Source: UNIVERSITY OF FLORIDA submitted to NRP
IDENTIFICATION AND CHARACTERIZATION OF A REGULON INVOLVED IN THE PERSISTENCE OF CANDIDATUS LIBERIBACTER ASIATICUS IN CITRUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012528
Grant No.
2017-67013-26531
Cumulative Award Amt.
$863,489.00
Proposal No.
2017-03060
Multistate No.
(N/A)
Project Start Date
May 15, 2017
Project End Date
May 14, 2022
Grant Year
2017
Program Code
[A1171]- Plant Biotic Interactions
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Microbiology and Cell Science
Non Technical Summary
Candidatus Liberibacter asiaticus (CLas), is a member of the alpha-proteobacteria group that has infected most of the citrus cultivars worldwide causing a disease called citrus greening. The devastating effects of this phloem restricted unculturable bacterium on all citrus varieties has caused a severe crisis in the industry, with no solution visible in the near future. As such, it is critical to further advance our understanding of CLas fundamental biology to facilitate the discovery of new and effective means to treat this recalcitrant disease. The long term goal of this project is to define and characterize the biological strategies used by CLas to evade defensive mechanisms and persist in the citrus host. As a step in that direction, the objective of this proposal is to characterize members of the LdtR regulon involved in peptidoglycan synthesis and remodeling. The presence of small molecules that decrease LdtR DNA binding in the cultivable Liberibacter crescens resulted in morphological changes impairing the bacteria tolerance to stress. We hypothesize that the transcriptional activation of peptidoglycan modification genes allows for the persistence of the pathogen in the citrus host. This hypothesis will be tested by (i) establishing the correlation between the expression of genes regulated by LdtR and the bacteria persistence in the citrus host, and by (ii) elucidating the biological consequences of LdtR-mediated regulation. With the completion of these objectives, the effects of peptidoglycan remodeling enzymes on the persistence in the host, will be thoroughly evaluated. The mechanisms to be studied are relevant in host-microbial interaction.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21209991100100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The long-term goal of this project is to define mechanisms used by Ca. L. asiaticus to evade and persist in the citrus host. As a step in this direction, the objective of this proposal is to identify and characterize members of the LdtR regulon involved in cell wall synthesis and modification.Objective 1. Establish the correlation between the expression of genes regulated by LdtR and the persistence of Ca. L. asiaticus in the citrus host. Based on our preliminary data, we hypothesize that the inactivation of LdtR with small molecules will result in decreased persistence in the citrus host.Objective 2. Elucidate the biological consequences of LdtR-mediated regulation. Based on our preliminary data, we hypothesize that LdtR acts as a global transcriptional activator of genes involved in peptidoglycan remodeling, cell division, and protein secretion, resulting in increased osmotic stress tolerance and evasion of host responses.
Project Methods
Objective 1. Establish the correlation between the expression of genes regulated by LdtR and the persistence of Ca. L. asiaticus in the citrus host. As a suitable method to culture Ca. L. asiaticus is not yet available, our approach, summarized in Figure 10 will be to:Determine the expression of genes regulated by LdtR in the citrus host during different stages of bacterial infection.Assess the viability and changes in gene expression of Ca. L. asiaticus in infected citrus plants upon treatment with small molecules that specifically inhibit the activity of LdtR.Determine the expression of ldtR in citrus plants during different stages of bacterial infection. In a recent study by Yan et al., 2013, the expression of ldtR mRNA was found to be up-regulated 5-fold in the leaves of infected sweet orange (Citrus sinensis) seedlings when compared to expression levels observed in infected psyllids (Diaphorina citri). We hypothesized that the LdtR regulon is differentially expressed during plant colonization as a requirement for active cell division, protein secretion, and/or peptidoglycan remodeling.Citrus sinensis "Valencia" inoculation. Nine month-old seedlings will be graft inoculated with budwood from infected trees. Prior to inoculation, the Ca. L. asiaticus infected source will be confirmed by PCR. Inoculated plants will be kept in a secure glass house approved by the US Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS).Determine the expression of genes regulated by LdtR during infection.Briefly, four leaves from each infected seedling (10 seedlings, 40 samples) will be collected every two months, for 8 months (T2, T4, T6 and T8, Fig. 11). The leaves will be processed immediately as we reported in Pagliai et al., 2014.Assess the viability of Ca. L. asiaticus and changes in gene expression in infected citrus plants, upon treatment with small molecules that specifically inhibit the activity of LdtR. In our laboratories we developed a quick assay using infected leaves of Citrus sinensis "Valencia" to determine the potential of the chemicals identified to affect the transcriptional activity of Ca. L. asiaticus (Pagliai et al., 2014).Treatment study. Benzbromarone will be tested at sub-inhibitory and inhibitory concentrations (as determined in vitro) for their potential to affect/eliminate Ca. L. asiaticus infection. The 17 month-old seedlings will be divided into 4 groups (10 seedlings per group) as follow: 1) control (vehicle, DMSO), 2) benzbromarone (1 mM / 0.604 mg/l) 3) benzbromarone (10 mM / 6.04 mg/l) and 4) benzbromarone (100 mM / 60.4 mg/l).Determination of Ca. L. asiaticus transcriptional activity. We will follow the active transcription of the ribosomal genes as an indication of metabolic activity. RNA extractions will be performed as described in 1.1.b. The cDNA will be synthesized using iScript cDNA synthesis kit (BioRad) following the manufacturer instructions. qRT-PCR will be performed using the Ca. L. asiaticus specific primers CQULA04F/CQULA04R (rplJ, (Wang et al., 2006) as well as rplL and 16S rRNA genes (Pagliai et al., 2014).Evaluate modifications in peptidoglycan composition upon treatment with chemicals and/or osmotic stress. Our recent data showed that in S. meliloti, mutations in Smc01768 resulted in shortened cells (mini-cells), while a similar phenotype could be induced in L. crescens by the addition of chemicals that inactivate the transcriptional regulator B488_10910 (Fig. 4, Pagliai et al., 2014).As described above, these experiments will be performed using L. crescens as a model organism since it is the closest culturable bacteria to Ca. L. asiaticus. To this end, L. crescens cells will be grown in presence or absence of 25 mM hexestrol, 50 µM phloretin, or 50 µM benzbromarone, at 26 ºC to early stationary phase (Pagliai et al., 2014). Osmotic stress conditions will be induced by the addition of 200 mM of sucrose or 150 mM NaCl to BM7 media (Pagliai et al., 2014).Purification of Peptidoglycan (PG). Cells will be harvested by centrifugation. The pellets will be washed once with 20 mM sodium acetate (pH 5.0), and resuspended in the same buffer on ice. Cells will be extracted for peptidoglycan as described by Dougherty (1985), and modified by Erbs et al. (2008). Briefly, the cells will be added dropwise to a solution of 10% sodium dodecyl sulfate (SDS), buffered with 20 mM acetate (pH 5.0), at 100 °C, and boiled for 30 min.Preparation of Muropeptides, HPLC, and LC-MS Analyses. The isolated PG will be degraded with muramidase mutanolysin (M1, Sigma-Aldrich), overnight at 37 ºC to generate a spectrum of muropeptides. The PG preparations will be analyzed for amino acid composition by GC-MS as described by Words (2000) and Xu et al.(1997).Live detection of peptidoglycan modifications upon chemical treatment. Live detection will be performed using fluorescent D-amino acids. This technique allows for the quick covalent incorporation and detection of fluorescently derivatized peptidoglycan components (Kuru et al., 2012). Using this approach, Siegrist et al. (2013) showed that the incorporation of D-aminoacid reporters in the peptidoglycan changed with the activity of L,D- transpeptidases in E. coli.Characterize the enzymatic activity of LdtP and its susceptibility to antibiotics.In most bacteria, D,D-transpeptidase activity is mediated by classical penicillin binding proteins (PBPs) which cross-link glycan chains by forming 4-3 peptide bonds connecting residues at the fourth and third positions of stem peptides. In contrast, L,D-transpeptidase activity forms the peptide bond at the third position of both stem peptides.Determination of L,D transpeptidase activity. The enzymatic activity will be determined by measuring the production of 3-3 cross-linked muropeptides. To establish the feasibility of this experiment, we have already purified Ca. L. asiaticus LdtP (Fig. 11). The muropeptides that will be used as substrates will be isolated from L. crescens sacculi as described in Cordillot et al. (2013).Effect of antibiotics on the LdtP activity. Recent reports on Ldt's from M. tuberculosis and E. faecium (Lecoq et al., 2013) have indicated a different sensitivity to B-lactam antibiotics. Since L,D transpeptidases show little sequence and structural conservation, we will determine the effect of imipenem, ceftriaxone, ampicillin, and streptomycin on LdtP enzymatic activity.Determine the effect of modifications in the peptidoglycan composition in the plant immune response. Citrus sinensis response to peptidoglycan. We will test that the observed changes in the plant immune response are directly related to modifications in the pathogen peptidoglycan. To this end, whole PG and the muropeptides (extracted in 2.1) from L. crescens will be tested for their ability to change PR1, CDR1, PDF2.2 and LYM2 gene expression. These host genes have been used successfully as molecular markers for the induction of plant immune responses (Bedini et al., 2005, Silipo et al., 2005; Albrecht and Bowman, 2012).Citrus sinensis response to chemical treatment. We will determine the expression of PR1, CDR1, PDF2.2, and LYM2 in all samples obtained in objective 1.

Progress 05/15/17 to 05/14/22

Outputs
Target Audience: Nothing Reported Changes/Problems:The extra year with not cost extension was important to complete the work proposed. Three articles were published with the results obtained after the pandemic finished. What opportunities for training and professional development has the project provided?The student Kaylie Padgett-Pagliai obtained her PhD in april of 2022 How have the results been disseminated to communities of interest?Three Scientific articles were published in scientific Journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Establish the correlation between the expression of genes regulated by LdtR and the persistence of Ca. L. asiaticus in the citrus host. Based on our preliminary data, we hypothesize that the inactivation of LdtR with small molecules will result in decreased persistence in the citrus host. We had demonstrated the importance of LdtR as a master regulator in Ca. L. asiaticus (Pagliai et al., 2018). This transcription factor is affected by zinc (Pagliai et al., 2018), controlling the expression of accessory regulatory protein like PrbP (Pan et al., 2019). The control of gene expression in response to the plant environment is probably one of the central biological roles of LdtR (Padgett Pagliai et al., 2022; Pan et al., 2021). This claim is based on several evidenced collected in our assays during this granting period. LdtR is regulating the expression of key genes encoding enzymes with proteolytic activity like Lon. This protease is an ATP dependent enzyme regulated (directly and indirectly) by this central transcription factor. The post translational regulation is controlled by direct protein-protein interaction with LotP. The expression of the LotP encoding gene is directly regulated by LdtR (Loto et al; 2017). We also provided direct evidence of the biological role of LotP by injecting purified protein into the tissues of healthy plants. The evidence collected suggested that LotP was able to interact with citrus proteins as well, probably with plastids encoded Lon proteases (Merli et al.,2021). Our conclusion is that the axis LdtR-LotP-Lon is critical for Ca. L. asiaticus to establish the infection and thrive in the phloem of the citrus tree. More important the effects of LotP can spread out the citrus greening toxicity beyond the physical location of phloem cells. Objective 2. Elucidate the biological consequences of LdtR-mediated regulation. Based on our preliminary data, we hypothesize that LdtR acts as a global transcriptional activator of genes involved in peptidoglycan remodeling, cell division, and protein secretion, resulting in increased osmotic stress tolerance and evasion of host responses. Structural modifications in the bacteria cell wall is a relevant characteristic to evade the host defensive mechanisms. The same enzymes modifying the peptidoglycan biochemistry can increase the ability of the bacteria to generate biofilms resisting to harsh conditions in the plant phloem (Pan et al., 2021). We collected evidence indicating that LdtR is central in coordinating the physiological abilities of Ca. L. asiaticus enhancing their ability to survive such conditions. We had purified and characterized the protein named LdtP, a DD-transpeptidase with esterase activity. The modifications of the cell wall envelope described in Coyle et al., 2018 could help the bacteria evade the plant defensive system. In summary, we had completed the objectives proposed describing several molecular mechanisms helping Ca. L asiaticus to be a successful citrus plant pathogen. Our contribution to better understanding this recalcitrant citrus pathogen help us to increase our basic knowledge toward the finding a definitive solution for the problem.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Padgett-Pagliai, K. A., Pagliai, F. A., da Silva, D. R., Gardner, C. L., Lorca, G. L., and Gonzalez, C. F. (2022). Osmotic stress induces long-term biofilm survival in Liberibacter crescens. BMC microbiology, 22(1), 1-16. PMID: 35148684
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Pan, L., Gardner, C.L., Beliakoff, R., Da Silva, D., Zuo, R., Pagliai, F.A., Padgett?Pagliai, K.A., Merli, M.L., Bahadiroglu, E., Gonzalez, C.F. and Lorca, G.L., (2021) PrbP modulates biofilm formation in Liberibacter crescens. Env Microbiol, 23(11), 7121-7138. PMID: 34431209
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Merli, M. L., Padgett-Pagliai, K. A., Cuaycal, A. E., Garcia, L., Marano, M. R., Lorca, G. L., and Gonzalez, C. F. (2021) Candidatus Liberibacter asiaticus Multimeric LotP Mediates Citrus sinensis Defense Response Activation. Front Microbiol, 12. PMID: 34421834


Progress 05/15/20 to 05/14/21

Outputs
Target Audience: 2020 was a challenging year for us as well as for the world due COVID 19 pandemic. All the programmed activities for broad communication in vivo were put on hold. We didn't have many opportunities to interact directly with general public. However, we kept our channels of discussion fully open via online tools. The regular in person meetings with the stakeholders were suspended during 2020. We expect to have, at least, one regular meeting scheduled for the fall of 2021 to discuss our progress. This will serve as a dual purpose, evaluate the achievements, and recover information to better focus the future research. Scientific community: We reach the scientific community via direct scientific collaborations with international groups and publishing our results in scientific journals. See publications section. Changes/Problems:Our assays suffered the consequences of the covid pandemic. We were able to recover and restart some of the assays. It is exppected to complete the analysis of the samples and repeat the assays interrupted. We are confident that we will be able to acomplish the goals proposed during 2021. What opportunities for training and professional development has the project provided?Dr Marcelo Merli finished his postdoctoral training in opur laboratory. Dr. Lucila Garcia (CONICET scholar from Argentina), completed a short term training in our laboratory. She is a co-author in one of our articles recently submitted. The graduate Student Kaylie Padgett- Pagliai is in her last semester and working on her PhD thesis to be presented in October-November of 2021. How have the results been disseminated to communities of interest?Our results were submitted to publication in scientific journals. What do you plan to do during the next reporting period to accomplish the goals?We have to recover from the effects of the COVID pandemic. We are completing the assessment of the assays, restaring some of the green house assays interrupted during 2020 and analyzing samples stored at -80C. We are confident that we will complete our assays before the extension period ends and publish the reamining results.

Impacts
What was accomplished under these goals? Objective 1: Sucrose stress with L. crescens was shown to induce specific genes transcription and iron uptake. In our assays survival to high sucrose concentrations could be affected LdtR and PrbP inhibitors with zinc and/or iron transport inhibitors. The survival of the cells could be related to a transcriptome responding to the biofilm formation. Our data suggests that prbP is primarily induced in early growth stages, thus regulating the initial stages of biofilm formation, while ldtR is highly induced in later stages. This rationale may also explain why the chemical inactivation of LdtR showed little correlation with the genes identified during sucrose shock. As anticipated, we revealed that sucrose has a significant effect on biofilm formation in the culturable L. crescens, increasing the overall biomass as well as mean and maximum thickness. Further qRT-PCR assays of the aforementioned samples revealed varying expression for a large number of transcriptional regulators with or without sucrose stress. In example, mocR's expression pattern changed from prbP in late phase biofilm growth. This suggests alternate regulation than in later phases. Contrastingly, ldtR expression changed minimally amongst most conditions while increasing expression in non-biofilm forming conditions, inducing a different subset of genes (Padgett-Pagliai et al., 2021 submitted) Following the formation of biofilm, inhibition of PrbP reduced the viability of L. crescens. These results support the role of PrbP in stress response, via interactions with heat stress responding proteins. The direct role of PrbP in essential biological processes, such as ribosomal biogenesis and metabolic homeostasis. Overall, our work demonstrates that PrbP is involved in transcriptional and post-transcriptional regulation of several key factors involved in stress responses, cell motility, cell cycle and biofilm formation. Furthermore, the high similarity of PrbP among Liberibacter spp suggests that a similar role of PrbP may exist in L. asiaticus (Pan et al., 2021 submitted). Objective 2: LotP, a member of the LdtR regulon, recently purified and characterized in our laboratory for the first time seems to play a pivotal role in the posttranscriptional control of the bacteria proteostasis network. We demonstrate that this protein could also affect the serine proteases of the citrus. We have evaluated the interactome of LotP - revealing a new protein-protein interaction targetd and its effect on citrus plant tissue integrity. We found that via protein-protein interactions LotP can enhance Lon protease activity, increasing the degradation rate of its specific targets. Infiltration of purified LotP strained citrus plant tissue causing photoinhibition and chlorosis after several days. Proteomics analysis of LotP tissues recovering after the infiltration revealed a large abundance of plant proteins associated with the stabilization and processing of mRNA transcripts, a subset of important transcription factors, and pathways associated to innate plant defense were highly expressed. Furthermore, LotP's interactions and substrate binding module suggest potential interactions with plant proteins, most likely proteases (Merli et al; 2021 submitted). We are recovering our infected plants and tracking the gene expression to complete the studies proposed in Obj. 2. These assays were delayed because of the Covid pandemic.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Marcelo L. Merli, Kaylie A. Padgett-Pagliai, Alexandra E. Cuaycal, Lucila Garcia, Maria Rosa Marano, Graciela L. Lorca, Claudio F. Gonzalez. Candidatus Liberibacter asiaticus multimeric LotP mediates Citrus sinensis defense response activation. Frontiers in Microbiology [submitted]
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Padgett-Pagliai, Pagliai FA , Gardner CL, Da Silva DR, Lorca GL, Gonzalez CF Analyses of the global stress response reveals a link between PrbP and LdtR in Liberibacter crescens. Microbial Biotechnology [submitted]
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Lei Pan L, Gardner CL, Reagan Beliakoff R, Da Silva D, Pagliai FA, Padget K, Merli ML, Bahadiroglu E, Gonzalez CF, Lorca GL. PrbP modulates biofilm formation in Liberibacter crescens. Scientific Reports [submitted]


Progress 05/15/19 to 05/14/20

Outputs
Target Audience:1. Our target is the general public, we reached this segment of our audience via different online tools. We have active participation on a variety of portals to educate our community regarding the importance of citrus for Florida. A variety of the portals used are more user friendly for the growers and the general public. The languaje used is precise and simple, this help people to achive a better undertsanding of the greening problems. http://citrusindustry.net/2020/05/06/new-citrus-greening-treatment-shows-promise/ https://www.thepacker.com/article/florida-researchers-find-success-citrus-greening-study http://blogs.ifas.ufl.edu/news/tag/claudio-gonzalez/ https://flrnet.org/ufifas-researchers-find-chemicals-that-treat-citrus-greening-in-the-lab-2/ 2. Stakeholders, we have informal but regular meetings with the growers and answering questions via email and phone calls. This is extremely important for us and for the growers, we have a mutual feedback to keep our research pace and up to date the progress regarding citrus greening. 3. Scientific community (publications and scientific meetings using posters and oral presentations). Changes/Problems:We observed a non significant change of gene expression when the plants were treated with sublethal concentration of the chemicals used in our treatments. As a consequence, we focused our attention in the analysis of the proteins expressed once the plants are challenged with diference concetrations of one important L. asiaticus protein. This protein demostrated to be one the central genes regulated by LdtR, the central regulator in L. asiaticus phisiology affected by one of the chemicals studied. This alternative allowed us to track important plant responses to the bacteria and evaluate the plant proteomic pattern. This indiret approach is valid to increase the sensitivity of the method used to put in evidence changes otherwise difficult to be observed. What opportunities for training and professional development has the project provided?The graduate student working on this project approved the qualifying test, she is starting the last phase toward her PhD. She did important progress in the analysis of the plant transcriptome and we are working on our next publication. A Scholar visitor from the University of Rosario (Argentina) worked during 2,5 months in our laboratory. We were able to add new skills and technologies to our group. An important reporter system to evaluate stress response in plants was optimized expanding our analytical capabilities. How have the results been disseminated to communities of interest?We are in constant contact with our growers and discussing the future needs to better focus our research to translate our experience to the sector. Citrus greening is also slowly entering the Iberian Peninsula. The PI offered a conference in Valencia and established contact with Dr. Jose Gadea from Instituto de Biología Molecular y Celular de Plantas (IBMCP), Valencia (IBMCP). We expect to establish further collaboration with Spanish research groups in the near future to work together in specific citrus greening topics of common interest. What do you plan to do during the next reporting period to accomplish the goals?We had an important setback that will affect our progress. The next report period will probably reflect the impact of Covid-19 and we will not have the amount of progress we demonstrated in our previous reports. The university of Florida's work was halted due to the pandemic affecting USA. This situation started approximately 12 weeks ago, the students, technicians and PI were not allowed to work on the campus, greenhouses and laboratories. The situation is still on going, UF is slowly going back to normal work in several steps, we are actually in Phase 2 of the 5 stages established by IFAS. We expect to going back to normal as sson as possible, but the work scheduled suffered an important delay. This is specially important when working with citrus plants that have to adapted, synchronized and they depend of many factors. We will require extra months of work and efforts to accomplish what it was proposed. Once this pandemics ends we will be in a better position to re-schedule our work. We are evaluationg our biological material permanently to have everything ready to go and minize the impact. Our plan is to keep working on the Goal #2, recovering the plants in the greenhouse to perform the tests described immediately we start working again. In the meantime we are investing our time in the analysis of qRT-PCR and proteomics. This analysis is carried out in silico, I'm in permanent contact with my students working on line to speed up the analysis of the data. We are actively working in our next publication.

Impacts
What was accomplished under these goals? Objective 1. Establish the correlation between the expression of genes regulated by LdtR and the persistence of Ca. L. asiaticus in the citrus host. We computed differential gene expression before and after treatments, controlling for seasonal differences accounted by the control trees. We obtained 34 down-regulated and 28 up-regulated genes for the Tolf treatment, while Benz induced the expression of 387 genes and down-regulated expression of 17 genes. Notably, most genes upregulated by Tolf were also induced by Benz, while little overlap on gene expression differences was observed for down-regulated genes, which suggests a consistent drug effect on the expression of citrus induced genes. In trees that received Benz, FET returned fifteen enriched pathways including phenylpropanoid biosynthesis, starch and sucrose metabolism, basic metabolic processes (lipids, nucleic acids and aminoacids), as well as pathways associated to biosynthesis/modification of the cell wall. On the contrary, no enriched pathways were identified for the upregulated genes in trees that received Tolf, possibly due to the lower number of genes significantly induced/repressed in this second treatment. Raw data was deposited in the Gene Expression Omnibus Data base (GEO). The data obtained was recently published (Gardner et al, 2020). The analysis of plants treated with sublethal concetrations of the chemicals in study did not produce the expected results. The variations observed generate non significant results between the groups of treatments. Objective 2. Elucidate the biological consequences of LdtR-mediated regulation. Several important pathways were identified in addition to the LdtP (transpeptidase). The results obtained are the subject of current analysis. It is anticipated that the genes identified are critical to affect the persistence of the bacteria in the citrus host as well as for interference with the plant defensive arsenal. Two articles will be published during the current year describing the results obtained.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zuo, R; de Oliveira, A.; Bullita, E.; Torino, M.I.; Padgett?Pagliai, K.A.; Gardner, Ch.L.; Harrison, N.A.; da Silva, D.; Merli, M.L.; Gonzalez, C.F. and Lorca, G.L. (2019) Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus. Env. Microbiol. 21 (12), 4822-4835. PMID: 31631506
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: D Zhang, DR da Silva, TJ Garrett, CF Gonzalez, GL Lorca (2020) Method Optimization: Analysis of Benzbromarone and Tolfenamic Acid in Citrus Tissues and Soil Using Liquid Chromatography Coupled With Triple-Quadrupole Mass Spectrometry Frontiers in Plant Science 11, 222 PMID 32210995
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gardner, Ch.; da Silva, D.R.; Pagliai, F.A.; Pan, L.; Padgett-Pagliai, K.A.; Blaustein, R.A.; Merli, M.L.; Zhang, D.; Pereira, C.; Teplitski, M.; Chaparro, J.X.; Folimonova, S.Y.; Conesa, A.; Gezan, S.; Lorca, G.L., and Gonzalez, C.F. (2020) Assessment of unconventional antimicrobial compounds for the control of Liberibacter asiaticus, the causative agent of citrus greening disease. Sci. Rep. 10 (1) 1-15 PMID 32214166
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Padgett-Pagliai KA, Merli ML, Gonzalez CF. August 17 2019. The Hunt for Citrus Survival. Microbiology and Cell Science Annual Graduate Symposium, Department of Microbiology and Cell Science, University of Florida, Orlando, Florida, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Padgett-Pagliai KA, Gardner CL, Silva DR, Pagliai FA, Pan L, Merli ML, Chaparro JX, Lorca GL, Gonzalez CF. September 2019. Identifying key mechanisms of interaction in L. asiaticus-citrus pathosystem. Plant Science Conference, Valencia, Spain.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Padgett-Pagliai KA, Merli ML, Lorca GL, Gonzalez CF. June 19 2019. The Significance of LotP, a Key Bacterial Effector, in Huanglongbing. American Society for Microbiology Microbe,San Francisco, California, USA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Gonzalez CF. Identifying key mechanisms of interaction in Liberibacter asiaticus-citrus pathosystem. Plant Science Conference, Valencia, Spain. September, 2019
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Pan L, da Silva D, Pagliai FA, Harrison NA, Gonzalez CF, Lorca GL. The ferredoxin-like protein, FerR, regulates PrbP activity in Liberibacter asiaticus. Appl Environ Microbiol 2019 85(4). pii: e02605-18. doi: 10.1128/AEM.02605-18. PMID:30552192


Progress 05/15/18 to 05/14/19

Outputs
Target Audience:Our publications (scientific articles) are directed to the scientific community with expertize in plant science, microbiology and molecular biology background. The published results were, in general, previously discussed with stakeholders. Our publications are usually summarized in layman terms in our webpage to inform the general audience regarding our progress. The information in our web page is up to date, the students involved in the research process are involved in the selection and keeping the information up to date. We use each opportunity, like regional meetings, to discuss the progress with peers and growers. The presentations in public meetings and symposiums, mostly at regional level, are directed to put in contact the scientific research performed with a broad audience where most of them are citrus growers from Florida. Since the citrus greening disease is of public knowledge, Floridians are usually very interested and engaged in the discussion. Changes/Problems:Working with L. asiaticus, a bacteria that cannot grow in vitro is associated to several challenges. We allways have to adjust the assays to minimize the variability and validate the results statistically. The experience in the work is a valuable tool to have and our team is prepared for it. We recently adjusted the time, formulation and concentrations of the chemicals being used for the treatment. Similarly, we continuously work in the otimization of sample processing and mRNA obtained from plants samples to improve the quality of our results. We have also included in our studies a deep characterization of one of the protein LotP of the LdtR regulon. We believe that after completion of this grant we will have described one of the most important mechanisms responsible of L. asiaticus pathogenicity. What opportunities for training and professional development has the project provided?Currently, a graduate student is working in her disertation in gene regulatory mechanisms in L. asiaticus. Similarly a posdoctoral fellow is gaining training in L. asiaticus pathogenesis. The undergraduate and graduate students involved in this project are continuously interacting with graduate students working on the same area in our department. Citrus greening disease is a major problem in Florida as well as in the world. We hosted a visiting scholar from Tucuman Argentina, a citrus producing region in South America. We are also collaborating with Dr Adrian Vojnov of the Cesar Milstein Institute in Buenos Aires. This group is producing nanobodies (lama antibodies) to track L. asiaticus proteins in the tree samples. Several undergraduate students are directly or indirectly learning from our research activities. How have the results been disseminated to communities of interest?We have a large group of growers in direct contact with our research group. We are receiving feedback and field observations that are extremely valuable for our project. Our published papers with a digest in layman's terms are up to date in our web page at UF. We also have a interaction forum in the page. However we need to improve the public participation in that forum. We will work on that involving undergraduate students in the process. What do you plan to do during the next reporting period to accomplish the goals?We are on track with our research, we expect to publish our results for the objective 1 before ending the 3rd period of granting. It is also expected to have published the results obtained with one of our target genes, identified as a critical protein in L. asiaticus pathogenicity.

Impacts
What was accomplished under these goals? Objective 1. Establish the correlation between the expression of genes regulated by LdtR and the persistence of Ca. L. asiaticus in the citrus host. Based on our preliminary data, we hypothesize that the inactivation of LdtR with small molecules will result in decreased persistence in the citrus host. We are currently working on the assays for the objective 1. We have 121 citrus plants infected with L.asiaticus (18 months old plants) in a greenhouse setting. The plants were individually infected via grafting. The rate of infection was established via qRT-PCR using the 16S rRNA gene as a target. L. asiaticus infection rate is not uniform in the plants, this also include those plants infected via grafting using explants obtained from the same L. asiaticus infected donor plant. After the grafting, the samples were collected from all infected plants every 20-25 days and analyzed via qRT-PCR. The determinations started 7 months after the grafting day. Based on the results obtained after tracking the infection rate of the plants during 9 months, the infected citrus plants were divided into three groups, high infection, medium infection and low infection. These three groups of plants were treated with sub lethal concentrations of the chemicals proposed and samples were collected every two months for 12 months. A preliminary analysis of the samples, by quantifying the gene expression by qRT-PCR, indicated that the expression of the host genes (PR1,PDF2, LysM2 and CDR1) were more affected by the treatments than the expression of the L. asiaticus genes regulated by LdtR. These plants were in treatment during several months and the processing and analysis of the samples with qRT-PCR is a labor intensive and tedious process. Our progress is on time with the analysis originally proposed in our Timeline table. It is expected to finish the mRNA extraction and verify the bacterial survival in the infected plants during the first semester of the 3rd year of funding. A new experiment designed to track the gene expression of the same genes in shorter period of times is ongoing. This new assay will provide details of the gene regulation during the first stages immediately after the application of the selected chemicals. We expect these results will allow us to clarify some discrepancies obtained in the preliminary results. Objective 2. Elucidate the biological consequences of LdtR-mediated regulation. Based on our preliminary data, we hypothesize that LdtR acts as a global transcriptional activator of genes involved in peptidoglycan remodeling, cell division, and protein secretion, resulting in increased osmotic stress tolerance and evasion of host responses. We evaluated the biochemical characteristics of CLIBASIA_01175, renamed LdtP (L,D-transpeptidase). Surrogate strains were used to evaluate its potential biological significance in gram-negative bacteria. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP. This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. The enzyme with moonlighting activity was associated to the modification of the bacterial cell wall and LPS; probably helping the bacteria to be unnoticeable by the defensive mechanism of the citrus host. The LdtR operon from L. asiaticus, which includes ldtP, was previously characterized and identified to be involved in the osmotic stress response. In agreement with this, we found that ldtP was strongly induced in L. crescens in the presence of culture media containing a high level of osmolytes, either NaCl or sucrose. Analysis of the lipid A moiety of the LPS showed alterations in structure. The E. coli quintuple mutant LD-TPase strain expressing LdtP showed a different pattern of lipid A composition compared to the wild type. This result, along with the demonstrated esterase activity in vitro, supports an additional role for LdtP in the modification of the cell LPS. Modifications of the LPS structure, specifically in the lipid A region, have been shown to have dramatic effects on bacterial survival and virulence and can alter host immune responses. Modifications of L. asiaticus lipid A by LdtP, probably due esterase activity, may also contribute to the survival strategy of this bacterium, allowing it to thrive in the phloem of the citrus host. This protein could be a critical target to direct antibiotic therapy to treat L. asiaticus infections.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Pan, L., da Silva, D., Pagliai, F.A., Harrison, N.A., Gonzalez, C.F. and Lorca, G.L. (2018) The ferredoxin-like protein, FerR, regulates PrbP activity in Liberibacter asiaticus. Appl. Environ. Microbiol. [Epub ahead of print] PMID:30552192
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Coyle, J.F., Pagliai, F.A., Zhang, D., Lorca, G.L. and Gonzalez, C.F. (2018) Purification and partial characterization of LdtP, a cell envelope modifying enzyme in Liberibacter asiaticus. BMC Microbiol.18:201-216. PMID:30497377
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Coyle, J.F., Lorca, G.L. and Gonzalez, C.F. (2018) Understanding the Physiology of Liberibacter asiaticus: An Overview of the Demonstrated Molecular Mechanisms. JMMB 28:116-127. PMID:30176677
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Padgett KA, Merli ML, Lorca GL, Gonzalez CF. The Significance of LotP, a Key Bacterial Effector, in Huanglongbing. 3rd Annual UF Plant Science Symposium, January 2019, University of Florida, Gainesville, Florida.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Padgett KA, Merli ML, Lorca GL, Gonzalez CF. Characterization of a Putative Pathogenic Determinant from Liberibacter asiaticus. 14th Annual Florida Genetics Symposium, Cancer and Genetics Research Complex, October 2018, University of Florida, Gainesville, Florida.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Padgett KA, Merli ML, Lorca GL, Gonzalez CF. Elucidation and characterization of a novel chaperone-interacting protein from Liberibacter asiaticus. Microbiology and Cell Science Annual Graduate Symposium, Department of Microbiology and Cell Science, University of Florida, August 2018, Orlando, Florida.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Merli ML, Padgett KA, Cuaycal AE, Gonzalez CF. Site-Directed Mutagenesis Study of the ATPase LotP from Liberibacter asiaticus. American Society for Microbiology Microbe, June 2018, Atlanta, Georgia.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pan L, da Silva D, Pagliai FA, Gonzalez CF, and Lorca GL. 2018. The ferredoxin-like protein, FerR, regulates PrbP activity in Liberibacter asiaticus. American Society for Microbiology microbe 2018, Atlanta, GA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Padgett KA, Merli ML, Cuaycal AE, Lorca GL, Gonzalez CF. Characterization of a Putative Pathogenic Determinant from Liberibacter asiaticus. 2018 ASM Florida Branch Meeting, October 2018, Orlando, Florida.


Progress 05/15/17 to 05/14/18

Outputs
Target Audience:1. General public, online using the web page and social media. 2. Stakeholders, regualr meetings with our stakeholders and advisors. 3. Scientific community (publications and scientific meetings using posters and oral presentations). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Citrus greening is a global problem, students from different part of the world (Sweden, Brazil and Argentina) are in permanent contact and participation to increase knowledge and adquire important skills to work with L. asiatius as well as with L crecens. Dr Lucila Garcia, from Argentina received training during 3 months in our laboratory improving her technicals skills to perform research in south America. Those techniques include, formulation of culture media to work with L. crescens, electroporation, gene cloning from infected citrus plants, protein purification and enzyme assays. Two students from Sweden will participate of research during summer of 2018. The student Janelle Coyle received a PhD in 2017, part of her work was subsidized by the actual project. Her thesis dissertation is at the University of Florida (on line system - will be avalible in a year). One article is completed and will be submitted for publication, a secon article is on review process. One graduate student started the program in 2017, this year she will actively participate in field studies and molecular assays in L. asiaticus. How have the results been disseminated to communities of interest?Published results were discussed with stakeholders. Our publications are usually summarized in layman terms in our web page to inform the general audience regarding our progress. We use each opportunity, like meetings, regional encounters to discuss the progress with peers and growers. What do you plan to do during the next reporting period to accomplish the goals?The progess made in the first year of work is progressing according our expectations.

Impacts
What was accomplished under these goals? Objective 1: LdtR is a master regulator of gene expression in Liberibacter asiaticus, one of the causative agents of citrus greening disease. LdtR belongs to the MarR-family of transcriptional regulators and it has been linked to the regulation of more than 180 genes in Liberibacter species; including cell motility, cell wall envelope, energy production, and gene transcription. Transcriptomic evidence suggested that LdtR is directly involved in the modulation of the zinc uptake system genes (znu). These assays were carried out in the closely related L. crescens used as a surrogate strain. We demonstrated that LdtR is involved in the regulation of one of the two encoded zinc uptake mechanisms in L. asiaticus, named znu2. LdtR binds zinc with higher affinity than benzbromarone, a synthetic effector inhibitory molecule, resulting in the disruption of the LdtR:promoter interactions. Using site-directed mutagenesis, electrophoretic mobility shift assays (EMSAs), and isothermal titration calorimetry, we identified that residues C28 and T43 in LdtR, located in close proximity to the Benz1 pocket, are involved in the interaction with zinc. These results provided new evidence of a high-affinity effector molecule targeting a key player in L. asiaticus' physiology and complemented our previous findings about the mechanisms of signal transduction in members of the MarR-family. Biochemical and in vivo assays confirms that the concentration of zinc that exerts an inhibitory effect on LdtR is physiologically relevant. The development of antimicrobial treatments (i.e. zinc) that do not affect the structure of the microbial community and are able to restore the nutrient balance of the plants by targeting specific proteins in Liberibacter species, such as LdtR, will provide new and safer options for the HLB eradication. Increasing our knowledge regarding the mechanisms used by L. asiaticus to adapt and survive inside the host will allow us to develop new and safer therapeutics to combat citrus greening disease. The evidence presented here, where zinc acts a high-affinity effector molecule for LdtR, indicates that the use of a combination of effector molecules targeting LdtR, such as benzbromarone, phloretin, or zinc, may be an efficient method to impair crucial pathways in L. asiaticus, providing a safer and novel strategies for the management of HLB infected trees. Objective 2: We used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_01175, renamed LdtP. The gene encoding LdtP as well as the protein were characterized by bioinformatics techniques, which supported the hypothesis that this protein functions as transpeptidase. LdtP was successfully cloned and purified. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP. This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. Taken together, this data indicates that LdtP is a novel protein involved in an alternative pathway for modification of the bacterial cell, potentially affording L. asiaticus a means to survive within the host. Transcriptional activation of ldtP was crucial to increase osmotic stress tolerance in the phylogenetically-related surrogate species, S. meliloti, during assay conditions (Pagliai et al., 2014). This suggests that the activity of LdtP may be essential to the survival of L. asiaticus in response to the high osmotic pressure sustained within the citrus phloem by increasing the rigidity of the cell wall through an increase in the abundance of 3-3 cross-links. Alternatively, L. asiaticus may also mask the peptidoglycan and LPS from detection by the citrus plant immune system by modification that improves viability and survival within the host. Targeting LdtP for the development of therapeutic agents against HLB is an approach that will have a significant effect on persistence of this pathogen in the host.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Pagliai, F.A., Pan, L., Da Silva, D., Gonzalez, C.F. and Lorca, G.L. 2018 Zinc is an inhibitor of the LdtR transcriptional activator. PlosOne 13(4) e0195746
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Janelle Coyle, Exploring molecular mechanisms of pathogenesis of Liberibacter asiaticus in the plant-microbe interaction
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pan L, Gardner CL, Pagliai FA, Gonzalez CF, and Lorca GL. 2017. Identification of ligand binding pocket in PrbP from Liberibacter asiaticus. V International Research Conference on Huanglongbing, Orlando, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pan L, Gardner CL, Pagliai FA, Gonzalez CF, and Lorca GL. 2017. Tolfenamic acid inactivates PrbP, a transcriptional accessory protein in HLB causative agent Liberibacter asiaticus. Florida Genetics Symposium, Gainesville, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pan L, Gardner CL, Pagliai FA, Gonzalez CF, and Lorca GL. 2017. Tolfenamic acid inactivates PrbP, a transcriptional accessory protein in HLB causative agent Liberibacter asiaticus. Materials Innovation for Sustainable Agriculture 2017 Symposium, Orlando, FL
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Merli ML, Padgett KA, Cuaycal Bastidas AE, and Gonzalez CF. 2018. Site-Directed Mutagenesis Study of the ATPase LotP from Liberibacter asiaticus. ASM microbe 2018, Atlanta, GA.