Progress 09/15/23 to 09/14/24

Outputs
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. What opportunities for training and professional development has the project provided?Clebson Tavares acquired important technical skills that included DNA extraction using different methods, cell culturing, and qPCR analysis. How have the results been disseminated to communities of interest?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. What do you plan to do during the next reporting period to accomplish the goals? 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.

Impacts
What was accomplished under these goals? Accomplishments: Objective 1. CLas viability in ACP cell culture medium optimized over 48 hours. Objective 2.Three continuous ACP cell lines (i.e. Dici1, Dici3, and Dici5) have been established and passaged more than 40 times each. CLas replication in Dici1 and Dici 3 cells over 4 days and Dici1 over several passages was documented. Majoractivities completed and experiments conducted for each objective: Objective 1 1.1. 1 CLas extraction from dissected guts of CLas-infected psyllids.Asian citrus psyllids were collected from CLas-infected plants reared in a greenhouse culture ofValencia sweet orange (Citrus sinensis(L.) Osbeck). Before gut dissection in 1x PBS, psyllids were disinfected with 70% ethanol for at least 30 seconds followed by water wash. Guts were dissected under a surface cleaned microscope and transferred to 1.5 mL microcentrifuge tubes. Upon completion of dissection, the guts were washed thrice with PBS followed by a brief centrifugation. Guts were then resuspended with ACP cell culture medium (no antibiotic included) and homogenized in 1 mL Wheaton homogenizer in a cell culture hood. Homogenate was then centrifuged at 1000 x g for 2 minutes; the supernatant was transferred to a new 1.5 mL microcentrifuge tube and CLas re-extracted from the pellet. 1.2 Viability of CLas in ACP cell culture medium. Gut homogenate from guts of ~70 CLas-infected psyllids was split into nine aliquots, placed in 2 mL microcentrifuge tubes (~70 µL), and incubated at 28 °C with 150 rpm shaking. Three microcentrifuge tubes were taken after 3, 24, and 48 h and immediately treated with propidium monoazide (PMATM, Biotium) following the manufacturer's recommendation. PMA is a dye that penetrates the cell membrane of dead cells (but not live cells), binding to DNA and rendering it unavailable for qPCR following exposure to blue light (465-475 nm wavelengths). The following treatments were tested for each time point, followed by incubation in the dark for 10 minutes and exposure to blue light for 15 minutes: i. Gut homogenate + PMA + light exposure, ii. Gut homogenate + light exposure, and iii) gut homogenate (90 °C/5 min) + PMA + light exposure. After light exposure, CLas was pelleted by centrifugation at 13,000 x g for 5 minutes, followed by a wash with 1X PBS. Pellets were resuspended with 20 µL of DNA elution buffer, boiled at 100 °C for 10 minutes, and a 2 µL sample was used for qPCR. Objective 2 2.1. Characterization of ACP Cell lines. All three cell lines were characterized morphologically. The transcriptomes of Dici1 and Dici3 have been sequenced, characterized. The putative origins of the cell lines were identified, as were the microorganisms infecting these cell lines.PCR/ sequencing and immunohistochemistry were then used to validate the identities of microorganisms infecting all three cell lines. CLas inoculation of Dici1 and Dici3 cells. CLas was extracted from dissected guts as previously described and used to inoculate (homogenate from ~ 10 guts) Dici1 and Dici3 cells seeded in 24-well plates with ~70% confluency. An equal volume of gut homogenate was stored at -30 °C for DNA extraction. Following inoculation, the 24-well plate was centrifuged at 1,500 x g for 10 minutes to bring CLas into close contact with the attached Dici cells. The CLas-containing medium was removed after 24 hours, the wells were washed with 1X PBS and fresh medium was added. Dici1 and Dici3 cells from CLas inoculated or control treatments were collected at 2, 3, and 4 days. Collected cells were washed with 1X PBS and stored at -30 °C. DNA was extracted using the Cetyltrimethylammonium bromide (CTAB) method and CLas was detected by qPCR. 2.2. CLas inoculation of Dici1 cells and replication over several cell passages.Three continuous ACP cell lines (i.e. Dici1, Dici3, and Dici5) have been established and passaged more than 40 times each.To evaluate whether CLas can persist and replicate in Dici cells, we inoculated Dici1 cells (~60% confluent) with CLas-containing gut homogenate (described in 2.1) followed by CLas detection over several passages. Fos this assay, we treated the CLas-containing gut homogenate with lipofectamine 3000 (according to manual instruction, Invitrogen) in an attempt to increase CLas entry into Dici1 cells, as this was previously shown to increase infectivity of a bacteria species in cell culture (Li et al., 2020). The negative control comprised of lipofectamine reagent without lipofectamine 3000. A total of 9 wells/treatment were seeded with Dici1 cells: 3 wells for cell collection at 2 days, 3 wells for cell collection at 6 days; cells in the remaining 3 wells were combined and passaged to a T12.5 flask (P1). At each passage (~90% confluency), half of the cells collected was used for DNA extraction and the other half was transferred to a new T12.5 flask. The medium at every collection time was collected and centrifuged at 13,000 x g, followed by DNA extraction through boiling and qPCR for detection of CLas in the medium. DNA from the Dici cells was extracted using the CTAB method followed by qPCR as described by Li et al. (2006). Data collected for each objective. Objective 1 Viability of CLas in ACP cell culture medium. The increase in Ct values of CLas+PMA treatment shows that CLas do not replicate in ACP cell culture medium over 48 hours, but remain mostly viably over the same period based on the small difference between CLas+noPMA and CLas+PMA treatments (Figure 1). As expected, the Ct values of boiled gut homogenate (CLas dead cells) were greater than the other treatments at all time points, suggesting that PMA binds to DNA of dead cells and decreases its availability for qPCR. Objective 2 Cell characterization.ACP cell lines are composed of morphologically diverse cells, with Dici1 and Dici5 mostly consisting of fibroblast/neuron-like cells and Dici3 consisting of fibroblast/neuron-like and epithelial-like cells. CLas inoculation of Dici1 and Dici3 cells.Both Dici1 and Dici3 cells were inoculated with gut homogenate from ~10 guts,which resulted in a Ct value of 28.22. Using the standard curve for CLas quantification by qPCR previously established in our lab (Y = -3.601X + 46.09), we estimate the copy number of CLas DNA used in the Dici cell inoculation at 1.37x106. Cells inoculated with CLas-containing gut homogenatehad lower Ct values than the control at all time points, suggesting that CLas enters both Dici1 and Dici3 cells. The slight increase in the Ct values in the Dici cells inoculated with CLas indicates that CLas did not replicate in Dici cells over the 4 days following inoculation. No apparent change in cell morphology was observed between CLas inoculated and non-inoculated cells. CLas inoculation of Dici1 cells and replication over several cell passages.To test whether CLas can persist and replicate in Dici cells over longer time scales, we inoculated Dici1 cells and performed qPCR for CLas detection over several passages. The Ct values of Dici1 cells inoculated with CLas were ~ 32 and 30 at 2 and 6 days, respectively while the negative control were around 36. However, once the cells were passaged to a T12.5 flask, the Ct values of inoculated cells significantly increased, becoming similar to that of non-inoculated cells. These results suggest that CLas enter Dici1 cells but does not persist or replicate over the reported time.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Wu, Ke., Vu, E., Ghosh, S., Mishra, R. & Bonning, B.C. Continuous cell lines derived from the Asian citrus psyllid, Diaphorina citri (Liviidae: Hemiptera) harbor viruses and Wolbachia.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Wu, K., Ortgiesen, G. J., Goodman, C. L. & Bonning, B. C. Optimized conditions for the long-term growth of primary cell cultures derived from the Asian citrus psyllid, Diaphorina citri (Liviidae: Hemiptera). In Vitro Cell Dev Biol Anim 59, 235240 (2023).
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Oral presentation: 1. Vu, E., Wu, K., Ghosh, S. & Bonning, B.C. Continuous Asian citrus psyllid cell lines: viruses and Wolbachia. 2024 International Congress on Invertebrate Pathology and Microbial Control and the 56th Annual Meeting of the Society for Invertebrate Pathology (2024).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Wu, K., G. J., Goodman, C. L. & Bonning, B. C. Continuous cell lines derived from Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). International Research Conference on Huanglongbing VII (2024).

Progress 09/15/22 to 09/14/23

Outputs
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. What opportunities for training and professional development has the project provided?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. How have the results been disseminated to communities of interest?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. What do you plan to do during the next reporting period to accomplish the goals?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.

Impacts
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.

Publications