A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs

Understanding Effector Secretion and Function of two Proteobacteria: Pseudomonas syringae and Candidatus Liberibacter asiaticus

Gram-negative bacteria employ secretion systems to deliver virulence factors, such as effectors, to target plant immunity in hosts. My research focuses on secreted effectors from plant pathogenic bacteria Pseudomonas syringae and Candidatus Liberibacter asiaticus. In chapter one, I focused on P. syringae type III secreted effector HopZ1a. I confirmed the relationship between HopZ1a-mediated acetylation and subsequent degradation of its target JAZs (JAZ10) in Arabidopsis thaliana. Next, I determined decreased susceptibility in JAZ10 plants mutated in acetylation sites during infection, indicating HopZ1a-mediated acetylation and subsequent degradation of JAZ10 affects bacterial growth. My findings in this chapter highlight the significance of HopZ1a’s acetylation modification of JAZs. In chapter two, I optimized the use of CLas Sec-delivered effector 1 (SDE1) (CLIBASLIA_05315) for direct tissue blot immunoassay (DTBIA). I also generated Liberibacter crescens (L. crescens) cell and lipopolysaccharide (LPS) specific antibodies to serve as a cocktail primary antibody for enzyme-linked immunosorbent assay (ELISA). Furthermore, L. crescens LPS structural analysis by the Complex Carbohydrate Research Center (CCRC) revealed the presence of very long chain fatty acid (VLCFA 27OHC28:0). It is possible this VLCFA is required for culturing of Liberibacters. In chapter three, I generated a functional model system to confirm CLas predicted secreted effectors using L. crescens. Our lab generated a list of predicted secreted effectors of CLas and we focused on some for detection biomarkers, and functional work was performed on one effector SDE1 (CLIBASIA_05315). Using this foundation of knowledge, I used SDE1 as my test subject for secretion in L. crescens. My research shows that L. crescens can serve as a tool to confirm and possibly study CLas secreted effectors

Understanding Effector Secretion and Function of two Proteobacteria: Pseudomonas syringae and Candidatus Liberibacter asiaticus

Gram-negative bacteria employ secretion systems to deliver virulence factors, such as effectors, to target plant immunity in hosts. My research focuses on secreted effectors from plant pathogenic bacteria Pseudomonas syringae and Candidatus Liberibacter asiaticus. In chapter one, I focused on P. syringae type III secreted effector HopZ1a. I confirmed the relationship between HopZ1a-mediated acetylation and subsequent degradation of its target JAZs (JAZ10) in Arabidopsis thaliana. Next, I determined decreased susceptibility in JAZ10 plants mutated in acetylation sites during infection, indicating HopZ1a-mediated acetylation and subsequent degradation of JAZ10 affects bacterial growth. My findings in this chapter highlight the significance of HopZ1a’s acetylation modification of JAZs. In chapter two, I optimized the use of CLas Sec-delivered effector 1 (SDE1) (CLIBASLIA_05315) for direct tissue blot immunoassay (DTBIA). I also generated Liberibacter crescens (L. crescens) cell and lipopolysaccharide (LPS) specific antibodies to serve as a cocktail primary antibody for enzyme-linked immunosorbent assay (ELISA). Furthermore, L. crescens LPS structural analysis by the Complex Carbohydrate Research Center (CCRC) revealed the presence of very long chain fatty acid (VLCFA 27OHC28:0). It is possible this VLCFA is required for culturing of Liberibacters. In chapter three, I generated a functional model system to confirm CLas predicted secreted effectors using L. crescens. Our lab generated a list of predicted secreted effectors of CLas and we focused on some for detection biomarkers, and functional work was performed on one effector SDE1 (CLIBASIA_05315). Using this foundation of knowledge, I used SDE1 as my test subject for secretion in L. crescens. My research shows that L. crescens can serve as a tool to confirm and possibly study CLas secreted effectors

Two serine residues in Pseudomonas syringae effector HopZ1a are required for acetyltransferase activity and association with the host co-factor

Gram-negative bacteria inject type III secreted effectors (T3SEs) into host cells to manipulate the immune response. The YopJ family effector HopZ1a produced by the plant pathogen Pseudomonas syringae possesses acetyltransferase activity and acetylates plant proteins to facilitate infection. Using mass spectrometry, we identified a threonine residue, T346, as the main autoacetylation site of HopZ1a. Two neighboring serine residues, S349 and S351, are required for the acetyltransferase activity of HopZ1a in vitro and are indispensable for the virulence function of HopZ1a in Arabidopsis thaliana. Using proton nuclear magnetic resonance (NMR), we observed a conformational change of HopZ1a in the presence of inositol hexakisphosphate (IP6), which acts as a eukaryotic co-factor and significantly enhances the acetyltransferase activity of several YopJ family effectors. S349 and S351 are required for IP6-binding-mediated conformational change of HopZ1a. S349 and S351 are located in a conserved region in the C-terminal domain of YopJ family effectors. Mutations of the corresponding serine(s) in two other effectors, HopZ3 of P. syringae and PopP2 of Ralstonia solanacerum, also abolished their acetyltransferase activity. These results suggest that, in addition to the highly conserved catalytic residues, YopJ family effectors also require conserved serine(s) in the C-terminal domain for their enzymatic activity

Two serine residues in P

Gram-negative bacteria inject type III secreted effectors (T3SEs) into host cells to manipulate the immune response. The YopJ family effector HopZ1a produced by the plant pathogen Pseudomonas syringae possesses acetyltransferase activity and acetylates plant proteins to facilitate infection. Using mass spectrometry, we identified a threonine residue, T346, as the main autoacetylation site of HopZ1a. Two neighboring serine residues, S349 and S351, are required for the acetyltransferase activity of HopZ1a in vitro and are indispensable for the virulence function of HopZ1a in Arabidopsis thaliana. Using proton nuclear magnetic resonance (NMR), we observed a conformational change of HopZ1a in the presence of inositol hexakisphosphate (IP6), which acts as a eukaryotic co-factor and significantly enhances the acetyltransferase activity of several YopJ family effectors. S349 and S351 are required for IP6-binding-mediated conformational change of HopZ1a. S349 and S351 are located in a conserved region in the C-terminal domain of YopJ family effectors. Mutations of the corresponding serine(s) in two other effectors, HopZ3 of P. syringae and PopP2 of Ralstonia solanacerum, also abolished their acetyltransferase activity. These results suggest that, in addition to the highly conserved catalytic residues, YopJ family effectors also require conserved serine(s) in the C-terminal domain for their enzymatic activity

A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs

Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family

Effectors secreted by the type Ill secretion system are essential for bacterial pathogenesis. Members of the Yersinia outer-protein J (YopJ) family of effectors found in diverse plant and animal pathogens depend on a protease-like catalytic triad to acetylate host proteins and produce virulence. However, the structural basis for this noncanonical acetyltransferase activity remains unknown. Here, we report the crystal structures of the YopJ effector HopZ1a, produced by the phytopathogen Pseudomonas syringae, in complex with the eukaryote-specific cofactor inositol hexakisphosphate (IP6) and/or coenzyme A (CoA). Structural, computational and functional characterizations reveal a catalytic core with a fold resembling that of ubiquitin-like cysteine proteases and an acetyl-CoA-binding pocket formed after IP6-induced structural rearrangements. Modeling-guided mutagenesis further identified key IP6-interacting residues of Salmonella effector AvrA that are required for acetylating its substrate. Our study reveals the structural basis of a novel class of acetyltransferases and the conserved allosteric regulation of YopJ effectors by IP6