Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 100% |
Much progress has been achieved in the control of newly emerging plant diseases by first understanding the various strategies used by the pathogens to cause disease and overcome native plant defenses, and then by interfering with key elements of their life cycles, infection strategies, pathogenicity determinants and/or counter-defenses. Genomic DNA sequencing allows the identification of all of the suspected genes in a pathogen's offensive and defensive arsenals. Functional genomics typically confirms suspected pathogen mechanisms for both offense and defense, and provides a firm basis for testing of disease control strategies in infected plants. With newly emerging pathogens, this often involves identification of key new pathogenicity genes, functional confirmation, and hypothesis testing to develop control measures. When a pathogen is uncultured, Koch's postulates cannot be completed, functional genomics becomes severely impacted, and hypothesis testing is extremely difficult and limited. This has been the case with Huanglongbing (HLB) and its associated agent, Ca. Liberibacter asiaticus (Las). A priority for functional studies is to culture Las, in order to leverage the abundant available genomic information and use it towards developing disease control and/or management methods. Limited progress has been made in attempting to culture Las. Only 2 labs (Davis and De La Fuente) have published substantial lab results indicating partial progress towards the goal. Lack of success has likely been due to multiple reasons, including: 1) missing required metabolites in media tested; 2) missing genes or pathways required for free-living growth in the specific Las strain used; 3) unrecognized toxic substances in some media used; 4) activation of phage lytic cycles genes in the Las genome, 5) a substantially weak Las outer membrane barrier, and 6) missing host gene regulation functions following separation of Las from an intracellular host environment. Any combination of these factors could prevent growth, even when a perfect combination is hit with a particular media formulation. What is perhaps most frustrating is that Liberibacter crescens (Lcr) can be readily cultured, and yet it has only a 20% larger genome than Las (1.5 Mbp for Lcr vs 1.26 Mbp for Las). Lcr is limited in that it may no longer be pathogenic or capable of growth in any host, despite dedicated efforts by several labs to inoculate it into different plants and insects. Koch's postulates have yet to be completed for Las or Lcr. Lcr has nevertheless been used (Gabriel lab) to develop a functional genomics toolkit for 1) Las gene expression; 2) characterization of Las phage gene promoters and key regulators, and 3) early and late gene reporter constructs capable of high throughput screening for small molecules that may affect phage gene expression, either directly or indirectly. The Lcr functional genomics system allowed identification of the Las prophage peroxidase as a likely critical lysogenic conversion gene that is needed by Las to both suppress citrus defense (peroxide) and citrus defense signaling (Gabriel lab). Las peroxidase is thus a potential target for Las control in citrus. Published comparative gene expression analyses (Gabriel lab) have shown that high levels of phage late gene expression, including lytic genes, occurs in Las-infected periwinkle, less in citrus, and almost none in psyllids. Since phage lytic replication does not occur in psyllids, indicating that all phage genes are under stringent repression in psyllids, but less so in citrus. The Las late gene reporter construct was used in the Lcr system to allow identification of a 27 kDa protein in psyllid extracts that directly binds to the late gene promoter and behaves as a strong repressor (Gabriel lab, unpublished). Since all Florida Las strains examined to date carry similar phage, it may be possible to artificially de-repress the phage lytic cycle and/or individual lytic genes in Las infected psyllids and citrus trees in the field and thereby cure both. This repressor and the promoter it binds to are thus two additional potential targets for Las control in psyllids, and expression of this protein is proposed here to help enable culturing of Las. More recently an additional key Las phage regulatory gene was identified by analysis of Las gene expression constructs in Lcr (Gabriel lab), helping to explain why Las phage particles readily form in periwinkle, but not in citrus, and providing another potential control target. Although Lcr has been genetically manipulated and is tractable for transformation, including knockout and knock-in mutations, Las has yet to be transformed so that not even a single genetically marked Las strain has been produced. Although antibodies have been produced that might be used to trap and concentrate Las (for observational, transformation, and culturing purposes), the specificity and threshold level of detection of the antibodies was not reported. This proposal is to: 1) coordinate simultaneous parallel culturing efforts in 4 labs (Duan, Davis, Killiny and De La Fuente) using a variety of complementary approaches and real-time data and deliverable sharing of what works and what does not, and using different Las sources (insect vs. plant); 2) to leverage what has been learned from functional genomic analyses of Lcr and the Las phage in continuing efforts to use Lcr as a culturable proxy (Gabriel) to engineer Lcr to be more Las-like in terms of its outer membrane to allow a more straightforward approach to obtaining antibodies with high specificity and affinity (Ma), and 3) to develop a phage vector (Jones & Gabriel) and/or conjugation system (Gabriel) so that Las can be genetically marked and engineered to enable growth on artificial media.
This is a Standard Research and Extension Project (SREP) focused on culturing Ca. Liberibacterasiaticus (Las), with direct applications expected in Huanglongbing (HLB) control and detection/diagnostics. Despite the enormous efforts that have gone into obtaining 5 complete genomic DNAsequences of the 3 different species of HLB bacteria, not one of the causal species has beencultured, and potential molecular targets for control cannot be functionally validated. Mostculturing efforts have focused on nutritional supplements and various media formulations in anattempt to supply presumably missing nutrients, but only to a single Las genotype or clonal group.This may be the wrong approach. One Liberibacter species, L. crescens (Lcr), was readily culturedmany years ago using very standard bacterial techniques. Its genome is 20% larger than all othersequenced Liberibacters, and has all genes needed for culture. It is also missing specific phage lyticgenes that can kill their bacterial hosts under stress conditions and are found in most Las strains andmay limit culture. Unfortunately, the single extant Lcr strain also appears to have lostpathogenicity and so is not useful for citrus or general plant pathogenicity assays. Continuedattempts to culture a single Las genotype or clone may be fruitless, and therefore multiple parallelapproaches are proposed here, including attempts to culture a phageless Las strain, a new citrusspecies from Colombia that may be related to Lcr, microfluidic chambers, improved chemicalscreens and those involving improved monoclonal antibodies, and both chemical and phagetherapies for HLB.
Objective 1: Concerted, parallel and coordinated efforts towards obtaining viable Las and/or Lca cultures (Castañeda, Davis, De La Fuente, Duan and Killiny). Published empirical approaches to Las culture and use of diverse nutritional resources will be continued (Davis, De La Fuente). These will be expanded in two additional labs (Duan, Killiny), using microarrays, combinatorial libraries, detailed phloem sap analyses from healthy versus infected citrus, fresh versus spent media, psyllid hemolymph, and honeydew. Data will be shared amongst all laboratories so that continual improvements in media formulations can be made. Inoculum derived from both psyllid (Davis, Killiny and Duan labs) and citrus, including phageless Japanese isolates (De La Fuente) will be tested. Phage lytic cycle inhibitors from psyllids will also be provided (Gabriel).Lca is tentatively expected to be imported on bud-sticks from infected material in Colombia; materials will be collected in Colombia (refer attached letter, Castañeda) and imported by Gabriel. Following grafting to clean citrus, material will be monitored for Lca and curated separately from Las infected materials in the Plant Containment Facility. If Lca can be maintained in citrus, Davis will travel to Gainesville to assist in attempts to culture from this material.Objective 2: Define the influence of physical environment in Las culturability (De La Fuente & Ma). All attempts to culture Las to date have used standard batch systems such as agar plates, test tubes, and flasks. Bacteria can behave much differently under different pressures than when grown in batch cultures. Las grows actively in phloem cells where nutrients are being transported by liquid flow, and pressures are high and fluctuating. The microfuidic chamber currently used in the De La Fuente lab allows experimental variation in growth pressures, temperatures, gas exchange and flow rates. Antibodies are needed in order to coat the microfluidic chambers and both increase initial titer of the inoculum and to observe Las behavior in these chambers. The Ma lab will generate high affinity monoclonal antibodies (MAbs) against two classes of cell surface antigens. Firstly, the variable O-antigen epitopes in the outer membrane structural lipopolysaccharide (LPS) will be used to develop species-specific antibodies. LPS will be extracted from Lcr, and high affinity, Liberibacter-specific antibodies will be screened using a library of synthetic, monoclonal antibodies generated by Dr. Xin Ge's laboratory at U.C., Riverside. Secondly, antigenic outer membrane proteins, such as abundant adhesins, will be identified for antibody development using the same library. MAbs will also be useful to develop non-PCR detection methods, such as ELISAObjective 3: Define the role of chemical signaling and co?factors in culturability of Las and/or Lca (Davis, De La Fuente, Gabriel & Killiny) One of the few examples of successful culturing of Las (Davis) was achieved when Las was co-cultured with another bacterium. Attempts at co-cultivation with Lcr will be made to identify unknown signals using a diffusion 'sandwich' approach in agar plates with macerated suspensions of infected and non-infected citrus and psyllids. In addition, filtrates from secreted compounds obtained from a metagenomics library; and addition of quorum sensing molecules (Killiny) and siderophores will be evaluated. Direct cultures of Las with Lcr engineered with conditional inhibitors (sucrose sensitivity or temperature sensitive lethal conditional mutations will be attempted. Microfluidic chambers are ideal for direct injection and testing of identified signals or co-factors for Las response (De La Fuente).Objective 4: Develop genetic tools that enable delivery of (missing) candidate growth factor genes identified and identified and partially characterized into Las (Gabriel & Jones) At least one likely necessary growth factor gene of Lcr has been identified (Gabriel lab) that is completely missing from the Las genome. Lcr has proven to be genetically tractable for gene knockouts and gene additions, and two compatible DNA shuttle vectors capable of conjugational transfer and phage packaging (cosmid) and transfection have been proven for use in Lcr (Gabriel lab). The standard functional tools developed for Lcr have not been applied to Las because such techniques are not readily applied to uncultured cells, primarily because of low cell density. Conjugation or phage transfection could overcome these issues, and both will be attempted. If co-cultures are successful (Objective 3), conjugational transfer of a vector carrying the growth factor gene plus marker could be likely achieved using an Lcr strain engineered to die if chemically or genetically induced (CRISPR-Cas9; Yang lab). T4-like phages from S. meliloti have been characterized and some, but not all key Las phage structural phage elements identified (Jones lab). Gabriel & Jones will attempt to create an in vitro Las phage packaging system for genetic manipulation of Las. If successful, such a phage system may be further developed (Gabriel & Yang) into a novel therapeutic agent for targeted killing of Las in infected tissue. Once cultured, Las would need to be reintroduced into psyllids, likely by injection (Killiny, Duan labs), and subsequently into citrus.Objective 5: Outreach (Alabi, Roberts, Vidalakis) The ability to successfully culture Las in vitro will be a huge milestone for the scientific community working on the citrus-HLB-ACP pathosystem. However, it is uncertain if growers and other stakeholders in the citrus industry will fully appreciate the importance of such a breakthrough. Hence, outreach activities will need to be developed and implemented to educate members of the public on the importance/ relevance of the project right from its inception. Even if not considering the different degrees of HLB epidemic in the three leading citrus-producing states, the proposed outreach plan will be implemented such that there is a cohesive message on the promise and outcomes of the project. At the project inception, the focus of the outreach activities will be to educate growers, industry stakeholders and members of the public on the importance of being able to culture Las in vitro. Subsequent activities will then focus on providing periodic project updates and breakthroughs to the stakeholders. Finally, news of successful culturing of Las will be announced via face-to-face, print and electronic media materials to be developed jointly by Vidalakis (California), Roberts (Florida) and Alabi (Texas) in consultation with the PD and other co-PIs. Specific outreach activities planned for the project include:
Target Audience
The target audience is primarily other researchers, most of whom are working on emerging plant pathogens, insect endosymbionts, bacteria-bacteria interactions, host-parasite interactions, and those working on culturing previously uncultured bacteria, including Liberibacters. However, the target audience also includes citrus industry representatives, particularly those who attend the International Research Conference on HLB. Last year, Co-Pi Vidalakis organized the Joint 21st Conference of the International Organization of Citrus Virologists (IOCV) and the 6th International Research Conference on Huanglongbing (IRCHLB) in Riverside , CA, March 10-15, 2019 (122 oral presentations and 181 posters with attendance of 568 from 23 countries. Our project website, set up by Roberts, provides up to date publication information generated as a result of this funding. https://plantpath.ifas.ufl.edu/citrusgreening/
Changes / Problems
Nothing Reported
Training & Professional Development
During this reporting period, we trained: two postdocs (Gabriel); one postdoc (Jones); 3) one graduate student (De La Fuente); and at USDA-ARS, Ft Pierce, one postdoc (Duan).
Dissemination Streams
Results to the scientific community over the last year (with project extension) have been fourteen (14) peer reviewed publications, as well as thirty (30) published abstracts representing presentations at scientific congresses (ICPP/APS and IRCHLB by Killini, Gabriel, Ma, Jones, De La Fuente, Vidalakis, Davis and Alabi labs). The overall goals of this Project have been outlined at numerous citrus grower conferences this year. Alabi gave monthly Master Gardner Training Classes, and bimonthly meetings of the Texas Citrus Pest and Disease Management Corp. Vidalakis organized the Joint 21st Conference of the International Organization of Citrus Virologists (IOCV) and the 6th International Research Conference on Huanglongbing (IRCHLB) in Riverside , CA, March 10-15, 2019 (122 oral presentations and 181 posters with attendance of 568 from 23 countries; distribution of educational materials via the weekly CCPP budwood shipment program to 3,500 customers, organized a high school Jamboree (iGEM) that received a bronze award, (https://2019.igem.org/ Team:Bio_Without_Borders) and a California Citrus Nursery Society Conference held 14 Nov., 2019 in Monterey, CA. Our project website, set up by Roberts, provides up to date publication information generated as a result of this funding. https://plantpath.ifas.ufl.edu/citrusgreening/
Next Reporting Steps
Nothing Reported
Target Audience
The target audience is primarily other researchers, many of whom are working on emerging plant pathogens, insect endosymbionts, bacteria-bacteria interactions, host-parasite interactions, and those working on culturing previously uncultured bacteria, including Liberibacters. However, the target audience also includes citrus industry representatives, particularly those who attend the International Research Conference on HLB. The tools used were technical oral presentations and posters presented at international and national scientific meetings. In addition, efforts were made starting in 2016 to inform citrus growers at grower conferences, and in this reporting period included monthly meetings in Texas and a Twitter feed (by Alabi), nearly monthly meetings in California and presentations in Brazil and Spain (by Vidalakis) A website (set up by Roberts) is now live that provides up to date publication information generated as a result of this funding.(https://plantpath.ifas.ufl.edu/citrusgreening/).
Changes / Problems
We are on track to meet all objectives, as described in Accomplishments report above. Since the experiments with such slow growing bacteria require a lot of patience , all experiments tend to run longer than expected. We plan to request a no cost extension for up to a year for completion of some tasks.
Training & Professional Development
During this reporting period, we trained: 1) at the University of Florida (UF), one graduate student (Gabriel), two postdocs (Gabriel); at Florida State, one postdoc (Jones); 3) at Auburn, one graduate student (De La Fuente); at UC Riverside, one graduate student (Ma), and at USDA-ARS, Ft Pierce, one postdoc (Duan).
Dissemination Streams
The overall goals of this Project have been outlined at numerous citrus grower conferences this year, including (by Alabi) monthly meetings in Texas, an invited talk given at the 2018 Agriculture and Applied Economics Association (AAEA) annual meeting in Washington, DC, the APS / ICPP meeting in Boston (co-authored by Vidalakis and Roberts) and the IRCHLB meetings; and (by Vidalakis) a presentation in Brazil, one in Spain, and 14 in California. A website set up (by Roberts) has gone live that provides up to date publication information generated as a result of this funding. https://plantpath.ifas.ufl.edu/citrusgreening/ Results to the scientific community over the last year have been seven peer reviewed publications (Gabriel, Killini, De La Fuente, Davis & Vidalakis labs), as well as eighteen presentations at scientific congresses (ICPP/APS and IRCHLB by Killini, Gabriel, Ma, Jones, De La Fuente, Vidalakis, Davis and Alabi labs).
Next Reporting Steps
We are on track to meet all objectives, as described in Accomplishments report above. <br><br>
<br>What was accomplished under these goals? Much progress has been made identifying and confirming the multiple genetic limitations to axenic growth that are inherent in the highly degenerate genome of Ca. Liberibacter asiaticus (Las). Methodology to establish long term, viable co-cultures of Las with "helper" bacteria have now been established in two labs from samples removed from both infected psyllids and infected citrus fruits, and confirmed repeatable in a third lab. The mature psyllids were then subsequently fed on healthy citrus seedlings and very preliminary results indicate the possibility of Las transmission to citrus. These plant transmission results must be confirmed, but progress in growing and maintaining viable Las for over a year together with the psyllid larval feeding technique has allowed the transmission to psyllids. Liberibacter crescens (Lcr), with a slightly less degenerate genome, is routinely grown in axenic culture and is now being studied into a model host-parasite system with the discovery that Lcr can infect and grow in Galleria moth larvae. Lcr has already been used in testing hypotheses about Las effector secretion and in incrementally improved culture media for Las, including buffers and specific additives, thereby significantly lengthening initially short Las survival and growth in co-cultures. Methods for effective introduction of DNA (genes) into Lcr should be applicable to Las in co-cultures, with the goal of allowing axenic Las cultures by introducing the missing Lcr genes that allow it to grow. Objective 1: Concerted, parallel and coordinated efforts towards obtaining viable Las and/or Lca cultures. Three labs (Davis, De La Fuente and Duan) have developed significant improvements in Ca. Liberibacter asiaticus (Las) co-culture longevity. Davis evaluated 90 different media combinations. Duan developed a techniques for establishing and maintaining viable co-cultures for well over a year, albeit at low Las titer (up to 106 cells/ml of co-culture). Remarkably, Duan reports successfully re-inoculating psyllids with a co-culture for the first time; the inoculated psyllids were allowed to mature, and confirmed to be infected with Las at low levels (Ct = 29 to 34). The isolation and co-culturing technique has been shared and independently confirmed by Gabriel. Each sub-culture resulted in progressively lower titers, and survival was limited to the first two subcultures. De La Fuente independently established and maintained three sequential Las subcultures of co-cultures sourced from citrus fruits, finding that adjusting the pH of grapefruit juice medium from 3.5 to 5.85 increased growth and viability of Las in co-cultures. Killiny is evaluating the use of the larvae of the waxy moth Galleria mellonella as a model culturing host for Las, and evaluating the chemical composition of its haemolymph. Liberibacter crescens (Lcr) has been increasingly utilized as an experimental proxy for Las culturing. Since Lcr growth in media has an atypically rapid death phase, Jones began mathematical modeling of the growth states of Lcr cell populations in batch cultures. The conclusions were: 1) Lcr rapidly loses viability during culture due to medium alkalinization; 2) this alkalinization can be mitigated by increasing the buffering capacity of the medium; 3) addition of ammonium chloride to Lcr cultures inhibits alkalinization and prevents culture death. Jones has designed an optimized medium that enhanced Lcr growth, culture density, and cell survival to late stationary phase. Objective 2: Define the influence of physical environment in Las culturability. The Ma lab has produced several antibodies targeting different Liberibacter cell components; at least one of these antibodies can bind to Las cells. The latter antiserum was sent to the Gabriel lab to help confirm the bacterial culture generated from in vitro cultivation. The Ma lab is also working to obtain monoclonal antibodies using targeted outer membrane proteins of Las. Although synthesized peptides failed to produce monoclonal antibodies, the targeted proteins were expressed in E. coli and the purified recombinant proteins are being used as antigens for monoclonal antibody generation. The De La Fuente lab demonstrated the ability of proxy Lcr to form biofilms in vitro and the culture conditions influencing the critical cell-surface adhesion growth phase. His lab also demonstrated that Lcr attaches to surfaces, and forms cell aggregates embedded in a polysaccharide matrix both in batch cultures and under flow conditions in microfluidic chambers. Biofilm structures have been observed during insect vector colonization by Las, likely helping with host retention, immune system evasion, and transmission. Objective 3: Define the role of chemical signaling and co-factors in culturability of Las and/or Lca. The Gabriel lab identified a protein that is found encoded only in the Wolbachia bacteria that infects psyllids and that suppresses the Las phage lytic cycle in psyllids. This protein was commercially manufactured and provided to the Davis, Duan and De La Fuente labs for evaluation and use as an additive in Las culture media. None of these labs have seen any effect on growth of Las in different media when this protein was added. This protein may not be sufficient to allow increased growth. The Killini lab studied changes in D. citri nymph metabolism (Las infected vector) due to their ability to acquire Las. Nymphs reared on CLas-infected Valencia sweet orange were higher in many metabolites, than those reared on healthy plants. They also demonstrated that Las and psyllid infections of citrus accelerated the conversion of α-ketoglutarate to glutamate, then to GABA, causing an accumulation of GABA in the cytosol. In silico analysis showed that the citrus genome possesses a putative GABA permease that connects the GABA shunt with the TCA cycle and supports the accumulation of succinate, fumarate, and citrate. Taken together, the GABA shunt appears to contribute to the flux toward succinate rather than an intact TCA cycle in Las infected citrus. Objective 4: Develop genetic tools that enable delivery of (missing) candidate growth factor genes identified and identified and partially characterized into Las. The Jones lab identified and isolated a new 40.4 kb lambda-like Lcr phage from chloroform extracts of Las infected psyllids. The phage sequence was determined from infected Lcr cultures. The phage inoculum retarded the growth of Lcr cultures but did not lyse them or reproducibly form plaques. Conditions needed to enhance phage production are being investigated, with a view to developing this phage as a DNA vector for Las. The utility of the Lcr model has also been expanded by the Gabriel lab with the recent demonstration that Galleria moth larvae can be infected by genetically (GFP) marked Lcr. The Gabriel lab also provided the first evidence that Lcr is naturally competent for direct uptake and chromosomal integration of both linear and circular plasmid DNA. Chromosomal integration of naturally acquired DNA (both linear and plasmid) in Lcr was achieved by homologous recombination within a non-essential target locus. Lcr cells simply mixed with linear DNA reliably yielded 3-4 natural transformants per µg DNA; 10-12 transformants by mixing closed circular (non-replicative) plasmid DNA, and 900 transformants by mixing broad host range shuttle plasmid pUFR071. Experiments to transform Las are currently underway. Objective 5: Outreach The overall goals of this Project have been outlined at numerous citrus grower conferences this year, including (by Alabi) monthly meetings in Texas and the APS / ICPP meeting in Boston and the IRCHLB meetings; and (by Vidalakis) a presentation in Brazil, one in Spain, and 14 in California. A website set up (by Roberts) has gone live that provides up to date publication information generated as a result of this funding. https://plantpath.ifas.ufl.edu/citrusgreening/ <br><br><b>Publications</b><br>
Target Audience
The target audience is primarily other researchers, many of whom are working on emerging plant pathogens, insect endosymbionts, bacteria-bacteria interactions, host-parasite interactions, and those working on culturing previously uncultured bacteria, including Liberibacters. However, the target audience also includes citrus industry representatives, particularly those who attend the International Research Conference on HLB. The tools used were technical oral presentations and posters presented at international and national scientific meetings. In addition, efforts were made starting in 2016 to inform citrus growers at grower conferences, and in this reporting period included(by Alabi) monthly meetings in Texas, a keynote address in Orlando, and (by Vidalakis) two presentations in Australia and eight in California. A website (set up by Roberts) is now live that provides up to date publication information generated as a result of this funding.(https://plantpath.ifas.ufl.edu/citrusgreening/).
Changes / Problems
Nothing Reported
Training & Professional Development
During this reporting period, we trained: 1) at the University of Florida (UF), one graduate student (Gabriel), two postdocs (Gabriel); at Florida State, one postdoc (Jones); 3) at Auburn, one graduate student (De La Fuente); at UC Riverside, one graduate student (Ma), and at USDA-ARS, Ft Pierce, one postdoc (Duan).
Dissemination Streams
The overall goals of this Project have been outlinedto stakeholders and growers at at numerous citrus grower conferences this year, including (by Alabi) monthly meetings in Texas, a keynote address in Orlando, the APS meetings and the IRCHLB meetings; and (by Vidalakis) two presentations in Australia and eight in California. A website (set up by Roberts) is now live that provides up to date publication information generated as a result of this funding. (https://plantpath.ifas.ufl.edu/citrusgreening/). Results to the scientific community over the last year have been nine peer reviewed publications (Killini, Gabriel & Ma labs), as well as eight presentations at scientific congresses (ICPP/APS by Killini, Gabriel, Ma, Jones, De La Fuente and Alabi labs). In addition, Gabriel presented overall project results at the NIFA sponsored Project Directors' & growers' meeting in Ft. Pierce this year.
Next Reporting Steps
We are on track to meet all objectives, as described in Accomplishments report above, and reported in Ft. Pierce at the Project Directors meeting. Following a query from a grower at the San Antonio meeting as to how we might accelerate progress towards the culturing goal---time being of the essence---an extensive discussion of this question occurred during a meeting of the entire research group at the IRCHLB Congress in Orlando. We concluded that adding an insect cell culture component to the group effort would be an important project adjustment, as this avenue for Las culture research had not previously been aggressively or adequately pursued. We concluded that Dr. Nabil Killiny, entomologist and co-PI of this project, had the ideal experience to meet this goal, particularly when working together with co-PI Prof. Mike Davis. Dr. Killiny initiated the Galleria moth hemolymph analyses and injections with Lcr and Las, as described in the accomplishment section above. We also agreed they should be better funded with at least a shared postdoc in their adjacent labs in Lake Alfred, and supplemental funding has been requested this year. <br><br>
<br>What was accomplished under these goals? Significant progress has been made identifying and confirming the multiple genetic limitations that are inherent to Ca. Liberibacter asiaticus (Las). The bacterium has a highly degenerate genome and a few special gene additions that allow adaptation to a parasitic lifestyle in both psyllids and citrus, but to date disallow growth outside of living cells and tissues, except transiently. Some of these limitations can be compensated by additions to culture media. Fortunately, Liberibacter crescens (Lcr), a different species of the same genus and with a slightly less degenerate genome, can be cultured, although it is not pathogenic, which limits its utility in testing hypotheses regarding Las virulence and pathogenicity. Nevertheless, genomic comparisons and development of genetic and microbiological tools to manipulate Lcr have facilitated progress in Las culturing, the highlights of which are 1) incrementally improved culture media for Las, including buffers, specific additives and particular Las gene regulators, thereby lengthening transient culture periods; 2) partial success with Las co-cultures; 3) success in feeding media to psyllids and providing a likely pathway for re-introduction of transiently cultured Las into psyllids and citrus, and 4) isolation of a potential phage delivery system for adding missing genes to Las. Objective 1: Concerted, parallel and coordinated efforts towards obtaining viable Las and/or Lca cultures. Three labs (Davis, De La Fuente and Duan) have maintained Ca. Liberibacter asiaticus (Las) cultures for up to two months using Las isolated from both psyllids (Davis and Duan) and citrus fruits (De La Fuente) sent from Florida (Gabriel), Texas (Alabi) and Colombia (Castañeda). Davis has improved growth of Las in media by using 1) DS2 insect basal culture medium; 2) addition of nucleotide triphosphates; 3) addition of freshly ground citrus leaf extract and 4) use of 10% reduced oxygen levels. Killiny is evaluating the use of the larvae of the waxy moth Galleria mellonella as a model host, and evaluating the chemical composition of its haemolymph. Due to its highly reduced innate immune system, many microorganisms are able to be cultured in the haemolymph of Galleria larvae. Duan reported co-cultures of Las with as yet unknown microbes visualized by electron microscopy at Las titers estimated at up to 107 cells/ml. All three labs report that co-cultures fluctuate in titer and finally die out. Killiny's published psyllid feeding technique is being used by all labs to reintroduce these transient Las cultures back into psyllids and to citrus. Liberibacter crescens (Lcr) has been increasingly utilized as an experimental proxy for cultures of Las. Since Lcr growth in media has an atypically rapid death phase, Jones began mathematical modeling of the growth states of Lcr cell populations in batch cultures using OD600, percent live/dead (SYTO9/propidium iodide staining) and hemocytometer counts. A sharp drop in viability of Lcr after day 5 correlated with a unexpectedly sharp rise in pH, and may be reflective of loss of Las culturability after 5 days. Metabolite changes associated with the sharp pH rise in Lcr cultures and using different buffering systems are being investigated. Objective 2: Define the influence of physical environment in Las culturability. The Ma lab generated two polyclonal antibodies targeting different antigens. The first was generated using heat-inactivated Lcr and found by ELISA to specifically bind to Lcr cells. This antisera was sent to De La Fuente. During the development of this antibody, an unusual lipopolysaccharide (LPS) content in Lcr was discovered, and Gabriel helped purify the LPS and sent it to the Complex Carbohydrate Research Center at the University of Georgia for characterization. The second polyclonal antibody was obtained by Ma using a Las outer membrane protein that was expressed in E. coli and also showed specific binding to Lcr. Ma also screened a monoclonal antibody library using a synthesized peptide fragment and purified full length protein. The De La Fuente lab overcame lack of attachment of Lcr cells to glass surfaces by replacing FBS in the media with methyl-β-cyclodextrine, as suggested by Davis, allowing clear observations of Lcr in microfluidic chambers. This resulted in increased Lcr cell viability and reduced Lcr intracellular oxidative stress (assessed by ROS production) and also the first demonstration of ability of Lcr to form biofilms in vitro and culture conditions influencing the critical cell-surface adhesion growth phase. Objective 3: Define the role of chemical signaling and co-factors in culturability of Las and/or Lca. The Gabriel lab identified a protein that is found encoded only in the endosymbiotic Wolbachia that infects psyllids that suppresses the Las phage lytic cycle. This protein has commercially manufactured and provided to the Davis, Duan and De La Fuente labs for evaluation and use as an additive in Las culture media. The Gabriel lab also identified two secreted proteins, a phage peroxidase and a chromosomally encoded peroxiredoxin that interrupted the downstream activation of host innate immunity and callose deposition. In addition, the peroxiredoxin protected against degradation of lipid membranes in planta, preventing subsequent accumulation of antimicrobial oxylipins that can also trigger the localized hypersensitive cell death response. Both enzymes are likely critical virulence factors for Las. The Killini lab confirmed the functionality of the Las solo LuxR quorum sensing response transcriptional regulator, which can regulate bacterial biofilm formation. Crude extracts from psyllids and citrus were found to activate Las-luxR. Acyl homoserine lactone (AHL) -like substances were found in insect extracts, but not in citrus; the plant derived extracts are likely to be structurally unrelated AHL mimics. Davis had already documented that Las forms a biofilm on the outer surface of the D. citri midgut, leading Killiny to speculate that this might be due to high levels of activation of LuxR in the insect hemolymph. By over-expressing several LuxI genes (which form AHLs) in citrus using a citrus tristeza virus (CTV) based vector system, Killiny demonstrated that citrus plants expressing LuxI showed highly localized, severe HLB symptoms, providing indirect evidence that (protective) biofilm formation by Las can be induced, possibly improving Las cultures. Objective 4: Develop genetic tools that enable delivery of (missing) candidate growth factor genes identified and identified and partially characterized into Las. The Jones lab transformed an Lcr strain with a plasmid expressing the Las ompA gene; in Sinorhizobium, OmpA can be a specific phage receptor. The resulting A3.1 strain grew normally and was used in a soft-agar-overlay screen for phage that might be specific for Las OmpA. Chloroform extracts of Las-infected or -uninfected citrus psyllids and of sweet orange were screened. A single sample from an uninfected psyllid (Ps5) produced zones of lysis in the plaque assays in multiple replicates. Lytic activity was observed only using A3.1, but not on wild type Lcr. Lytic activity from Ps5 has been retained through multiple rounds of propagation in A3.1. The phage is not virulent enough to fully lyse liquid cultures. Phage DNA from the lytic zones will be amplified and Illumina-sequenced. Objective 5: Outreach The overall goals of this Project have been outlined at numerous citrus grower conferences this year, including (by Alabi) monthly meetings in Texas, a keynote address in Orlando, the APS meetings and the IRCHLB meetings; and (by Vidalakis) two presentations in Australia and eight in California. A website set up (by Roberts) has gone live that provides up to date publication information generated as a result of this funding. <br><br><b>Publications</b><br>
Target Audience
The target audience is primarily other researchers, many of whom are working on emerging plant pathogens, insect endosymbionts, bacteria-bacteria interactions, host-parasite interactions, and those working on culturing previously uncultured bacteria, including Liberibacters. However, the target audience also includes citrus industry representatives, particularly those who attend the International Research Conference on HLB. The tools used were technical oral presentations and posters presented at international and national scientific meetings. In addition, efforts were made starting in 2016 to inform citrus growers at grower conferences, including one in Florida, one in Texas, six in California and one in Brazil. A website is being set up, hosted by the University of Florida in Florida.
Changes / Problems
As mentioned in the research plan for the next reporting period, time is of the essence in the effort to culture, given the dire situation of the Florida citrus industry. Following an extensive discussion of how to accelerate the research during a meeting of the entire research group at the IRCHLB Congress in Orlando, we concluded that adding an insect cell culture component to the group effort would be an important project adjustment, as this avenue for Las culture research had not previously been aggressively or adequately pursued. We concluded that Dr. Nabil Killiny, entomologist and co-PI of this project, had the ideal experience to meet this goal, particularly when working together with co-PI Prof. Mike Davis. We also agreed they should be better funded with at least a shared postdoc in their adjacent labs in Lake Alfred. Some funds may be shifted internally to support this added component to Objective 1 , and an additional funding request will be made if NIFA funds are available.
Training & Professional Development
During this reporting period, we trained: 1) at the University of Florida (UF), one graduate student (Gabriel), 1 visiting postdoc (Gabriel), and one postdoc (Gabriel); at Florida State, one postdoc (Jones); 3) at Auburn, one graduate student (De La Fuente); at UC Riverside, one graduate student (Ma), and at USDA-ARS, Ft Pierce, one postdoc (Duan).
Dissemination Streams
Although this project has just begun, the overall goals of this Project have been outlinedto stakeholders and growers at numerous citrus grower conferences, including one in Florida, one in Texas, six in California and one in Brazil. A website is being developed, to be hosted in Florida, expected to be up, running and advertised to stakeholders in May, 2017. Results to the scientific community to date have been two peer reviewed publications (Killini & Gabriel), as well as four presentations at scientific congresses (APS and ISMPMI; Gabriel). In addition, eight presentations were presented to growers and the scientific community at the IRCHLB Congress in Orlando (1 by Jones & Gabriel; 1 by Killiny and 6 by Gabriel), as well as communicated to NIFA and growers at the San Antonio meeting by Gabriel.
Next Reporting Steps
We are on track to meet all objectives, as described in Accomplishments report above, and reported in San Antonio at the Project Directors meeting. These efforts will be continued as described. Following a query from a grower at the San Antonio meeting as to how we might accelerate progress towards the culturing goal---time being of the essence---an extensive discussion of this question occurred during a meeting of the entire research group at the IRCHLB Congress in Orlando. We concluded that adding an insect cell culture component to the group effort would be an important project adjustment, as this avenue for Las culture research had not previously been aggressively or adequately pursued. We concluded that Dr. Nabil Killiny, entomologist and co-PI of this project, had the ideal experience to meet this goal, particularly when working together with co-PI Prof. Mike Davis. We also agreed they should be better funded with at least a shared postdoc in their adjacent labs in Lake Alfred. Some funds may be shifted internally to support this added component to Objective 1 , and an additional funding request will be made if NIFA funds are available. <br><br>
<br>What was accomplished under these goals? Objective 1: Concerted, parallel and coordinated efforts towards obtaining viable Las and/or Lca cultures (Castañeda, Davis, De La Fuente, Duan and Killiny). Functional genomics using L. crescens (Lcr) as a proxy for Las has revealed at least four independent reasons that would cause failure to culture Las: 1) absence of a phage repressor (host sourced); 2) lack of ATP in media; 3) lack of dNTPs in media, and 4) failure to remove or detoxify methylglyoxal, a potent and highly toxic byproduct of glycolysis. Attempts to culture Las using traditional culture techniques by Davis continue, now consistently informed by the group's efforts, including the above identified obstacles. As a result, major modifications have already been made to the base media used for culture of Lcr (and attempts to culture Las). Killiny performed metabolomic comparative analysis of the phloem sap of curry leaf tree (Bergera koenegii), orange jasmine (Murraya paniculata), and Valencia sweet orange (Citrus sinensis). Diaphorina citri can infest the three hosts while Las cannot grow in curry leaf tree. Curry leaf tree was the lowest in most of the metabolites as well as total metabolites. The nutrient inadequacy of the phloem sap in curry leaf tree, especially the amino acids could be the reason behind the limitation of Las growth on this host. The screening of the Torrey Pines small molecule scaffold library is complete and the positional scanning libraries are now being surveyed by Duan for additional compounds that may improve growth. De La Fuente obtained APHIS permits to receive infected material from Japan and Colombia, in order to expand the number of Liberibacter strains that are tested for culturability. Gabriel has sent monthly shipments of Florida Las infected fruits to De La Fuente for culturing attempts using methodology and Las sources different from those used by Davis. Protocols are being developed to quantify Las cell viability in plant samples using rtPCR. qPCR-PMA did not work well when samples were cloudy due to plant material. Objective 2: Define the influence of physical environment in Las culturability (De La Fuente & Ma). An essential step for increasing the utility of microfluidic chambers is the availability of anti-Las antibodies.Ma has prepared lipopolysaccharide (LPS) extracts and heat-inactivated cells of BT-1 and sent these extracts to two companies for polyclonal antibody development. There appear to be only two LPS biosynthetic gene differences between Lcr and Las; both involving Lipid A. A gene knockout in the most critical one of these has been made in Lcr by Gabriel,This knockout will also be used by Ma to produce LPS for polyclonal antibody production. Ma also selected a protein that is associated with the outer membrane of Las (not a porin) and is screening a monoclonal antibody library using synthesized peptides for antibodies that bind to this antigen with high affinity. De La Funete has overcome one of the main limitations of using microfluidic chambers; a modification in the media greatly improved attachment of Lcr to glass surfaces. The BM7 medium modification is based on replacing fetal bovine serum (FBS) with methyl-β-cyclodextrine. FBS is also expensive, so finding a media modification that allows growth without FBS is useful. Objective 3: Define the role of chemical signaling and co-factors in culturability of Las and/or Lca (Davis, De La Fuente, Gabriel & Killiny). All Florida Las strains examined to date carry prophages which appear to be inactive in psyllids and repressed in citrus. A promoter element that controls both the Las lytic cycle and (lethal) holin gene expression was used to drive a reporter gene that allows easy detection of chemicals or proteins that regulate or interfere with the genetic element. That reporter was used by Gabriel to identify a protein by LCMSMS that is found only in endosymbiotic Wolbachia that infect psyllids that suppresses both holin and the phage lytic cycle. This protein has been expressed in vitro and physically binds the promoter, and will be commercially manufactured to be used as an additive in Las culture media. The typical LuxR bacterial "quorum sensing" system consists of two components, LuxR and Acyl-Homoserine Lactone (AHL) inducer. The Las genome contains a luxR gene but lacks luxI that produces AHL. Killini has confirmed the functionality of the Las-LuxR by constructing a luxR gene promoter fused with a GFP reporter. Several AHLs as well as extracts from the insect vector and citrus host plants have been found to activate Las-luxR. Using GC-MS and TLC Killini detected AHL-like substances in insect extracts. Because of this specific binding, Las forms a biofilm on the surface of D. citri midgut. As a response to infection by Las, citrus plants may induce the production of AHL mimic(s), which would bind to LuxR and trigger cell aggregation and possibly limit bacterial growth and movement in planta. Killiny also expressed Las-LuxR in citrus using the CTV-based vector system. These citrus plants showed evenly distributed severe HLB symptoms and higher bacterial titer when infected with Las. Objective 4: Develop genetic tools that enable delivery of (missing) candidate growth factor genes identified and identified and partially characterized into Las (Gabriel & Jones) Lcr has now been demonstrated by Gabriel to import isotopic sucrose in a time- and dose-dependent manner, enabling use of the sacB marker eviction system. Subsequently two marker interruption mutations were achieved in Lcr that make Lcr more Las-like. This enables unlimited numbers of sequential knockout mutations in Lcr. In addition, Gabriel is currently attempting a series of conditional knockout mutations using this system with genes suspected of being critical for enabling Lcr growth in media, each time evaluating different rescue strategies to keep the resulting mutant strains alive. Using both these knockout tools and knock-in vectors, two Las peroxiredoxins and a peroxidase were identified, cloned and expressed in Lcr and all were demonstrated to be functional in Lcr, providing targets for high throughput bioassays for identification of chemical inhibitors of these critical pathogenicity factors. These studies also serve to validate use of Lcr as a proxy for Las until Las can be cultured. Lcr strain BT-1 is being developed as as a trap species for isolation of Liberibacter phages by Jones. Material has been collected or received by collaborators from multiple plant and psyllid tissue samples and these are being tested for plaque formation on lawns of wild type Lcr BT-1. A solid-media, Lcr-soft-agar-overlay protocol was developed for these phage plaque assays. These samples are currently being tested after an Lcr-culture-enrichment step. Gram-negative bacterial outer membrane proteins commonly serve as bacteriophage receptors. One strategy that may permit selection of Las phages is to use the Las psy62 outer membrane porin as a receptor to replace the Lcr BT-1 porin. Three different plasmid constructs for insertion mutagenesis of the Lcr porin have been made by Jones and introduced into Lcr. Jones has also engineered expression constructs that substitute the Las porin open reading frame for that of Lcr. Objective 5: Outreach (Alabi, Roberts, Vidalakis) Although this project has just begun, the overall goals of this Project have been outlined at numerous citrus grower conferences, including one in Florida, one in Texas, six in California and one in Brazil. A website is being developed, to be hosted in Florida, expected to be up, running and advertised to stakeholders in May, 2017. <br><br><b>Publications</b><br>