Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 999 - Citrus, general/other | 1130 - Entomology and acarology | 80% |
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 20% |
Candidatus Liberibacter asiaticus (CLas), is the presumed causative agent of citrus greening, which has devastated citrus production in Florida and now threatens all citrus growing regions in the U.S., including Texas and California. There is currently no cure or durable remedy to combat citrus greening. While the ability to culture CLas in vitro would provide huge benefits for analysis of CLas biology and for fulfillment of Koch's postulates to confirm that CLas causes citrus greening, attempts to culture CLas in the absence of other bacteria have failed. Our long-term goal is to provide a reliable, insect cell culture-based method for culturing of CLas bacteria.The goal of this proposal is to identify an ACP cell culture system for in vitro culture of CLas. Our objectives are: 1) test for CLas replication in axenic culture using optimized insect cell culture media, 2) assess hemipteran insect cell culture systems for CLas replication, and 3) establish cell lines from CLas-positive psyllids. Once a CLas culture is established, we will test for ACP transmission of cultured CLas to healthy citrus, and whether inoculation of healthy citrus results in citrus greening disease.Upon completion of this project, we will have a culture system that serves as an essential research tool for increased understanding of CLas biology and for effective, rapid screening of antimicrobial agents against CLas. Outreach activities will facilitate public understanding and grower adoption of antimicrobial strategies for CLas management.
Our long term goal is to delineate CLas molecular interactions with the psyllid vector. The objective of this project is to assess the utility of improved insect cell culture systems for culturing of CLas. Our central hypothesis is that optimized ACP cell culture systems can be exploited for in vitro culture of CLas. The objectives for the proposed research are:
Once a CLas culture is established, we will test for ACP transmission of cultured CLas to healthy citrus, and whether inoculation of healthy citrus results in citrus greening disease.Upon completion of this project, we will have a culture system that serves as an essential research tool for increased understanding of CLas biology and for effective, rapid screening of antimicrobial agents against CLas. Outreach activities will facilitate public understanding and grower adoption of antimicrobial strategies for CLas management.
?Asian citrus psyllid adults will be collected from CLas-infected plants reared in a greenhouse culture of Valencia sweet orange.Isolation of CLas from ACP midguts will be conducted as described by Dale et al. (2006) for isolation of insect endosymbionts.We will test CLas for resistance to antibiotics.Briefly,eight antibiotics (ampicillin, chloramphenicol, kanamycin, oxytetracycline, polymyxin B, rifampicin, streptomycin, and tetracycline) will be tested at three concentrations (50, 500, and 1000 ppm) in culturemedium as described by Fujiwara et al (2018).Prior studies demonstrated resistance to oxytetracycline fordifferent CLas isolates (Fagen 2014 L.c. gen.nov; Fujiwara 2018). Characterization of the antibiotic resistance of CLas isolated from Florida ACP, will facilitate production of CLas-only cultures.Having recently screened 10 different insect cell culture media for their ability to support the survival and replication of ACP-derived primary cell cultures, we have identified Shields and Sang, and CLG#2 media supplemented with 9% FBS as optimal for cell attachment and replication. We will test these two media and a 1:1 combination of the two media (GSS medium) for their ability to support CLas replication. Experiments will initially be conducted at 28ºC, the optimal temperature for insect cell growth, but temperatures of 29 and 30ºC will also be assessed, based on increased growth rate of CLso at these temperatures in axenic culture (Sena-Velez 2019).We will build on prior work demonstrating improved performance of Liberibacter (CLas or CLso) in culture, by testing supplements shown to positively impact in vitro growth. We will assess the potential benefit of ammonium chlorideand ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer to avoid the alkalinization shown to be detrimental to CLso growth (Sena-Velez 2019). We will also assess the use of α-ketoglutaric acid (αkg) as a carbon source, choline chloride and citrate supplementation. These reagents contributed to improved recovery of CLso from culture (Sena-Velez 2019; Cruz-Munoz, 2018). We will also assess the use of citrus juice, shown to prolong CLas viability in culture (Parker 2014).We will test primary ACP embryonic cultures generated for their ability to support CLas replication. CLas prepared as described in 1.2.1 will be added to ACP primary cell cultures and cultures observed daily using a phase contrast microscope for survival of ACP cells and for bacterial growth, potentially from co-introduced bacteria. Viability of CLas will be tested using a LIVE/DEADBacLightBacterial Viability Kit (ThermoFisher). Addition of CLas to primary cultures may result in three distinct outcomes: 1) ACP cells die due to CLas replication in the culture medium, exhausting nutrients required by the cells, 2) ACP cell growth is reduced or stopped due to infection with CLas, 3) ACP cells continue to grow; CLas does not replicate. Aliquots of cell culture medium will be collected daily for assessment of CLas abundance by ethiduium monoazide (EMA) treatment (Foliminova et al. 2010) and qPCR with the HLBas/HLBr/HLBp primer and TaqMan probe as previously described (Li et al. 2006). Sequencing of 16S RNA will be used for identification of bacteria present (Parker 2014). Any of the ACP cell cultures that test positive for CLas by qPCR will be visualized and photographed in a Morgani 268transmission electron microscope (TEM) equipped with an AMT digital camera (Andrade et al. 2019).While we expect that primary ACP cell cultures will eventually result in continuous ACP cell lines, this process can take many months. A total of 40 or more passages are typically required before a cell line can be considered "continuous". To fast-track this process, we will use thec-mycproto-oncogene to generate a continuous ACP cell line from ACP primary embryonic cultures. Primary cell cultures will be transfected with the plasmid pcDNA3c-myc using lipofectin transfection reagent for introduction ofc-mycinto the cell genomic DNA. This plasmid also carries a neomycin resistance gene allowing for selection of transformed cells as described previously (Kitagishi 2011).Once actively dividing cells are acquired, PCR will be used to confirm integration of the gene. PCR amplification and sequencing of the COI gene will be used to confirm the identity of resulting cell populations. Once immortalized ACP cells have been acquired and amplified, CLas replication will be assessed in the immortalized ACP cell line.Wewill continue to maintain and establish new primary cell cultures, with continuous exposure to the mutagen, nickel sulphate at5 µg/ml as described in Preliminary Data. While use of this approach resulted in phenotypically altered and immortalized human embryonic kidney cells in 70-100 days(Tveito et al., 1989), the process may take longer given the relatively slow division rate observed for ACP primary cell cultures.We will test three continuous hemipteran cell lines, described previously, for their ability to support CLas replication: DmII, AC20, and AtE. While CLas is likely to replicate the best in ACP cell lines, testing of these continuous hemipteran cell lines for their ability to support CLas replication is worthwhile. Even low rates of replication would provide a valuable resource as the continuous ACP cell lines are being developed.We will use the methods that we have optimized for establishment of ACP primary cell cultures for establishment of primary embryonic cell cultures from CLas-infected ACP. Both eggs and ovaries will be used for establishment of primary cell cultures.Our current system results in 50% of cultures derived from 100 eggs / embryos yielding a dividing colony of cells within a period of two months (Preliminary Data). Given that CLas is detected in 3 to 6% of ACP eggs (Pelz-Stelinski et al2010), we will generate >20-fold more primary ACP cell cultures than completed for establishment of the ACP cell line to optimize chances for success. After 6 months, primary cultures will be tested for the presence of CLas by PCR. Positive cultures will be visualized and photographed using TEM.On acquisition of primary cell cultures harboring CLas, we will use thec-mycproto-oncogene and / or nickel sulphate-mediated mutagenesis to generate immortalized cell lines.
Target Audience
The target audience includes citrus growers throughout the United States (Florida, Texas, and California), the agro-chemical industry focused on citrus production,and consumers of citrus products. We engaged with the target audience through extension and outreach presentations at industry meetings.
Changes / Problems
Technical challenges with cultures, including delays in inoculating insect cultures due to the unavailability of sufficient inoculum and initial low viability of cell lines caused delay in optimizing and confirming replication for multiple passages (objective 2 and culturing cells from CLas-positive psyllids in objective 3.
Training & Professional Development
Clebson Tavares acquired important technical skills that included DNA extraction using different methods, cell culturing, and qPCR analysis.
Dissemination Streams
A manuscript was published, and another one was submitted. Multiple presentations were made at professional conferences. Extension materials with results have been shared with growers stakeholders.
Next Reporting Steps
The ACP cell lines will be maintained, and the culturing conditions will be further optimized to ensure they are continuously available for CLas study. Microscopy will be conducted to visulize cells with CLas to confirm host status. Test for CLas replication in Dici3 and Dici5 cell lines over several passages. Continue assessing CLas replication in the inoculated Dici1 cells for more passages. Test for CLas replication in ACP cell culture medium for an extended period. Inoculate Dici cells with a greater copy number of CLas DNA and perform multiple inoculations over time in an attempt to increase the ratio of CLas infected cells.
Target Audience
The target audience includes citrus growers throughout the United States (Florida, Texas, and California), the agro-chemical industry focused on citrus production,and consumers of citrus products. We engaged with the target audience through extension and outreach presentations at industry meetings.
Changes / Problems
Cells treated with NiSO4displayed signs of cellular stress and even experienced elevated cell death when compared to untreated controls. Surviving cells were able to recover only after NiSO4was removed from the medium, suggesting that NiSO4 at 5 µg/mL is toxic to ACP cells. Therefore, all NiSO4-treated ACP cultures were eventually reared in CLG#2 only medium. Infection of cell lines with CLas was delayed due to supply difficiulties. The protocol for the extraction of CLas requires centrifuges tubes with 0.58 um filter membranes. These filters were unavailable for five months during the middle of the year. Filters were received in September, enabling progress on CLas extraction and cell infection to continue.
Training & Professional Development
Experienced technical staff have engaged in hands on )one-on-one) traing with a technical staff member, one graduate student, and one postdoctoral scientist. Training conssted of techniques for cuturing cells to facilitate rapid screeng of cell lines concurrently.
Dissemination Streams
Both written and oral updates of the cell cultures investigated in this project havehave beendelivered to the Florida citrus industry dueing a series of outreach presentations and workshops , including the FLorida Citrus Expo. We have used the Science for Citrus Health (SCH) website (https://ucanr.edu/sites/scienceforcitrushealth/) as our main web-based medium for disseminating new information related to culturing CLas in an ACP cell line. The SCH team meetsbi-monthly to discuss the next tasks relevant to the goals of the group.The SCHteam (current lead members: P. Lemaux, L.L. Stelinski, and Ed Stover) is comprised of outreach specialists, postdocs and graduate students. The group produces outreach/extension documents and organizes/delivers events. To date, we have completed the webinar portion of the cell culture projectcontinue to work ondeveloping a podcast for the website. During the last quarter we published an information flier about the Science for Citrus health website on the California Research Board E-News mailer and the Florida Citrus Industry magazine newsletter to increase grower traffic to the site. We also published a new snapshoton the website.
Next Reporting Steps
Future Work: Over the next year, our primary focus will be on maintaining and establishing continuous cell lines for our research. This involves continuing to passage all cell lines until we successfully establish one or more continuous lines. To ensure the preservation of promising cell lines, we will maintain multiple backups that are passaged on a staggered schedule. Additionally, we will create frozen cell stocks every 5 passages to enable quick recovery in case of any unexpected setbacks. In parallel, we will complete the transcriptomic study of Dici1 and Dici3, which is a significant part of our ongoing research efforts. In the coming year, we will prepare a manuscript detailing the creation of ACP cell lines. Furthermore, we will provide cell cultures to partners and collaboratorsto study CLas-ACP cell interactions, ACP virus replication, and pesticidal proteins. <br><br>
<br>What was accomplished under these goals? We tested the ability of ACP primary cultures to support CLas replication. We fast-tracked the production of a continuous ACP cell line by 1) transforming primary cell cultures with the c-myc proto-oncogene, and 2) continuously exposing cultures to the mutagen nickel sulfate. We also tested whether three additional hemipteran cell lines (AC20, AtE, and DMII) supported CLas replication. We hypothesized that primary and continuous ACP cell cultures would support CLas replication. Generate an immortalized ACP cell line using c-myc proto-oncogene To generate an immortalized ACP cell line using c-myc proto-oncogene, we followed a previously described protocol that showed immortalization of a honey bee cell line after transfection by a plasmid (i.e., pcDNA-cmyc) encoding human c-myc proto-oncogene(Kitagishi et al. 2011). Approximately 3 × 150 0-3 days old ACP eggs were used to produce three primary cultures in T12.5 flasks containing CLG#2 medium using methods described in the original proposal. Two days later each culture produced approximately 300 cell clusters, with each cluster containing approximately 20 cells (i.e., ~ 6,000 cells/flask). These three cultures received three different treatments. One primary culture was used as an untreated control; the other two were transfected with 3.25 µg of pcDNA3-cmyc plasmid complexed with Lipofectamine 3000 at either the high or the low ratios as recommended by the manufacturer (Invitrogen). One day after transfection, G418, an antibiotic that specifically selects transfected cells, was added to the medium to achieve a final concentration of 200 µg/mL. This antibiotic would be present in all subsequent media. The cells in all three cultures gradually died in the following months. There were no surviving cells in any of the transfected cultures 63 days later. There are at least two possibilities that might account for the lack of G418-resistant ACP cells. Firstly, the transfection efficiency might have been too low to generate G418-resistant cells. Secondly, the promoter (i.e., SV40 promoter) driving the G418 resistance gene may not function in ACP cells. Because determining the exact causes of the negative results would require significant resources and some ACP primary cultures reared in CLG#2 showed promise, we decided to focus on producing more primary cultures and screening those to find fast-growing cultures that could be passaged later. We produced 100 primary ACP cultures within 12 months using approximately 12,000 0-3 days old ACP eggs. The CLG#2 medium was better at supporting the long-term growth of ACP cells than any other media tested, including Shields and Sang(Wu et al. 2023). Eleven primary cultures reared in CLG#2 medium reached near confluency within 7.5-15 months and were passaged to new flasks. Subsequent passaging was performed whenever a culture reached ~ 80% confluency. Five of eleven cell lines are promising, having exceeded passage number 10 by early August 2023. Of these five lines, Dici3 appears to be the most promising one with an estimated cell doubling time of 168 hours. Six additional cell lines that are passaged monthly or every 2-3 months have lower passage numbers and thus are not listed here. Cells in different cell lines exhibit diverse morphology. Cells in the adherent cell lines share some similarities in that they consist of differentiated and round, less differentiated cells that usually aggregate together. Importantly, many differentiated cells in these three lines show distinct morphologies. Most cells in Dici1 are spindle-shaped or stellate-shaped cells with fine, elongated processes, resembling fibroblasts. In contrast, many cells in Dici3 and Dici6 have more rectangular cell bodies and lack elongated processes. Other cells in Dici3 have a more fibroblast-like morphology. By comparison, most cells in the Dici2 line exist as suspended clumps of undifferentiated cells. The fact that the cells show distinct morphology and growth rates in different lines suggests they may have different origins. This could be beneficial in two ways. Firstly, it would increase the chance of obtaining a continuous cell line. Secondly, it would potentially facilitate the study of CLas-ACP cell interaction because it is likely that only certain types of cells (e.g., gut epithelial cells) contain receptors for CLas. These cell lines provide us with the ability to screen for the best one that promotes CLas entry/ replication. TheDici1 line crashed at passages 12, 18, 22, and 23 in the hands of four people in two different labs that have been maintaining the line.The exact causes of the crash remain unknown, although it is hypothesized that cellular stress, possibly related to reovirus, may have caused the crash.Measures to reduce stress and prevent crashes include the use of the prewarmed medium/solutions and only passaging the cells in the active phase in which the confluency of differentiated cells reaches at least 80%, which may take up to three weeks.No major crashes have occurred since the modified passaging method was adopted on a backup Dici1 culture. In the upcoming funding year, we will continue to maintain and passage existing ACP cell lines to obtain a continuous line. We will also regularly make frozen cell stocks (~ every 5 passages). Of note, a frozen stock of Dici1 was successfully revived, suggesting that ACP cells can be readily revived if necessary. This is important because it will allow us to continue investigating CLas-ACP cell interactions even if continuous lines are unavailable. Generate an immortalized ACP cell line using nickel sulphate Ten ACP primary cultures in CLG#2 medium were treated with NiSO4(5 µg/mL) following a protocol previously used to immortalize human kidney epithelial cells(Tveito et al. 1989). No difference in cell growth or morphology was observed between cultures treated with NiSO4and untreated cultures for the first 2-3 months. Then, cells treated with NiSO4displayed signs of cellular stress and even experienced elevated cell death when compared to untreated controls. Surviving cells were able to recover only after NiSO4was removed from the medium, suggesting that NiSO4 at 5 µg/mL is toxic to ACP cells. Therefore, all NiSO4-treated ACP cultures were eventually reared in CLG#2 only medium. Summary of Accomplishments: Forty-one primary cultures wereestablishedin CLG#2byOctober 2022. Eleven Asian citrus psyllid cell lines were created between October 2022 and May 2023.Some primary cultures were pooled together for their first passaging due to low confluency.Five cell lines have exceeded passage number 10. Most cell lines are composed of adherent cells with diverse morphology. Eight promising cell lines, with 6 over passage 10, were identified with estimated cell doubling times of 7 to 14 days. Dici1a and Dici3 are the current front runners. Dici3 is the most consistently replicating line with an estimated cell doubling time of 168 hours. Maintaining existing lines and screening of fast-growing cell lines is ongoing. <br><br><b>Publications</b><br>