EXPRESSION AND PURIFICATION OF PHYTOPHTHORA INFESTANS (MONT.) DE. BRAY RXLR EFFECTOR

The RXLR-class effectors play an important role in the pathogenesis of pathogens. They not only can enhance the pathogenicity of the pathogen, but also can be identified as a non-toxic protein to induce cell hypersensitive response (HR) by resistance protein of the host plant. In this study, the gene of RXLR effector was amplified by PCR, then the target gene and expression vector pET-28a were ligated and transformed into BL21(DE3) competent cells to obtain the pET-28a-RXLR recombinant expression plasmid. The protein was induced expressed by IPTG, purified by Ni affinity chromatography and detected by SDS-PAGE. The recombinant expression plasmid pET-28a-RXLR was successfully constructed and expressed in E. coli to produce a protein sized 18.4 kDa, consistent with the theoretical value. The high purity RXLR effector was successfully obtained in this study, laying a foundation for its pathogenicity study. Introduction Plant-pathogenic oomycetes can cause a variety of diseases in crops, and are especially destructive to economic crops, resulting in enormous economic losses (Kamoun et al. 2015). Phytophthora infestans is an oomycete that has been studied extensively and is an essential pathogenic factor causing reduced production or no production of potato (Fry et al. 2015). The pathogen carries hundreds of effector proteins that are secreted into the apoplast or into the host cells. The effector proteins secreted in the host cells are called cytoplasmic effectors, primarily two classes, Crinkler (CRN) proteins and RXLR proteins (Haas et al. 2009, Raffaele et al. 2010). According to gene sequencing and bioinformatics analysis, P. infestans has more than 500 RXLR effectors (Haas et al. 2009), all including a conserved sequence motif Arg-Xaa-LeuArg (RXLR) consensus sequence, in which Xaa represents any amino acid. It is mainly located in gene sparse and repetitive regions, and it evolves faster than genes in dense regions. Therefore, by rapid adaptation RXLR effectors can avoid recognition by R protein (Du et al. 2018). Current findings suggested that RXLR effectors can affect autophagy (Dagdas et al. 2016), protein degradation and stability (Gilroy et al. 2011), kinase/phosphatase signaling (Petra et al. 2016), transcription (McLellan et al. 2013), RNA binding and small RNA biogenesis (Jing et al. 2016), protein secretion (Du et al. 2015), endoplasmic reticulum (ER) stress-mediated immunity and Brassinosteroid signaling (Jing et al. 2016). RXLR effectors can act both as virulence factors to suppress plant basal immunity, and act as non-toxic factors to activate homologous resistance genes (R genes) in the host. However, the biological activities and molecular mechanisms of most RXLR effectors remain elusive. In this study, present authors cloned the RXLR gene by prokaryotic expression technique, ligated to the expression vector pET-28a and transformed into BL21 (DE3) competent Escherichia coli for protein expression. Then the effector was purified by Ni affinity chromatography and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) detection. It was aimed to obtain the RXLR effector and lay a foundation for subsequent research. *Author for correspondence: .