Activity Analysis Of Sulfotransferase RaxST From Xanthomonas Oryzae Pv.Oryzae

Xanthomonas oryzae pv. oryzae (Xoo), a gram-negative pathogens, causes rice bacterial blight, which dramatically decreases the rice yield. To prevent the disease, further study on the characteristics and mechanisms of pathogenesis of different Xoo The plant immune system, is essential for improving rice yield.Rice with Xa21is resistant to Xanthomonas oryzae pv. oryzae (PXO99), while rice without Xa21is susceptible to PXO99. These showed that Xa21can specifically recognize microbe-associated molecular pattern (MAMP) of PXO99. The N-terminal and C-terminal protein segment of PXO-03968(Ax21) had been separated from the active ingredient component of PXO99bacterial cultural broth. The sulfated polypeptide (axYs22),N-terminal of Ax21, which can be recognized by Xa21, was also found in cultural broth, and tyrosine sulfation is essential for its activity. RaxST is considered to catalyze sulfation of Ax21or axY22in PXO99, with3’phosphoadenosine5’-phosphosulfate (PAPS) as sulfate donor.Twenty eight hydrophobic amino acids were found at the N-terminus of RaxST, which may promote the association of RaxST to the membrane bilayers, and therefore may affect the purification and activity assay of RaxST. In this study, we constructed full length RaxST(fRaxST) expression vectors (pET24a-KanP-RaxSTand pMALC2E-fRaxST), and truncated RaxST (tRaxST, without hydrophobic sequence) expression vector (pMALC2E-tRaaxST). The pET24a-KanP-RaxST plasmid vector is transferred into competent PXO99by electrical transformation, and recombinant strains were selected by antibiotics kanamycin. Meanwhile pMALC2E-fRaxST and pMALC2E-tRaxST were transferred into competent E. coli BL21(DE3) by heat shock method for expression and further purification of RaxST from E. coli. fRaxST and tRaxST were expressed using different hosts, and purified with affinity chromatography. It is not clear about the real substrate for RaxST. Ax21, axY22and the peptide smaller than axY22(octapeptide) are all possible substrates. Based on this hypothesis, pMALC2E-PXO-03968prokaryotic expression vector for expression of Ax21is constructed, and it was subsequently expressed and purified. Octapeptide (AENLSYNF) and axY22were commercially synthesized.Kinetic constants of RaxST were different with different source of RaxST and substrates. fRaxST purified from PXO99catalyzes sulfation of axY22with Vm and Km of10.5(±0.5) μM/min and0.26(±0.04) mM, respectively. fRaxST and tRaxST expressed in E. coli BL21(DE3) has no detectable activity with Ax21or octapeptide as substrates. tRaxST fromshows sulfation activity with axY22as substrate, with Vm and Km of7.12(±0.9) p.M/min and3.10(±0.2) mM, respectively.Studies on RaxST here provided some theoretical basis for the immune interactions between prokaryotes and their hosts, and information exchanges between prokaryote cells.