Genomic Organization And Sequence Dynamics Of The Avrpiz-t Locus And Functional Analysis Of Cysteine Residues Of AvrPiz-t Protein In Magnaporthe Orvzae

Plants mount effective immunity to pathogens by utilizing resistance (R) genes which are mostly dependent on the co-existence of pathogen derived avirulence (Avr) genes. The rice blast R gene Piz-t interacts in gene-for-gene relationship with the Magnaporthe oryzae Avr gene AvrPiz-t. In this study, we determined and compared the genomic sequences of the AvrPiz-t locus in an avirulent and a virulent isolates (which were named as AvrPiz-t-ZB15and avrPiz-t-70-15, respectively), and analysised the function of Avr Piz-t. Sequence analysis found that the AvrPiz-t-ZB15region is120,966bp whereas its allelic avrPiz-t-70-15region is146,292bp in length. Except for the absence of two predicted genes in the avrPiz-t-70-15region, extreme sequence similarity and good synteny with respect to the gene order and content were observed in predicted protein-coding regions at the AvrPiz-t locus. Nevertheless, frequent presence/absence and highly dynamic organization of transposable elements (TEs) was identified, representing the major variation of the AvrPiz-t locus between different isolates. Moreover, TEs constitute27.3%and43.2%of the genomic contents of the AvrPiz-t-ZB15and avrPiz-t-70-15regions, respectively, indicating that TEs contribute largely to the organization and evolution of the AvrPiz-t locus. The finding that the AvrPiz-t gene embeds within a TE-rich genomic region indicates that M. oryzae might employ a strategy by using diverse TE families to regulate the instability of certain Avr genes to fight against the rice R genes from an evolutionary point of view.Previous study found that AvrPiz-t could suppress the BAX-mediated programmed cell death in tobacco. In this study, we further observed that AvrPiz-t can suppress the BAX mediated cell death of rice protoplast using a transient assay. Avr Piz-t encodes a small predicted secreted protein (108amino acids) without known function. Sequence analysis of AvrPiz-t revealed that its predicted mature form AvrPiz-t90consists of4cysteine residues, which is predicted to be involved in intra-and intermolecular interactions. To further investigate the significance of these4cysteine residues in the function of AvrPiz-t, we utilized a site-mutagenesis approach to generate4constructs encompassing mutations at each cysteine residue. Complementation tests revealed that these4derived AvrPiz-t mutants lost avirulence function to Piz-t, which indicated that these4cysteine residues are critical for the avirulence function of AvrPiz-t.