| Plants possess a two-layer innate immune system to protect themselves from most microbial pathogens. The first layer of innate immunity is called PAMP-triggered immunity (PTI) that the recognition of pathogen-associated molecular patterns (PAMPs) is mounted by the pattern recognition receptors (PRRs). The second layer of the immune system called effector-triggered immunity (ETI), which is activated upon recognition of pathogen-secreted avirulence (Avr) effector proteins by cognate plant resistance (R) receptors. We previously characterized the AvrPiz-t gene cognate to the rice blast R gene Piz-t using a map-based cloning strategy in Magnaporthe oryzae. We have identified 12 potential host targets of AvrPiz-t, designated as APIPs, from rice using a yeast two-hybrid (Y2H) method. The solution structure of AvrPiz-t was previously determined by NMR. In this study, we will present the functional characterization of AvrPiz-t and APIP12.To investigate the role of catalytic amino acids and basic amino acids on the surface of the folded protein predicted by the solution structure in the function of AvrPiz-t, we utilized a site-mutagenesis approach to generate variants with mutations at each residue. Complementation tests revealed that the mutations of H25A and N64A in predicted deubiquitination catalytic center led to the loss of avirulence function of AvrPiz-t to Piz-t. On the contrary, the mutation at the adjacent amino acid of Q22A did not affect its function, indicating that amino acids of the catalytic center of AvrPiz-t are critical for the avirulence function of AvrPiz-t. In the Y2H interaction assay, the interactions between the AvrPiz-t variants containing mutations at each of four amino acid residues in the catalytic center, i.e., C23A, H25A, A41V and C70A, with APIP2, APIP6 and APIP10 are significantly weakened or completely blocked. We thus speculated that the physical interaction between AvrPiz-t and these three APIPs is critical for its avirulence function. Nevertheless, the Q22A and D89A mutations which did not lead to the loss of avirulence function in the complementation tests did not change the interaction with all of 12 APIPs, suggesting that these two amino acid residues are not essential to either the interaction or the avirulence function. In subcellular localization studies, AvrPiz-t is localized in the chloroplast and the mutations described above did not alter the subcellular localization of AvrPiz-t.2. APIP12 is a homologue of nucleoporin protein Nup98 without the conserved domain of Phe-Gly repeats and has no orthologue in other plants. APIP12 interacts with AvrPiz-t in yeast and its middle portion binds to the effector strongly. The interaction between AvrPiz-t and APIP12 is validated by a glutathione S-transferase (GST) pull-down assay. Both knockout and knockdown of APIP12 cause more susceptibility to virulent isolates of M. oryzae. The expression of some pathogenesis-related (PR) genes is reduced in APIP12-knockout mutants, suggesting that APIP12 is required for the accumulation of transcripts of PR genes upon the infection. It is worth noting that neither knockout/knockdown nor overexpression of APIP12 attenuates Piz-t resistance. APIP12 is not interact with LRR, NT and NBS domains of Piz-t in the yeast two hybrid, suggest that APDP12 is not necessary to Piz-t mediated resistance. Transient expressions of APIP12 in tobacco and rice protoplasts indicate APIP12 is localized within unknown structures in cytoplasm. APIP12 was found to be localized with AvrPiz-t when they are co-transformed into tobacco, suggesting that APIP12 may change the subcellular position of AvrPiz-t. In addition, APIP12 interact with APIP6 or APIP8 in the Y2H assay. Taken together, our results demonstrate that APIPI2 is a target of AvrPiz-t and is involved in the basal resistance against M. oryzae in rice. |
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