Structural Basis Of The DNA Binding Activity Of Pcg2 And MoSub1 From Rice Blast Fungus Magnaporthe Oryzae

Rice blast fungus Magnaporthe oryzae, the first one of the top 10 plant fungal pathogens, is the most destructive pathogen of cultivated rice. Due to the significance of rice on world food safety, as well as some unique biological characters of this fungus, M. oryzae has become an important model organism for studying interactions between pathogens especially filamentous fungi and plant hosts. Therefore, it is not only beneficial to the prevention and control of rice blast disease by understanding the pathogenic molecular mechanism of M. oryzae, but also of guiding significance for the study of other pathogenic fungi. DNA binding proteins, which are involed in the regulation of DNA replication, transcription, recombination, repair, rearrangement, and many other biochemical processes, affect the growth and development process, as well as the pathogenic infection process of pathogens. In this study, the structural basis of the DNA binding mechanism of Pcg2 and MoSubl from rice blast fungus has been studied.Pcg2, one of APSES transcription factors in rice blast fungus, has the ability to bind to cis-element MCB and SCB. Pcg2 plays significant roles in regulating developmental processes, for that the â–³pcg2 deletion mutant loses the pathogenicity on rice. Results from sedimentation velocity-analytical ultracentrifugation analysis demonstrate that Pcg2 DBD can form different protein-DNA complexes in a concentration-dependent manner, and that at low concentration the 1:1 complex predominates but an additional protein can be accommodated onto the MCB DNA to form a 2:1 complex at higher concentration. The recombinant expressed C-terminal truncation Pcg21-424, containing Pcg2 DBD and ANK repeats, forms a dimer in the solution. Because of the instability of Pcg21-424, a Pcg2 DBD Q82C mutant was designed based on the crystal structure of Pcg2 DBD-MCB complex to make an artifical dimer. Then DNA complexes of Q82C mutant with SCB, UN1 and BT1 were prepared and used for crystallization. The crystal structure of apo Q82C mutant has been determined, and the crystallization of the DNA complexes is ongoing. In addition, the effect of phosphorylation of Pcg2 DBD on its DNA binding activity has been analysized by surface plasmon resonance (SPR) technology using phosphomimetic mutant S123E. Results show that phosphorylation of the S123 may have no significant effect on the DNA binding activity of Pcg2 DBD alone.MoSubl is the homolog of human transcription co-activator PC4 or yeast Subl. These three proteins, sharing a highly conserved ssDNA binding domain, show some differences on their domain organization and biochemical functions. The structural basis for these differences are still unknown by now. F77 and W89 in PC4 have been proved to be two key amino acid residues to interact with ssDNA. The F77 are conserved in all PC4-like proteins, while W89 is substituted with tyrosine(Y) in PC4 orthologous proteins of seven species including MoSubl in M. oryzae. To understand the consequence and reveal the molecular details of this substitution, the crystal structure of PC4 CTD W89Y mutant in complex with ssDNA was determined. The comparison of this structure with the structure of PC4 CTD-ssDNA complex and that of MoSubl-ssDNA complex was made. Results show that a hydrophobic patch around W89 that favours interactions with two nucleotides is not formed in the PC4 W89Y mutant and MoSubl. Therefore, the conserved Y74 in MoSubl or W89 in PC4, is not only the key residue in making specific interactions with DNA but also required to determine the DNA binding mode of PC4-like proteins. The relationship between the differences in DNA binding mode and their biological functions need to be further studied. Moreover, the effect of phosphorylation on the ssDNA binding activity of MoSubl was explored using phosphomimetic mutant and phosphorylated samples by CKII kinase. Results demonstrate that, unlike PC4 or Sub1, phosphorylation of MoSubl does not affect the affinity of MoSubl binding ssDNA. Meanwhile, we also found that phosphate ion may also have effect on the formation of MoSub1-ssDNA complex. Further study is ongoing.All these studies provide the structure basis for us to understand the molecular details of the interaction between Pcg2 and the cis-element as well as that between PC4-like proteins and ssDNA in depth. Besides that, these results also provide structure clues for us to comprehensively reveal their biological functions in growth, development and pathogenic process of plant fungal pathogens.