Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 206 - Basic Plant Biology | 4010 - Bacteria | 1100 - Bacteriology | 50% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 50% |
The goal is to develop management strategies which boost plant defense to protect citrus from HLB by exploiting the virulence mechanisms of Candidatus Liberibacter asiaticus (Las) and how Las manipulates plant defense. The virulence mechanisms of Las are largely unknown. Understanding the virulence mechanisms is important for HLB management. Importantly, we identified an enzyme salicylate hydroxylase encoded by sahA of Las, which breaks down salicylic acid (SA) and its derivatives. SA plays a central role in plant defenses. Degradation of SA is likely one important strategy of Las to suppress plant defense. In order to achieve the goal of this study, the following objectives will be conducted. Objective 1. To characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors. Objective 2. To test different compounds in controlling HLB and characterize their mechanisms in controlling HLB.
PROCEDURES: Objective 1. To characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors Working hypothesis: The five putative virulence genes lsv1, lsv2, lsv3, lsv4, and lsv5 are important for Las to cause HLB disease symptoms and the SA hydroxylase is important for Las to suppress plant defense. 1.1 Characterization of putative virulence genes of Las We propose to further analyze the five identified putative virulence genes and how they contribute to the HLB disease symptoms. For this purpose, transgenic plants of grapefruit (Citrus paradisi) have been constructed. Further analysis of those transgenic plants will be conducted. To investigate the cellular localization of the potential virulence factors, specific antibody for each virulence factor protein combined with goat anti-mouse Alexa Fluor 488 will be used for immunofluorescence confocal laser-scanning microscopy analysis in the HLB-diseased citrus leaves. Healthy leaves will be used as control. This can be further verified by constructing a fusion protein of the virulence factor with green fluorescent protein (GFP) using the pGD binary vector series that allow Agrobacterium tumefaciens-mediated expression of genes in fusion with GFP. Empty vector with GFP will be used as control. Nicotiana tabacum will be used since it is amenable to agro-infiltration experiments. Furthermore, N. tabacum is the host of Ca. Liberibacter spp. transmitted by Cuscuta spp. (dodder) (Francischini et al. 2007). 1.2 Illustration of the role of salicylate hydroxylase, which is encoded by Las and has the potential to suppress plant defense responses Expression study of SA hydroxylase Our preliminary analysis indicates that SA hydroxylase is able to degrade SA using the crude extract of E.coli expressing SA hydroxylase. In order to further understand its role, purified SA hydroxylase will be used to test its effect to degrade SA as described previously (White-Stevens and Kamin 1972). We have successfully expressed SA hydroxylase in E.coli which was confirmed using Western blot. Effect of SA hydroxylase on SA accumulation and plant defense To further test whether SA hydroxylase affects SA accumulation in planta, transgenic expression of sahA in grapefruit will be conducted as described above using the pTA7001. SA purification and quantification will be done as described previously (Lahey et al. 2004). Endogenous SA in the transgenic, Las infected and control trees will be compared as described above. In order to further detect the effect of SA hydroxylase on SA accumulation, Xanthomonas axonopodis pv. citri A and Aw strains, which cause disease and hypersensitive response (HR) (Burnings and Gabriel 2003) involving significant SA accumulation on grapefruit duncan respectively, will be used.Briefly, healthy plants, transgenic plants, and Las infected plants will be infected with A and Aw strains at 5X108 CFU/ml with water as control as described in our previous study (Guo et al. 2010; 2011; Li and Wang 2010).SA accumulation will be measured as described above.Disease and HR development will be monitored.Gene expression of multiple defense related genes including PR-1 and PR-5 will be measured.This experiment will also test the effect of HLB on citrus to X. axonopodis pv. citri infection. 1.3 Identification of inhibitors of SA hydroxylase In order to inhibit the SA hydroxylase encoded by Las and restore the SA level in planta, we plan to identify the putative inhibitors (chemical or small molecules) of SA hydroxylase. For small molecule inhibitors, we will screen the small molecule library using structure-based design as described in our previous study (Akula et al. 2011). The SA hydroxylase protein (43 KD) has been expressed (Fig. 2). Inhibition of the enzyme activity of SA hydroxylase will be conducted as described in our previous study (Trivedi and Wang 2010). We will also test different metal ions (e.g. Cu2+, Zn2+) in inhibiting SA hydroxylase. Objective 2. To test different compounds in controlling HLB and characterize their mechanisms in controlling HLB Working hypothesis: Management of HLB could be done by inducing plant defense by restoring SA level in planta using different chemicals including SA hydroxylase inhibitors, and SA functional analogs (ASM, INA and BTH). In order to test the effect of different chemicals including SA hydroxylase inhibitors, SA functional analogs (ASM, INA and BTH), BABA, 2-DDG in controlling HLB, the following combinations will be tested (Table 1) with water as control. It is expected that more chemicals will be tested once identified in our current research. We will select two groves for this test. For each grove, three different groups of trees will be selected that include non-symptomatic, less symptom (low disease incidence 0-1), severe symptom (high disease incidence 3-4). For each symptom group and each chemical, we will select 20 trees with five trees as a replicate unit. We will monitor the symptom, disease incidence and fruit production. This experiment will be repeated for three years. For the treatment with positive effect on controlling HLB, we will compare the Las population before or after treatment using EMA-qPCR as described in our previous study (Trivedi et al. 2009), the gene expression of pathogenicity related genes (PR1, PR5), PP2 and callose synthase CalS1, starch accumulation and phloem blockage as described in our previous study (Kim et al. 2009). Determination of SA will be conducted as described elsewhere (Verberne et al. 2002) by using a high-performance liquid chromatography system (Halim et al. 2004). Jasmonic acid (JA) will be measured as described previously (Mueller and Brodschelm 1994). Those selected chemicals will also be tested in greenhouse. For each treatment, four trees will be used as a replicate. Similar tests will be conducted for the greenhouse experiments as described. Two year-old seedlings will be used for greenhouse study. We will also test whether those treatments could prevent the seedlings from being infected by Las through psyllid transmission.
Target Audience
Citrus growers, scentific community, students
Changes / Problems
None
Training & Professional Development
Training of two Ph.D. students and two postdocs.
Dissemination Streams
Publication of peer reviewed papers, presentations, posters, and news releases.
Next Reporting Steps
N/A
Target Audience
citrus growers, researchers, students, and citrus consumers
Changes / Problems
No.
Training & Professional Development
We provided opportunities for training and professional development for three students and five postdocs.
Dissemination Streams
publications, and presentations
Next Reporting Steps
Continue to study the virulence mechanism of the HLB pathogen and test novel HLB control approaches. <br><br>
<br>What was accomplished under these goals? HLB is threatening citrus production worldwide, and there is no cure for infected trees. Management strategies targeting diseased trees at different stages of colonization by Las are needed for sustainable citrus production in HLB-endemic regions. We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost on mildly-diseased trees. We tested thermotherapy on severely-diseased trees and assessed tree protectors to prevent feeding by ACP, thus preventing Las from being transmitted to new plantings that replaced HLB-moribund trees. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors β-aminobutyric acid (BABA), plus Ascorbic acid (AA) and potassium phosphite with or without salicylic acid (SA), slowed down the progression of HLB and reduced disease severity by approximately 18%, compared to the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared to the untreated control. Thermotherapy treatment (55°C for 2 min) exhibited a suppressive effect on growth of Las and progress of HLB in severely diseased trees for two to three months after treatment. The tree protectors prevented feeding by ACP, and therefore young replant trees remained healthy and free from infection by Las over the two-year duration of the experiment. Taken together, these results may contribute to a basis for developing a targeted approach to control HLB based on stage of host colonization, application of plant defense elicitors, N fertilizer, compost, thermotherapy, and tree protectors. There is potential to implement these strategies in conjunction with other disease control measures to contribute to sustainable citrus production in HLB- endemic regions. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants. In our study, eight plant defense activators and three antibiotics were evaluated in three field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that four trunk injections of several activators, including salicylic acid, oxalic acid, acibenzolar-S-methyl, and potassium phosphate, provided significant control of HLB by suppressing 'CandidatusLiberibacter asiaticus' titer and disease progress. Trunk injection of penicillin, streptomycin, and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of 'Ca.L. asiaticus' titer and HLB symptom expressions than plant defense activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of pathogenesis-related (PR) genesPR-1andPR-2genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction ofPR-2orPR-15gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision making regarding developing an effective HLB management program. <br><br><b>Publications</b><br>
Target Audience
Scientific community, citrus growers, graduate students
Changes / Problems
There are no major changes.
Training & Professional Development
Three Ph.D. students and four postdocs have been trained on the virulence mechanism of Candidatus Liberibacter asiaticus
Dissemination Streams
Five papers have been published. Five postershave been presented in scientific meetings. 6 oral presentations have been given.
Next Reporting Steps
Continue to study the virulence mechanism of Candidatus Liberibacter asiaticus. Control citrus HLB by targeting the interactions of Candidatus Liberibacter asiaticus and psyllids with citrus using a CRISPR-Cas system. Characterize the effect of trunk injection of bactericides to control Huanglongbing Characterize the Sec-dependent effectors in citrus huanglongbing <br><br>
<br>What was accomplished under these goals? Study the virulence factors of Las and identification of their putative targets. Evaluation of the spatiotemporal dynamics of oxytetracycline and its control effect against citrus Huanglongbing via trunk injection. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB. Our data indicate that Las encodes a functional salicylate hydroxylase, which suppresses plant defense by degrading salicylic acid (SA). Evaluation of plant defense inducers for the control of citrus Huanglongbing. Characterization of Sec-translocon dependent extracytoplasmic proteins of Las. Identification of SA hydroxylase inhibitors to counter the bacterial virulence mechanism. Targeted genome editing of sweet orange using Cas9/sgRNA and TALEN. <br><br><b>Publications</b><br>
Target Audience
Scientific community, citrus growers, and consumers
Changes / Problems
Nothing Reported
Training & Professional Development
Trained two Ph.D. students and seven postdocs. Weekly presentations and discussion were held. The students and postdocs presented their results at scientific meetings, and published the results in refereed journals.
Dissemination Streams
By publications, and presentations at scentific meetings.
Next Reporting Steps
Continue the research as planned in the CRIS project. <br><br>
<br>What was accomplished under these goals? Study the virulence factors of Candidatus Liberibacter asiaticus (Las) and identification of their putative targets. The interaction of SDE5 and its targets have been confirmed by BiFC and Pull-down assay. To study the function of function of the SDE5 targets, we are overexpressing the target gene and silence the target gene using RNAi. Evaluation of the spatiotemporal dynamics of oxytetracycline and its control effect against citrus Huanglongbing via trunk injection. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB. Our data indicate that Las encodes a functional salicylate hydroxylase, which suppresses plant defense by degrading salicylic acid (SA). Evaluation of plant defense inducers for the control of citrus Huanglongbing. Five small molecule compounds were identified that inhibit the ATPase activity of SecA, a key component of the Sec pathway, of Las in nano molar concentrations and showed antimicrobial activities. Development of a microemulsion formulation for antimicrobial SecA inhibitors for application purpose. Analysis of SEC-translocon dependent extracytoplasmic proteins of Candidatus Liberibacter asiaticus Las contains a complete Sec-translocon, which has been suggested to transport Las proteins including virulence factors into the extracytoplasmic milieu. In this study, we characterized the Sec-translocon dependent, signal peptide containing extracytoplasmic proteins of Las. A total of 166 proteins of Las_psy62 strain were predicted to contain signal peptides targeting them out of the cell cytoplasm via the Sec-translocon using LipoP, SigalP 3.0, SignalP 4.1, and Phobius. We also predicated SP containing extracytoplasmic proteins for Las_gxpsy and Las Ishi-1, Lam, Laf, Ca. L. solanacearum (Lso), and L. crescens (Lcr). For experimental validation of the predicted extracytoplasmic proteins, Escherichia coli based alkaline phosphatase (PhoA) gene fusion assays were conducted. A total of 86 out of the 166 predicted Las proteins were experimentally validated to contain signal peptides. Additionally, Las_psy62 lepB (CLIBASIA_04190), the gene encodes signal peptidase I, was able to partially complement the amber mutant of lepB of E. coli. This work will contribute to the identification of Sec-translocon dependent effector proteins of Las, which might be involved in virulence of Las. Identification of SA hydroxylase inhibitors to counter the bacterial virulence mechanism. <br><br><b>Publications</b><br>
Target Audience
Scentific community, citrus industry, citrus growers, students, regulators
Changes / Problems
Nothing Reported
Training & Professional Development
This project has trained three students and six postdocs.
Dissemination Streams
Publications, presentations (oral and posters), seminars, consultation, websites
Next Reporting Steps
Control citrus HLB by targeting the interactions of Candidatus Liberibacter asiaticus and psyllids with citrus using a CRISPR-Cas system. Characterize the Sec-dependent effectors in citrus huanglongbing. Control of citrus Huanglongbing by exploiting the virulence mechanisms of Candidatus Liberibacter asiaticus and inducing plant defense. Characterize the effect of application of beneficial bacteria on management of Huanglongbing. <br><br>
<br>What was accomplished under these goals? Understanding the virulence mechanism of Las and management of HLB Key findings Our data indicate that Las encodes a functional salicylate hydroxylase, which suppresses plant defense by degrading salicylic acid (SA). Evaluation of plant defense inducers for the control of citrus Huanglongbing (published on Phytopathology) Five small molecule compounds were identified that inhibit the ATPase activity of SecA, a key component of the Sec pathway, of Las in nano molar concentrations and showed antimicrobial activities. We developed the formulation for their application (Accepted by PLOS One). Targeted genome editing of sweet orange using Cas9/sgRNA and TALEN. Characterization of Sec-translocon dependent extracytoplasmic proteins of Candidatus Liberibacter asiaticus. Identification of antimicrobial peptidomimic compounds against Lipid A using pharmacophore search method. Impacts Two peer-reviewed papers were published or accepted. Identification of the salicylate hydroxylase gene led to further research to control HLB by screening salicylate hydroxylase inhibitors and SAR elicitors by re-activating plant defense via restoring SA level in planta. Patent issued for the antimicrobial compounds identified in our study. Patent filed for using plant defense inducers to control HLB. <br><br><b>Publications</b><br>
Target Audience
citrus growers, scientists, policymaker, students, general public
Changes / Problems
No major changes/problems
Training & Professional Development
conduct the most advanced experiments, present at scientific meetings e.g., APS annual meeting, and present at weely lab meetings, weekly discussion of projects, help improve skills in manuscript writing
Dissemination Streams
Presentations at meetings, e.g.. APS annual meeting, publications in peer reviewed journals, serve as the expert to lead the group discussion for the citrus industry
Next Reporting Steps
Conduct research and extension activities as planed. Specifically, we will focus on the following objectives: Characterization of putative virulence genes of Las Illustration of the role of salicylate hydroxylase, which is encoded by Las and has the potential to suppress plant defense responses Identification of inhibitors of SA hydroxylase To test different compounds in controlling HLB and characterize their mechanisms in controlling HLB <br><br>
<br>What was accomplished under these goals? · We found that Las encodes a functional salicylate hydroxylase, which suppresses plant defense by degrading salicylic acid (SA). · Five small molecule compounds were identified that inhibit the ATPase activity of SecA, a key component of the Sec pathway, of Las in nano molar concentrations and showed antimicrobial activities. We optimized the solvents for their application. We conducted the transcriptional and microscopic analyses of citrus stemand root responses to Candidatus Liberibacter siaticus infection We designed specific QRT-PCR primers to detect Candidatus Liberibacter <br><br><b>Publications</b><br>