The Role Of Pseudomonas Syringae Effector Avrpto In Promoting Bacterial Virulence And Eliciting Plant Immunity

The use of the type III secretion system (T3SS) to deliver a suite of effector proteins into the host cell cytoplasm is a common virulence strategy employed by many Gram-negative bacteria during host infection. Studies of bacterial effectors' functions inside the host cell reveal that many effectors have evolved to utilize and/or interfere with the eukaryotic host machinery to promote bacterial virulence. Pseudomonas syringae pv. tomato effector AvrPto is one of the best-characterized plant bacterial effectors. AvrPto is a modular protein with two distinct virulence determinants: the CD loop in the core structure and the phosphorylated C-terminal domain (CTD). The CD loop structure is required for the ability of AvrPto to interfere with pattern recognition receptor complexes to suppress host's pathogen associated molecular patterns (PAMPs)-triggered immunity (PTI). This domain is monitored by the tomato resistance protein complex Pto/Prf via the direct binding of Pto to AvrPto. The CTD of AvrPto is phosphorylated by a yet unknown host kinase, whose activity is conserved in many plant species. I describe here that the phosphorylated CTD contributes additively to AvrPto virulence together with the CD loop. The mechanism by which the CTD promotes bacterial virulence is unknown; however, it is different from that of the CD loop. The CTD is specifically recognized by a novel resistance only found in Nicotiana species; therefore, two distinctive host recognition mechanisms have evolved to monitor the two domains of AvrPto. These two virulence domains are functionally conserved in certain AvrPto homologs from other P. syringae pathovars, indicating that they might target host processes that are conserved in many plant species. Detailed characterization of AvrPto homologs has revealed a possible advantage of modularity for bacterial effector, which is the ability to avoid host recognition but still retain partial virulence. I investigated the host kinase activity, Avk for AvrPto kinase, that phosphorylates the AvrPto CTD. Avk activity is enhanced upon treatments with PAMPs, and T3SS effectors could suppress this induction. Using functional protein microarrays, I screened for plant kinases that could phosphorylate the CTD in vitro and identified calcium-dependent protein kinases (CDPKs) as potential Avks. Further characterization of Avk activity revealed that it is dependent on Ca2+, which supports the role of CDPKs as Avks. Thus, AvrPto has evolved to be a suitable substrate of a conserved plant kinase family for its activation; however, more work needs to be directed toward the investigation of whether or not a member of this kinase family are the virulence target of the AvrPto CTD.