Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 100% |
The lack of ability to culture "Candidatus Liberibacter asiaticus" (CLas) has precluded the use of genetics and gene transformation approaches to identify gene functions involved in CLas viability or virulence. Those are critical tools that will ultimately be needed in development of a fully integrated strategy to control CLas and eventually develop the capability to stop HLB spread by the Asian Citrus Psyllid (ACP) and the resulting economic devastation to citrus agriculture. This project aims to develop means to culture CLas in vitro using two novel, parallel, complementary and integrated strategies that also integrate proteomics, metabolomics and community sequencing approaches. This will ultimately lead to successful culture of CLas under both planktonic growth conditions and as biofilms.
The first major goal of this project is to develop a system to culture, in vitro, "Candidatus Liberibacter asiaticus" (CLas), the causative agent of Citrus Greening Disease, or Huanglongbin (HLB). A second major goal is to then make that system readily available and adopted by the national stakeholders, particularly citrus breeders and other researchers, thereby enabling genetics-based research that will have an enhanced capability to identify means to control the transmission of CLas. A third goal, which will be a natural offshoot of the other goals, is to develop rapid and easy methods to isolate and cultivate new strains of CLas from groves across the nation as well as adapting or mutating strains of CLas within regions of outbreaks or reintroductions that may occur even if means to control CLas are indeed developed. Culturing of new strains like that will be critical long term strategies to control HLB in the face of a changing, adapting pathogen. In order to accomplish those goals, four major objectives have been developed, as outlined directly below. The PD/CoPDs involved as major contributors to efforts to accomplish each objective are indicated in parentheses. The major hypotheses underlying each objective are also listed.
The original proposal outlined 3 major research objectives to be accomplished within a 5-year timeframe, plus an additional objective related to extension and outreach. One of those objectives was eliminated at the request of NIFA. For one of the major goals of the project, development of planktonic growth conditions and a planktonic culture system ofCLas, we are limited by the biology of the system, where some things just take a long time to occur within the biological system. Thus, the timeframe originally proposed, where 2 - 3 years to establish conditions for host cell-free metabolic activity (i.e., not necessarily cell division) ofCLas and about 5 years to develop a reliable culture system forCLas in the absence of host cells, is not likely to change much, but we are likely to be well on the way to meeting that goal by the end of the second year of the project. Indeed, we should have a good idea at that point (two year mark) how likely the project is to be a success. Thus, a two year proof of concept effort will be undertaken, which will allow enough research to provide proof of concept that the overall project is likely to be a success. If such is demonstrated, then an additional ~3 years of funding will be requested in a follow-on application.
Methods applied to accomplish each Objective are outlined below:Objective 1 (biofilm culture method): Proof of CLas existence in biofilms in psyllids has been obtained, suggesting that identification of conditions for culture of CLas under biofilm growth are within reach with the right approach. Using methods well established by Co-PI Beyenal, CLas bacteria will be isolated from biofilms growing in insect and potentially plant tissues, and will be the subject of a battery of experiments designed to identify conditions required for their growth in in vitro biofilms using an approach that enables separation of individual microbial community members (even species found only within biofilms) and analysis of their contribution to biofilm establishment, growth, protection and metabolism. This, in combination with detailed proteomic and metabolomic analysis of the test cultures, will identify those metabolic components required for biofilm growth in vitro.Objective 2 (host cell-free and planktonic culture): Establishment of planktonic growth will be achieved by first determining responses of CLas to nutrients for which the pathogen is a predicted auxotroph. By matching pathogen nutrient requirements to the organism's natural metabolic capacity, replication of CLas in an experimentally amenable culture system will be achieved. A major step in establishment of in vitro planktonic growth will be establishment of a cell culture model for CLas infection, which will offer i) a model to analyze interactions between CLas and insect vector tissue, and ii) a cultivation system to generate enough bacteria for physiological analysis (essential to the process of designing a host cell free cultivation system).Objective 3 (extension and outreach): The project website, scientific and extension publications and conference talks/posters, as well as cold-calls to potentially interested parties, will raise awareness of the new technology and enable the rapid dissemination of methods and developed cultures.
Target Audience
The target audience for this project includes researchers working to find methods to combat Huanglongbing, regulators, and the citrus industry in general.
Changes / Problems
Nothing Reported
Training & Professional Development
One graduate student graduated with a PhD with this project being part of her dissertation work. Four post-doctoral scholars worked on the project, all engaged in interdisciplinary research.
Dissemination Streams
We have published our method for culturing CLas in the mixed biofilm cultures, as well as other results related to culturing of the bacterium within leaf discs and other information related to growth conditions needed for Liberibacter species to grow in their hosts. We have presented these results at international and national conferences as well, including conferences like the California Citrus Conference, which target the industry members, not just academics.
Next Reporting Steps
Nothing Reported
Target Audience
Citrus industry, citrus breeders, other researchers working on HLB disease.
Changes / Problems
Obtaining permits to work on the Asian citrus psyllid and CLas in Washington State was a large challenge in the first year of the project. The process took much longer than we were told it would take, but we now have (finally) permission to work with the live insects and bacteria at WSU. This delayed some of the experiments we planned to perform, but other efforts were applied instead to keep the work moving forward (such as by using insects freshly killed in Florida and then shipped to WSU cold so that we could work on method development for metabolite profiling, proteomics and other research activities).
Training & Professional Development
Several post-doctoral researchers, staff scientists and graduate students are working in a collaborative, multidiscipinary project.
Dissemination Streams
Several presentations were made at the IRCHLB conference in 2017. These are listed under the Products area. In addition, a poster describing the project, titled "Culturing the HLB-Causing Bacterium" was produced and presented by Steve Futch at the 2016 Citrus Expo in Ft. Myers, FL, as well as at the IRCHLB conference in 2017.
Next Reporting Steps
The project team will move forward with plans as outlined in the original revised proposal, with additions based on feedback that we received after our annual Project Meeting from our Advisory Board. Two of our advisory board members, Steve Lindemann and Harold Browning, were present at that meeting and gave us some great suggestions forexperiments that we could include either this year or in the follow-on proposal that will be submitted later this year. They generated an Advisory Board Review Summary, that included those recommendations. The experiments that were recommended for effort under the current grant term are outlined below. We will integrate those suggestions as best we can in current efforts as long as they do not cause delay in other project areas. From Lindemann and Browning: We find the integration of work on Candidatus Liberibacter solanacearum (CLso) and potato psyillid (PoP) to have very significant potential to improve the rate at which this project makes progress, due to the much shorter timetables of potato and potato psyllid generation cycles. As no additional resources are currently available to incorporate this model system into the current project, we suggest a few experimental routes to incorporate these models that require only modest investment of resources. These approaches will generate preliminary data that supports larger, funded experimental efforts in the CLso/PoP model system. Inclusion of CLso into the comparative genomic analysis proposed in question 3 below. This approach will increase the strength of the CLas ortholog analysis and be independently useful for identifying hypothetical medium conditions differentiating CLso from CLas cultivation. Design equivalent qPCR assays for quantitation of CLso as have been generated for CLas. Specific and sensitive detection of CLso is a prerequisite of future cultivation design and monitoring. As has been accomplished by the Omsland lab for CLas, this assay should target a gene specific to CLso in singlet within the genome and be accompanied by construction of a plasmid standard for quantitating copy number of the unknown target within a sample. Such a design will benefit strongly from the comparative ortholog analysis described above. Begin in situ cultivations of CLso in parallel with those performed with CLas. We envision that these experiments will yield similar information driving hypothesized medium conditions for CLso without requiring large outlays of resources. PoP microenvironment characterization for CLso habitats. In a manner similar to that done for the Asian citrus psyllid (ACP), we suggest pH, DO, and ORP be characterized for individual PoP insects. As systems biology approaches currently being employed for CLas involve multiple 'omic techniques and can be very expensive, we do not recommend this level of analysis for the potato or PoP CLso habitats without additional external support. We feel that this level of investment at this stage may significantly hinder parallel progress being made on the CLas/ACP system. We propose that major effort be centered upon generating a robust comparative analysis of the genomes of members of genus Liberibacter, in the context of its sole cultivated representative, L. crescens. This will provide equivalency in analysis across all of the available genomes and allow evidence-based estimation of overall annotation quality and the probability that any given annotation is correct. Specifically, we suggest that all available Liberibacter genomes be subjected to identical gene model prediction techniques and comprehensive evidence generation from multiple independent approaches. With this robust genome analysis in hand, apples-to-apples genome comparisons can be made to identify differences between members of the genus and much higher-confidence in silico metabolic models can be constructed. These metabolic models can then serve as tools for modeling approaches. Making comparative genomic analyses and computational models available to other research teams would have value far beyond the scope of this project. We consider that the most significant experiments to be performed in the next three months are the following: Demonstrate passage-to-passage transfer of CLas-positive cultures, if possible. This can either occur within leaf disc cultures or in vitro - the important aspect being to demonstrate serial cultivation of a CLas-containing enrichment. We suggest that initial inocula be derived from infected psyllids, as the burden is significantly higher, and the number of potential competitor/contaminant species limited. The aforementioned comparative genomic analysis of genus Liberibacter should be performed, generating new hypotheses for the next proposal. Initial design of CLso detection systems parallel to Omsland lab strategies (single copy, selection of a gene with narrow phylogenetic range) for CLas detection should be completed, culminating in proof-of-concept tests that can be included in the proposal as preliminary data supporting a robust CLso experimental strategy. If possible, microelectrode profiling of live potato psyllids to identify DO and pH gradients. Additionally, upon receiving permission for insect work at WSU, profiling of CLas-infected psyllids for DO and pH to determine if these physicochemical parameters change upon infection. This experiment may be involved and require significant time investment, and so it is unclear to us whether it is feasible to perform this work before June 1. In our view, emphasis should be placed upon the following activities for the rest of the year: Identifying medium supplements that improve the growth of CLas in planta in leaf disc experiments and in defined psyllid feeding experiments. These should be hypothesis-driven in so far as they are possible and may illustrate commonalities in growth mode across multiple host organisms (e.g., possible evidence of psyllid nitrogen limitation in the presence of CLas may explain limitations in yield for CLas growth in planta). Establishing solid genomic bases for multi-omic studies of ternary interactions between host, pathogen, and commensals. This should initially be accomplished by cleaning up the comparative genomics of genus Liberibacter, as originally discussed. However, it should also extend into generation of well-annotated references (in comparison with other members of the genus, insofar as it is possible) for endosymbiont strains (e.g. Wolbachia spp, Carsonella sp.) which incorporates genome information both generated through this project and from other sources. This resource, coupled with improved psyllid genomes being developed, will allow comprehensive evaluation of each organism's response to CLas infection, and may uncover very important interspecies metabolic interactions that may be important to mimic in cultivation or may require co-isolation of endosymbionts, at least initially. Defined feeding experiments for ACP to identify impacts upon 1) CLas burden and 2) psyllid, CLas, and endosymbiont responses (via transcriptomics, metabolomics) to different nutrient conditions. These experiments may identify key limiting nutrients that restrict CLas' ability to proliferate in vivo, which are key candidates for essential nutrients that need to be provided in vitro. Use of the nanosampler method to harvest highly-pure hemolymph samples for inoculation of 1) uninfected leaf discs and 2) fully host-free enrichments. <br><br>
<br>What was accomplished under these goals? Development of a method to culture Candidatus Liberibacter asiaticus (CLas) will have a very large impact, indeed it will be a game change, on our ability to fight this devastating disease. At the present time, potential therapies (antimicrobials, other means to kill CLas) cannot be tested directly on the bacteria. This project is developing such culturing strategies, approaches and methods. The project has great early success that indicates we are well on our way to development of new culturing strategies that will be effective in growing CLas in vitro, thereby enabling all of the technologies needed to finally and successfully fight HLB. To date, significant findings and accomplishments include: 1) a better understanding of the microenvironment that the bacteria live in within the psyllid insect host, 2) the ability to grow the CLas bacteria through several culture transfers in media based on an analysis of the genome sequence of CLas relative to other bacteria and the host organisms, 3) a method that allows for growth of the bacteria in host tissues maintained in vitro, 4) infection of non-host eukaryotic cells with CLas and evidence for replication/growth within those cells. These major results were attainable through many smaller efforts, which are outlined below. OVERALL PROJECT DEVELOPMENT AND SUPPORT The Gang and Killiny Labs work with the other labs and provides logistical/chemical analysis support for all aspects of the project. Central to this role are significant efforts to better understand the metabolism of CLas relative to its hosts and associated endosymbiont neighbors (Ca. Proftella and Ca. Carsonella species) as well as ACP-associated Wolbachia species. Develop methods for psyllid metabolomic, lipidomic and proteomic analysis. We can routinely quantify ~1000 proteins and hundreds of metabolites and lipids in ACP samples. Differences between uninfected and infected psyllids are readily detectable. We have also developed MALDI-MS tissue imaging methods to analyze individual insects, leading to identification of specific compounds associated with Liberibacter infection. A lipid, dioleoyl phosphatidylcholine, is an example of a specific metabolite in ACP that was found mainly in the abdomen (gut) of the infected psyllid. This information is important for the project because it helps to verify or indicate which parts of metabolism are affected by CLas infection (metabolism in either the insect or bacterium), which information will be very helpful in understanding what components of metabolism are not functioning in CLas and therefore must be supplied either by the host in vivo or by us in artificial medium in vitro. Metagenomic analysis of endosymbionts in ACP. Driven by the hypothesis that ACP's endosymbionts may contribute to CLas growth and infection within ACP, we performed metagenomic sequencing and found that Candidatus profftella, Candidatus carsonella and Wolbachia are the main non-CLas bacteria present in ACP. We are currently working on metabolic network reconstruction of this microbial community based on the endosymbionts' genome sequences, which provide information about gene, protein and metabolic pathway differences among these bacteria as well as the hosts ACP and citrus. Such information has already informed initial culturing efforts (see below). OBJECTIVE 1 The Beyenal Lab is responsible for most of the work related to Objective 1, the establishment of a system for culture of CLas-containing biofilms. Characterize the physiochemical conditions inside ACP. To design the culture medium to grow CLas biofilm and free living/planktonic CLas, it is crucial to understand its growth conditions in the host. Since CLas replicates inside of the ACP body, determining oxygen levels and pH in the medium surrounding CLas in ACP's body will be helpful to design the culture medium and conditions for culturing CLas. For this goal, we used microelectrodes with a tip diameter of less than 20 µm to measure the local oxygen level and pH inside of the psyllid's abdomen. Measurements were conducted on multiple psyllids and suggested that aerobic conditions exist within the psyllid abdomen with alkaline pH conditions (7.5 to 9.0). These results suggested that oxygen supply and alkaline pH could be needed for CLas biofilm culture. Design media and examine the growth of CLas in mixed microbial cultures. Multiple media were designed based on (1) CLas genome information, (2) chemical composition of phloem sap, and (3) growth conditions of insect tissue. Due to the difficulties to obtain live ACP as the inoculum in Washington State, we initially started with infected citrus plants as CLas inoculum for our CLas-biofilm culture. All microorganisms (not just CLas) in the CLas-infected citrus were extracted and inoculated into the tested media. Our initial data suggested that CLas is able to replicate in the media in the presence of its microbial community. We have established culture media that can sustain the CLas cells for several transfers (so far tested up to 4). OBJECTIVE 2 The Omsland Lab is responsible for most of the work related to Objective 2, the establishment of a system for host cell-free culture of CLas. Evidence for CLas infectivity of or co-culture with cultured host cells. Based on the possibility that CLas could replicate intracellularly or rely on attachment to a host cell for replication, we have explored the ability of CLas to be cultured in co-culture with Vero (green monkey kidney epithelial) cells. Vero cells have been used to isolate parasitic bacteria and viruses for decades due to their poor innate immune functions, thus increasing the likelihood that parasites can exploit this cell type as a host. Non-infected or CLas-infected plant tissue was homogenized and the homogenate used to inoculate Vero cells. Fluorescence In Situ Hybridization (FISH) was used to detect CLas based on FISH probes designed to detect CLas 16S rDNA or the gene nttA. Vero cells inoculated with CLas-positive homogenates showed positive FISH results, which are currently being verified using qPCR. Establishment of leaf-disc cultures to study CLas metabolic responses in situ, and. Because CLas naturally replicates in citrus leaves, the leaves logically contain all nutrients CLas requires for replication. To identify physicochemical conditions (e.g., pH and dissolved oxygen, see above) and nutrients that can trigger or stimulate CLas replication, we have established a method for using leaf discs excised from CLas-infected citrus to probe such responses. By incubating leaf discs from CLas-infected plants under specific conditions, qPCR can be used to measure responses in CLas DNA replication as an indicator of conditions that stimulate CLas cell division. Quantification of CLas DNA in leaf discs incubated in the presence of different glucose concentrations serve as proof-of-principle for our "leaf-disc method" and preliminary data for the identification of glucose as a physiologically relevant carbon source for CLas. We are currently screening other conditions and nutrient combinations. Development of a robust protocol for quantification of CLas. Current methods for enumerating CLas in infected tissues are largely based on detection of CLas 16S rDNA. While fairly specific, PCR primers designed to detect 16S rDNA have a relatively high likelihood of binding to non-CLas sequences. Therefore, we have established a protocol based on detection a conserved hypothetical gene that appears to be limited to different strains of Liberibacter asiaticus. Importantly, our protocol can be used to detect CLas in tissues provided by Co-PI N. Killiny demonstrating that the CLas strain(s) used by the Killiny Lab can be effectively detected and enumerated using this protocol. OBJECTIVE 3 Efforts under this Objective will begin once culturing methods have been developed. <br><br><b>Publications</b><br>