Performing Department
Horticultural Sciences
Non Technical Summary
Psyllids have emerged as vectors of bacterial pathogens that cost millions of dollars annually in the U.S. The tomato psyllid Bactericera cockerelli transmits 'Candidatus Liberibacter solanacearum'threatening solanaceous crops, and Diaphorina citri transmits 'Ca. L. asiaticus', which is decimating the citrus industry. Today, the main management strategy relies on vector control, and pesticide use has increased dramatically. However, new control strategies are urgently needed.Bacterial effectors are proteins secreted by pathogens into the host cells helping the pathogen colonize the host and/or suppress the plant/vector immune system. They play key roles in promoting pathogenicity during the interaction of pathogens with plants and arthropods. To counteract infections, plants and insects evolved immune receptors to recognize these effectors and trigger immune responses. Programmed cell death is a defense response upon recognition of pathogen effectors preventing the pathogen from spreading in the host and vector. In this proposal we will identify liberibacter effectors and their targetproteins in plants and psyllids. Using a variety?of approaches, we will evaluate the role of these interactors in plant infection or pathogen transmission and whether we can use these interactions to disrupt plant infection or pathogen transmission. This work is original because it studies in parallel plant and insect immune systems.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Our long-term goal is to identify mechanisms underpinning the interactions among plants, vectors, and pathogens, so that approaches to disrupt these interactions are developed for the sustainable control of plant diseases. Toward this end, we use tomato, tomato psyllid (Bactericera cockerelli), and 'Candidatus Liberibacter solanacearum' (Lso) as a model system. Advantages of this system include: (1) tomato is a host of the vector and the pathogen, and is suited to study plant-microbe interactions; (2) tomato psyllids colonize solanaceous crops and model organisms; and (3) two Lso haplotypes (LsoA and LsoB) are associated with the vector and hosts in the U.S. Differences in the tomato responses to the Lso haplotypes were identified. Specifically, we propose in this project to study the interactions between the Lso haplotypes and tomato and tomato psyllids.Our overall objective is to identify key genes involved in the interaction between Lso and the plant host and/or the vector that leads to effective pathogen acquisition and plant infection, and to disrupt these processes.Our central hypothesis is secreted Lso proteins can control plant and insect immune responses and contribute to the success of the pathogen to colonize and infect the plant and the vector. This hypothesis is supported by the preliminary data obtained by our group. We plan to test our central hypothesis objectively by pursuing three specific objectives:Objective 1. Identify Lso secreted proteins and evaluate their role in inducing or reducing host defenses Hypotheses: (1) Sec-dependent and non-classical secreted proteins are encoded in the Lso genome, and (2) differences between the Lso haplotypes exist for some of these proteins. Further, (3) some of these Lso "effectors" are preferentially expressed in the plant or vector host, and (4) they can manipulate plant and vector immune responses.Objective 2. Identify tomato proteins interacting with Lso secreted proteins and evaluate their role in Lso infection. Hypotheses: (1) Lso secreted proteins interact with host proteins and manipulate plant defenses, and (2) effector differences between Lso haplotypes are linked to differences in Lso pathogenicity.Objective 3. Identify psyllid proteins interacting with Lso secreted proteins and evaluate their role in Lso infection. Hypothesis: Lso secreted proteins interact with insect proteins and manipulate vector defenses.
Project Methods
The different methods use in this project are the following for each objective.Objectif 1:We identified putative secreted proteins using SignalP and Secretome for Lso haplotype B (analysis of LsoA proteins with Secretome will be performed). The protein sequences will be compared among the different Lso haplotypes as well as with other Liberibacter and Rhizobiaceae species available in NCBI and classified as Lso haplotype-specific, Lso-specific, Liberibacter-specific. For each candidate, the gene sequence will be validated by sequencing the LsoA and LsoB strains maintained and characterized in our laboratory (see CKC_05701 correction). Other bioinformatics analyses, such as interproscan, will be conducted to assign a putative function to the proteins. Key milestonea candidate list will be generated.We will validate the functionality of the signal peptide. The predicted signal peptide (Sec-dependent) or the full length (NCSP) sequence of each candidate will be cloned in-frame fusion with the PhoA gene in E. coli. If differences in the sequence of the SP between LsoA and LsoB exist, both will be tested. For each candidate, secretion will be first performed using the bacteria grown in plates in the presence of 5-BCIP. For those identified as secreted, the bacteria will be grown in liquid media; the media will be filtered before adding 5-BCIP. Candidates with functional signal peptides in plate and liquid assays will be moved forward. Then, the candidates will be cloned and expressed in E. coli with a His tag(Champion pET, Takara). After induction, the secretion of the effector will be evaluated by western blot analysis using filter-sterilized bacterial medium and anti-his antibodies . This test will validate that the effector is secreted by bacteria.Key milestonesecreted candidate effector will be identified.Validated secreted protein genes will be cloned into the pEG101 vector. N. benthamiana leaves of will be agroinfiltrated to determine if the protein induces cell death. Co-infiltrations with Bax and PrfD1416Vwill be performed independently to determine if the protein can suppress Bax- and/or PrfD1416V-induced cell death. ROS assays will be performed from leaf discs transiently expressing effectors and increase of cytosolic Ca2+ concentration will be evaluated in the N. benthamiana SLJR15. The expression of the candidate protein in the assays will be validated by Western via the HA-tag present in the pEG101 vector. Finally, the subcellular localization of the effector will be determined using the YFP fused to the effector. If differences between the proteins encoded by LsoA and LsoB exist, all assays will be performed for both proteins.Adequate positive and negative controls will be included such as empty vectors, Pto (suppresor of Prf induced cell death)and results will be compared and statistically analyzed. Similarly, the ability of the candidates to suppress cell death will be tested in yeast: yeast will express the candidate effector constitutively (pGilda vector) and Bax under the control of a galactose-inducible promotor (pJG4-5 vector). Bax will be induced to test if the effector can suppress Bax-induced cell death. Bcl, a gene that can inhibit Bax-induced cell death, will be used as positive control.Key milestoneSuppression/induction of cell death, ROS and Ca2+ in planta.Objectif2: Identification of putative interactors in tomato: The top-tier candidate effectors (secreted proteins, highly expressed in plants, able to disrupt cell death, ROS or Ca2+) will be cloned into the bait vector. First, autoactivation of the reporter genes in the Y2H system by the effector will be ruled-out. Only baits passing this test will be used to screen the library. Positive clones will be evaluated in QDO which selects for interactions; the other Y2H reporter genes will be tested as well. Putative interactors will be sequenced, and their identity determined by blast searches. The prey vector will be tested for autoactivation and if the putative interactor is only a partial ORF, the complete ORF will be cloned into the prey vector and the interaction with the effector will be tested. Interaction validation: The interaction will be validated by coimmunoprecipitation (Co-IP). Proteins will be expressed in N. benthamiana with Flag and HA (vectors pMDC32-FLAG and pMDC32-HA), purified and their interaction will be tested by Co-IP.The interaction will also be validated by BiFC. BiFC will be used to evaluate the subcellular localization of the effector and the interactor. Interaction analysis: For each validated interactor. 1. If differences exist between LsoA and LsoB for the effector, the interaction between the interactor and the gene from the other haplotype will be tested. 2. The strength of the interaction will be measured using the α-Galactosidase Quantitative Assay as described in the Yeast Protocols Handbook using PNP-α-Gal Solution to compare the strength of the protein interaction with LsoA and LsoB encoded proteins if those are different. 3. If the interactor is part of a protein family, the interaction with other members of the family will be tested as we did for RAD23. 4. If the homolog interactor is identified in psyllids, the interaction will be tested. The interaction will be validated by pull-down assay.Objectif 3: Identification of putative interactors in psyllids: The top-tier candidate effectors (secreted proteins, highly expressed in psyllids, able to disrupt immunity) will be cloned into the bait vector. We will use the approach described in Obj. 2 to screen the psyllid library and identify interacting proteins: rule-out autoactivation of the Y2H system by the bait, screen the library, validate the interaction using different reporter genes, if necessary, the full-length interactor will be cloned and its interaction with the effector validated in Y2H. Validation and localization: To validate the interaction we will perform Co-IP and BiFC in Sf9 insect cells (Thermofisher). Protein localization, BiFC, and Co-IP in insect cells are on-going with vectors pIWnYM and pIWcYC (vectors used for BiFC and for Co-IP with the FLAG and myc tags).