A Bacterial Effector Co-opts Calmodulin to Target the Plant Microtubule Network

The bacterial pathogen Pseudomonas syringae depends on effector proteins secreted by its type III secretion system for the pathogenesis of plants. The majority of these effector proteins are known suppressors of immunity, but their plant targets remain elusive. Using Arabidopsis thaliana as a model host, we report that the HopE1 effector uses the host calcium sensor, calmodulin (CaM), as a co-factor to target the microtubule- associated protein 65 (MAP65), an important component of the microtubule network. HopE1 interacted with MAP65 in a CaMdependent manner, resulting in MAP65-GFP dissociation from microtubules. Transgenic Arabidopsis expressing HopE1 had reduced secretion of the immunity protein PR-1 compared to wild–type plants. Additionally, Arabidopsis map65-1 mutants were immune deficient and were more susceptible to P. syringae. Our results suggest a virulence strategy in which a pathogen effector is activated by host calmodulin to target MAP65 and the microtubule network, thereby inhibiting cell wall-based extracellular immunity. Includes supplementary materials

A Bacterial Effector Co-opts Calmodulin to Target the Plant Microtubule Network

The bacterial pathogen Pseudomonas syringae depends on effector proteins secreted by its type III secretion system for the pathogenesis of plants. The majority of these effector proteins are known suppressors of immunity, but their plant targets remain elusive. Using Arabidopsis thaliana as a model host, we report that the HopE1 effector uses the host calcium sensor, calmodulin (CaM), as a co-factor to target the microtubule- associated protein 65 (MAP65), an important component of the microtubule network. HopE1 interacted with MAP65 in a CaMdependent manner, resulting in MAP65-GFP dissociation from microtubules. Transgenic Arabidopsis expressing HopE1 had reduced secretion of the immunity protein PR-1 compared to wild–type plants. Additionally, Arabidopsis map65-1 mutants were immune deficient and were more susceptible to P. syringae. Our results suggest a virulence strategy in which a pathogen effector is activated by host calmodulin to target MAP65 and the microtubule network, thereby inhibiting cell wall-based extracellular immunity. Includes supplementary materials

A phytobacterial TIR domain effector manipulates NAD\u3csup\u3e+\u3c/sup\u3e to promote virulence

The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD+) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD+ and if the activity is essential for HopAM1’s suppression of plant immunity and contribution to virulence. HPLC and LC-MS were utilized to analyze metabolites produced from NAD+ by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1’s intrinsic enzymatic activity and virulence contribution. HopAM1 is catalytically active and hydrolyzes NAD+ to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1’s E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. HopAM1 manipulates endogenous NAD+ to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1’s TIR domain possesses different catalytic specificity than other TIR domain-containing NAD+ hydrolases and that pathogens exploit this activity to sabotage NAD+ metabolism for immune suppression and virulence

A phytobacterial TIR domain effector manipulates NAD\u3csup\u3e+\u3c/sup\u3e to promote virulence

The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD+) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD+ and if the activity is essential for HopAM1’s suppression of plant immunity and contribution to virulence. HPLC and LC-MS were utilized to analyze metabolites produced from NAD+ by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1’s intrinsic enzymatic activity and virulence contribution. HopAM1 is catalytically active and hydrolyzes NAD+ to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1’s E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. HopAM1 manipulates endogenous NAD+ to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1’s TIR domain possesses different catalytic specificity than other TIR domain-containing NAD+ hydrolases and that pathogens exploit this activity to sabotage NAD+ metabolism for immune suppression and virulence

Pseudomonas syringae Pathogenesis and Suppression of Plant Immunity

The Gram-negative bacterial plant pathogen Pseudomonas syringae requires a type III protein secretion system (T3SS) to cause disease. In this thesis, I describe three projects that I was involved with during my PhD studies. The first is on the T3E HopE1. This T3E uses the calcium sensor-calmodulin (CaM), as a co-factor to target the microtubule-associated protein 65 (MAP65). HopE1 interacted with MAP65 in a CaM-dependent manner, resulting in MAP65-GFP dissociation from microtubules. Additionally, Arabidopsis map65-1 mutants were immune deficient and were more susceptible to P. syringae. These suggest HopE1 effector is activated by host CaM to target MAP65, thereby inhibiting cell wall-based immunity. The second is focused GRP7, a plant target of the T3E HopU1. We found that GRP7 transgenic plants were more resistant to hemi- or biotrophic pathogens and induced levels of PRs after pathogen infection. Importantly, the increased resistance of GRP7 transgenic plants was dependent on salicylic acid (SA)-mediated immunity. The interaction between GRP7 and immunity-related RNAs was inhibited by HopU1 in a manner dependent on HopU1’s ADP-RT activity. I performed RNA immunoprecipitation-sequencing experiments and found that many RNAs interacted with GRP7 including many more immunity-related. Our research reveals the broad role of GRP7 in plant immunity. The third is focused on P. syringae’s ability to alter Arabidopsis histone modification. P. syringae to reduce H3K9 levels was dependent on a functional T3SS and is correlated with the suppression of immunity-related gene expression. Moreover, chromatin immunoprecipitation experiments showed that promoters of immunity-related genes had reduced H3K9ac levels in plants infected with P. syringae . Many T3Es can reduce H3K9 levels. This was likely indirect through their general ability to suppress plant immunity. Arabidopsis hda5 mutants were more resistant to P. syringae and showed increased production of callose deposition. These mutants also no longer exhibited decreased levels of H3K9ac associated with immunity-related genes upon infection with P. syringae suggesting that the reduction of H3K9ac levels in plants infected by P. syringae was due to HDA5. Therefore, HDA5 acts as a negative regulator of plant immunity and P. syringae can directly or indirectly employ it to aid in immunity suppression