarystructure prediction and tertiary structure modeling could provide guidance for subsequentprotein expression, purification and crystallization. （3）We designed different truncation constructs based on the character of CRN effectorand the analysis of bioinformation because the full-length protein could not express by thesoluble native protein, The following criteria were considered for selecting fragements to beexpressed and purified:①avoid disrupting predicted secondary structure elements, especially β-sheet, butα-helix can be cut apart in some cases;②preserve the protein domain as much integrity as possible;③as little disordered regions as possible to facilitate solubility and crystallizability;④omitt the signal peptide sequence and avoid presenting the terminal hydrophobicresidues.（4）Finally, we got the high purity target protein through truncations design, usingdouble-His tag, adding urea and Tween-20during washing process of affinity chromatography,increasing the concentration of imidazole in washing buffer, ion-exchange chromatographyand gel filtration chromatography.（5）The major bottleneck of crystal growth of PC080truncations was to obtain theprotein solution having good stability and properties. Since the growth rate of amorphousprecipitat was faster than the rate of crystal growth in the crystallization process. Eventually,we did not get an idea result, though the purified protein by various means were tried tocrystalize.The expression vectors we used in the experiment had pET28a, pET28a-SUMO, pMal,and pGOOD6p-1(GST),which were used to screen the recombinant strain; and thepurification methods used in later experiment had affinity chromatography(6×His tag), ionexchange chromatography, gel filtration chromatography. |
PDF Full Text Request