The function of the phytoplasma effector SWP12 depends on the properties of two key amino acids

Phytoplasmas are insect-borne bacterial pathogens capable of secreting effectors into host cells and interfering with host plant defense response processes. Previous studies have found that the Candidatus Phytoplasma tritici effector SWP12 binds to and destabilizes the wheat transcription factor TaWRKY74, increasing wheat susceptibility to phytoplasmas. Here, we used a Nicotiana benthamiana transient expression system to identify two key functional sites of SWP12 and screened a series of truncated mutants and amino acid substitution mutants to determine whether they inhibit Bax-induced cell death. Using a subcellular localization assay and online structure analysis websites, we found that structure rather than intracellular localization probably affects the function of SWP12. D33A and P85H are two inactive substitution mutants, neither of which interacts with TaWRKY74, and P85H does not inhibit Bax-induced cell death, suppress flg22-triggered reactive oxygen species (ROS) bursts, degrade TaWRKY74, or promote phytoplasma accumulation. D33A can weakly suppress Bax-induced cell death and flg22-triggered ROS bursts and degrade a portion of TaWRKY74 and weakly promote phytoplasma accumulation. S53L, CPP, and EPWB are three SWP12 homolog proteins from other phytoplasmas. Sequence analysis revealed that D33 was conserved in these proteins, and they exhibited the same polarity at P85. Transient expression in N. benthamiana showed that these proteins could inhibit Bax-induced cell death and suppress ROS bursts. Our findings clarified that P85 and D33 of SWP12 play critical and minor roles, respectively, in suppressing the plant defense response and that they play a preliminary role in determining the functions of homologous proteins.