Progress 09/01/20 to 08/31/24
Outputs
Target Audience:The activitiesmainly targeted to impact the plant pathology and microbiology scientific community by advancing the tools and knowledge required for studying plant pathogens and to increase our knowledge on HLB diease pathosystem. In addition, our work targeted tha citrus industry by providing them a new tool to estimate the disease severity in the trees. We toght and trained growers on this tool through extension meetings and field days. Changes/Problems:We did not make changes in the work plan, however, some of the treatments (2-DDG, SEORs) did not provide HLB tolerance. We encountered technical challanges in generating CRISPR mutations. What opportunities for training and professional development has the project provided?The project has provided research training and mentoring opportunities to students, postdocs and technician. The students, postdocs and staff were routinely mentored by the PIs in laboratory techniques and scientific inquiries. We also provided training opportunities for growers in measuring the tree canopy density through hand on activities in growers meetings (Citrus Expo, OJ Break) and dedicated field days. How have the results been disseminated to communities of interest?We organized a field day for Growers in Florida, in which we taught growers how to use light interception to determine tree health. We provided hand-on instruction for growers on how to measure light interception to determine the canopy density of the tree using a light meter and PAR-meter. Demonstrated measurements on two group of trees that received different nutrition treatments. We also organized training on how to measure canopy density using the cell phones during an OJ Break growers' activity. The growers went to the field and practiced this measurement. Our work was published in extension publications and trade hournals. We also created aYouTube instructional video for growers "Canopy Assist: An Evaluation Tool for Citrus" https://www.youtube.com/watch?v=sdnkDO-Mu9c. 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: Wehave made fundamental contributions to the understanding of the host-pathogen biology of HLB. Our work showed that CLas is not equally distributed within the tree, but it rather accumulating in the sink tissues, especially seed coats. We showed that both the pathogen and the tree respond differently when the pathogen is in either the source or the sink tissues. In the source tissues, once infected with CLas, the tree's defense system generates callose, a material that plugs the phloem to prevent pathogen movement, and that this generation inhibits the sugar movement in the tree. The tree also generates reactive oxygen species, involved in a plant's defense systems. This chronic immune response leads to the blockage and collapse of the phloem system. However, CLas responds to the generation of callose and reactive oxygen species by eliminating them, allowing bacteria to replicate and move throughout the tree once again.In thesink tissues, where CLas accumulates, there is no accumulation of callose and ROS. We conducted transcriptomic and proteomic analyses of the host response in the source and sink tissues, and we identified very different responses in these tissues.We then moved on to study the differences of these host-pathogen dynamics between susceptible and tolerant varieties. We compared susceptible sweet orange to the tolerant SugarBelle variety, and we identified three complementary mechanisms of tolerance in SugarBelle: (i) increased carbohydrate availability induced by greater CO2 fixation, mild effect in carbohydrate export, and local accumulation of callose in the phloem; (ii) activation of immune response, and (iii) increased investment in the xylem structure.These resuluts showed that thetolerant varieties possess a stronger immune response that is leadingto reduced and more balanced defense responses. These results were also verified in genetically modified plants that over-express the key immune regulatorNONEXPRESSOR OF PATHOGENESIS-RELATED GENES1(NPR1). We discovered that transgenic expression of the ArabidopsisNPR1gene in citrus repressesCLas-induced over-accumulation of both callose and ROS. This prevents the occlusion of the phloem pores, resulting in greatly reduced HLB symptoms in infected plants. Significantly, virus-induced gene silencing of the citrus ortholog ofNPR3, a negative immune regulator, also resulted in reduced callose and ROS accumulation uponCLas infection and enhanced HLB tolerance. Furthermore, we found that these functions ofNPRgenes in regulating pathogen-induced callose and ROS accumulation are conserved in Arabidopsis. These finding showed that this chronic immune response that is causing disease is the result of a weak immune system in the commercial scion varieties. The work suggests that improving the scion immunity through gene editing and traditional breeding will decrease HLB severity. Objective 2:Our study advances a basic understanding of how Las may adapt to different host environments and manipulate host defense and revealed regulatory components that may contribute to transcription reprogramming.We carried out a comprehensive transcriptomic analysis of CLas using the leaf midrib and vascular tissue-enriched seed coat tissues of citrus. Considering the specific colonization niche of CLas and its uneven/generally low titer in citrus, we explored a variety of methods with the aim of enriching bacterial cells and/or RNA from infected plant samples. These efforts eventually enabled us to detect up to 70% and 99% genes encoded in the Las genome from midrib and seed coat, respectively. We were then able to compare the expression profiles of CLas genes in these two different citrus tissues with the insect vector (psyllids). These comprehensive analyses established a fundamental understanding of how CLas colonizes different hosts and host tissues, representing a major step forward in HLB biology. The datasets are also valuable for the research community. Using GO term analysis and hierarchical clustering, we developed a dynamic transcriptional landscape of CLas in insect/citrus hosts. Comparative analyses allowed us to detect global transcription reprogramming in CLas when the bacterium colonized citrus. For example, genes involved in motility such as the flagellar machinery are significantly downregulated in both citrus tissues than in the psyllids. More importantly, differences in gene expression were also observed in CLas when colonizing midrib vs seed coat. In particular, the midrib and seed coat samples had 143 and 287 total differentially expressed genes (DEGs) when compared to psyllid samples respectively, with 76 DEGs shared in both samples. Ninety-one genes were specifically upregulated in seed coats, suggesting tissue-specific interactions of CLas with citrus. For example, CLas in seed coat samples undergoes drastic reprogramming at the RNA and protein level with significant upregulation of genes involved in transcription, translation, and protein turnover. In addition, genes involved in the glutamine metabolism pathway and the TCA cycle converting succinate to oxaloacetate were also upregulated. These findings highlight tissue type-dependent metabolic states of CLas in planta. These studies revealed changes in CLas cellular processes that were undetectable in previous studies and discovered an adaptation of CLas to different citrus tissues. Genes specifically upregulated in the vasculature-enriched seed coat tissues include virulence factors such as two Sec-dependent effectors (SDEs) and a peroxidase. Since Las was found to more efficiently suppress callose deposition in seed coats than in midrib, these genes may contribute to this difference. We expressed these genes inNicotiana benthamianaand confirmed that one SDE and the peroxidase were indeed able to suppress bacteria-triggered callose deposition. This experiment identified virulence factors that may play a role in manipulating host defenses in a tissue-specific manner. The full coverage of CLas gene expression profile allowed us to analyze cis-elements in the promoter region that might be responsible for the gene expression dynamics when the CLas colonizes citrus. From this analysis, we identified a LuxR motif that was enriched in the promoters of SDE genes. A similar motif is also enriched in the SDE gene promoters in Candidatus Liberibacter solanacearum (CLso), the pathogen causes the zebra chip disease of potato. This finding suggests a potential role of LuxR-type transcription factors in regulating Ca. Liberibacter virulence in planta. Objective 3: We generated citrus tristeza virus CTV constructs and screened three differentSIEVE ELEMENT OCCLUSION RELATED(SEOR) virus induced genes. We used virus induced gene silencing to downregulate the gene levels, but unfortunately there was no difference in phenotype after infection with HLB. We also tested tree responses after foliar spray with a callose inhibitor 2-deoxy-d-glucose (DDG) on HLB trees. Unfortunately, we didn't see a reduction in HB symptoms. Objective 4: Our work was presented to the citrus industry in trade jounals (Citrus Industry) and in different extension events (Citrus Expo, OJ Break, Field days and theInternational Research Conference on Huanglongbing (IRCHLB)).In May 2023, we introduced the Canopy Assist program, and lunched a website (https://crec.ifas.ufl.edu/extension/canopy-assist/) where growers can submit their canopy pictures for evaluation of the canopy density, and we determine their tree health according to the density. This method is employed with photos taken from smartphone. We published a YouTube video to introduce this Canopy Assist program to growers (https://www.youtube.com/watch?v=sdnkDO-Mu9c).
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Amit Levy, Taylor Livingston, Chunxia Wang, Diann Achor, Tripti Vashisth. 2023. Canopy Density, But Not Bacterial Titers, Predicts Fruit Yield in Huanglongbing-Affected Sweet Orange Trees. Plants. 2(2):290. https:// doi: 10.3390/plants12020290.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Taylor Livingston, Tripti Vashisth, and Amit Levy. 2023. A Simple Low-Cost Method for Accurate Canopy Density Evaluation in Citrus. HortScience 58(7):747-749. https://doi.org/10.21273/HORTSCI17112-2337.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
8. Jacobo Robledo, Stacy Welker, Ilana Shtein, Bernardini Chiara, Christopher Vincent, Amit Levy. 2024. Phloem and Xylem Responses Are Both Implicated in HLB Tolerance of Sugarbelle. Phytopathology 114:441-453 https://doi.org/10.1094/PHYTO-05-23-0148-R
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Poulami Sarkar, Jorge Santiago Vazquez, Mingxi Zhou, Amit Levy, Zhonglin Mou, Vladimir Orbovi? (2024). Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene. Transgenic Research 33(1-2):59-66. doi: 10.1007/s11248-024-00380-2
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Amelia H Lovelace, Chunxia Wang, Amit Levy, Wenbo Ma (2024) Transcriptomic profiling of ' Candidatus Liberibacter asiaticus' in different citrus tissues reveals novel insights into Huanglongbing pathogenesis. Molecular Plant Host Interactions (Accepted) doi: 10.1094/MPMI-08-24-0102-R
- Type:
Other Journal Articles
Status:
Published
Year Published:
2021
Citation:
Amit Levy. Tripti Vashisth. 2021. Citrus Canopy Health is Highly Important for HLB Tolerance. Citrus Industry. August 2021, pp 10-13.
|
Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The proposed activities in this reporting period were targeted to impact the plant pathology and plant biology scientific community by advancing the tools and knowledge required for studying plant pathogens. In addition, our extension work to evaluate disease severity targeted the citrus industry stakeholders. Changes/Problems:Due to the Covid-19 pandemic there were significant delays inobtaining the chemicals needed for the work. Most critical were the chemicals that were needed for our field trials, to test inhibitors of callose, which were significantly delayed. These field trials also suffered from two strong hurricanes that hit our area during 2022 and damaged the trees. Finally, we identified a citrus factor that is necessary for HLB progression but have faced difficulty in producing the CRISPR citrus mutant plants. What opportunities for training and professional development has the project provided?The project has provided research training and mentoring opportunities tostudents, postdocs and technicians. The students and staff were routinely mentored by the PIs in laboratory techniques and scientific inquiry. We also trained growers how to measure canopy density, so they can determine the health of their trees How have the results been disseminated to communities of interest?We presented the 'canopy density' measurement technique to growers in an OJ break extension event at Citrus Research and Education Center in Florida. We also published a YouTube video instructing growers on this technique. What do you plan to do during the next reporting period to accomplish the goals?Identification of candidate genes associated with HLB tolerance will be finished in 2023 and these will be tested for HLB resistance/tolerance in 2023-24. Field trials with callose inhibitors will continue for a second year during 2023-24.
Impacts
What was accomplished under these goals?
Objective 1 (Phloem dynamics in CLas-infected fruits): We compared the responses to HLB infection in two citrus cultivars, the tolerant Sugar Belle® mandarin (SB) and the susceptible Pineapple sweet orange (PA). We didn't see a difference in CLas and Callose accumulation in the fruit. However, the infected SB showed increased CO2 fixation capacity and minor changes in carbohydrate export, xylem structure, and callose deposition in the phloem, unlike PA. SB also exhibited an activated plant defense response, indicated by the upregulation of papain-like cysteine proteases (PLCPs), shedding light on the complex mechanisms of HLB tolerance. We carried out comparative transcriptomic and proteomic analyses on the healthy and infected seed vasculatures to determine the molecular factors behind the high titer of CLas in this tissue and identified 32 important genes and 26 proteins which were significantly regulated in the seed vasculatures upon CLas infection. Several peroxidase family genes and proteins were highly upregulated, along with 1,3 β-glucanases in the infected seed vasculatures. In addition, there are nine significant hormone-signaling genes found in the DEGs, which includes Gibberellin-regulated family protein (GRPs) and other SA-regulated genes upregulated in infected seed vasculatures. Interestingly, most of the defense related genes and proteins are also highly regulated. Objective 2 (Identify novel CLas-phloem interactions in HLB progression): CLas transcriptomes were analyzed from midrib samples, seed coat samples, and insect samples. Samples cluster based on the host type after PCA analysis suggesting that the transcriptomic profiles shift based on where CLas is in the plant and insect host. A total of 124 DEGs were detected in seed coat samples (91 upregulated & 22 downregulated) when compared against midrob samples. GO enrichment of DEGs in seed coat samples versus midrib samples exhibited enrichment in upregulated genes associated with the TCA cycle, primary metabolism, and glutamine metabolic process. This may suggest that CLas is more metabolically active in seed coat vasculature than in midribs. Of the 27 CORE Sec-dependent effectors (SDEs) identified in CLas, 2 exhibited significantly higher expressed in seed coat samples compared to midrib samples (CLIBASIA_04410 and CLIBASIA_05330). Additionally, a peroxidase had higher expression in seed coat tissues than in midrib tissues. Transient expression of these candidate effectors in Nicotiana benthamiana exhibit a moderate reduction in ROS burst after flg22 protection compared to the negative control. However, whether this corresponds to callose suppression is still being investigated. It is also worth noting that while the individual effects of these effectors are subtle, CLas may deliver these SDEs together resulting in a cumulative effect for immune suppression. We are now conducting a dual transcriptomic analysis, to connect effectors activity with plant response. Objective 3 (Reduce phloem plugging to reestablish sugar transport): We screened sweet orange seedlings infected with CTV-RNAi vectors targeting two more genes that were upregulated after CLas infection. One gene showed no effect, but another one , showed a delay and attenuation of HLB disease symptoms, suggesting this gene downregulation could be used to generate HLB tolerance. These genes are known to be also required for phloem-viruses infection, and we showed that in the CTV-RNAi line there is an inhibition of CALLOSE SYNTHASE 7 and PP2 phloem proteins genes, and lower phloem callose plugging, which may explain the broad inhibition of these proteins on phloem diseases progression. We generated CRISPR/Cas9 constructs to edit this gene, and transformed sweet orange with these constructs, but did not get any gene edit events yet. We are now planning to repeat this experiment with new constructs. We are also conducting the field trials with the callose inhibitor 2-deoxy-d-glucose (DDG). These field evaluate the effect of DDG on tree health, callose formation and carbohydrate translocation in sweet orange trees. According to first year of field trial, DDG treated trees showed improved leaf area and number. Improvement in leaf characteristics suggests improvement in tree health. No improvement in yield was observed in the first year with the treatments however DDG treatments had numerically higher fruit production. The second year of treatments is now initiated. In addition to described protocols, a rigorous leaf monitoring system will be employed. We anticipate seeing promising results in year 2 of trial. Objective 4 (Engage stakeholders and disseminate our findings): Basic work from this project showed that CLas is hardly present in the leaves, and therefore plant response (such as phloem plugging) play the main role in determining the disease severity, rather than CLas titers. We previously showed that the percentage of photosynthetically active radiation interception in the canopy (%INT) was positively correlated with fruit yield. However, %INT measurements were still too expensive and complicated for the growers. We found that we can measure the canopy density also with simple cellphone pictures, and we developed a new program, CanopyAssist (https://crec.ifas.ufl.edu/extension/canopy-assist/), to help growers determine their trees health. This program is based on the finding that the density of the canopy is correlated with the yields. The higher your tree's canopy density, the higher the yields, so measuring the canopy density can evaluate the health status of the trees. This method is employes with photos taken from smartphone. The grower takes four pictures from underneath the canopy, with the phone camera facing up to the sky, so it will take a picture of the canopy from four corners of the tree. After taking the pictures, they upload them into the server and we analyze the canopy coverage using artificial intelligence analysis to separate pixels from foreground (canopy) and background (Sky), and to find the percent coverage of each photo. This number tells if the trees are on the healthy side, of on the severely sick side. We have started the program very recently, and already received pictures uploads from growers. We are now testing the efficiency of the analysis with the grower's data, to be sure we are providing accurate evaluation of tree health. With more data coming in during the coming year, we hope to improve the program analysis so that we can predict actual yields.
Publications
|
Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Our project target audiances is the citrus industry stakeholders and the scientific community. We presented and discuss relevant results with citrus growers in grower's meetings, field days and other extension activities. Grower's relevant data generated during this project was published in a citrus trade journals, and manuscripts with scientific data were submitted to research journals. We also presented the project in a podcast aimed at the citrus community. Changes/Problems:We had some problems with infecting sweet orange trees with our CTV strains, and we are still trying to solve this issue.The main changes from the plan occurred due to the supply chain issues, and we couldn't get many products needed for carrying out the experiments.The biggest effect was on our field trials. We could not get any callose inhibitor (DDG), and trials could not start on time, and when they started, we could not conduct them as planned. This is an ongoing issue, and we are constantly waiting for more product to arrive. What opportunities for training and professional development has the project provided?T he work of the project team has provided training and mentoring for a graduate student, two post-docs and several technical workers. We presented a poster in the 'Plant Vascular meeting 2022" in Berlin, entitled "Candidatus Liberibacter asiaticus, the causal agent of citrus greening disease, inhibits plant defenses to enable its movement" we had three oral presentations and a poster presentation at scientific conferences as related to the delivery of therapeutics to the phloem tissue of citrus. The first was at an antimicrobial summit in California organized by theCitrusResearchBoard(CRB), the second oral presentation was at the 2nd congress for the International society for citrus Huanglongbing and phloem-colonizing bacterial pathosystems and the third presentation was at the 1stretreat for Virologist at the University of Florida. Although there was difference in the content of presentation, all presentations were titled "Using Citrus tristeza virus (CTV)-based vector as a platform for the management of Huanglongbing (HLB)". The poster presentation was at the American Phytopathological Society (APS) titled "Mapping effect of insertion position and length of truncated RNA on Citrus tristeza virus induced gene silencing". How have the results been disseminated to communities of interest?We organized a field day for Growers in Florida, in which we taught growers how to use light interception to determine tree health.We provided hand-on instruction for growers on how to measure light interception to determine the canopy density of the tree using a light meter and PAR-meter. Demonstrated measurements on two group of trees that received different nutrition. What do you plan to do during the next reporting period to accomplish the goals?Lastly, we are using the CTV vector in order to downregulate genes that we identified in our proteomic and transcriptomic analyses, to determine their role in HLB development.?
Impacts
What was accomplished under these goals?
The goal of our project is to increase our understanding on the Candidatus Liberibacter asiaticus (CLas)- phloem interactions, during HLB disease. These studies are challenging due to the phloem being a narrow tissue buried inside the stem, and the low and inconsistent titers of CLas in tissues studied so far. We have developed a novel system, using the citrus seed coats, which contain a extraordinary high amounts of CLas. The project is allowing us to identify bacterial genes that are important for CLas virulence, and citrus genes that are involved in disease progression and immune responses. Additionally, we are testing new treatments to reduce the disease progression, including downregulating genes that are involved in symptoms development and applying chemical treatments that will result in unplugging the phloem. Lastly, we are delivering our findings to the citrus stakeholders through grower meetings and field days, while providing training to new student and postdocs. Manipulating the HLB related bacterial and host genes, and applying the treatments to unplug the phloem, will be used to increase HLB tolerance, and increase our capacity to control HLB by integrating strategies to keep plant healthy through genetic engineering and/or chemical treatments. Objective 1 (Phloem dynamics in CLas-infected fruits): We discovered how the bacteria and citrus tree engage in a "back-and forth" reactionary relationship. Welearned that once infected with the CLas bacteria, the tree's defense system starts to generate callose in the phloem, a material that essentially "plugs" the phloem, and reactive oxygen species, that induce defense and can cause cell death. Ourresearch found that theCLas bacteria responded to the generation of callose and reactive oxygen species by actually reducing them, allowing bacteria to once again transport throughout the tree. This back-and-forth repetitive relationship of callose plugging and reactive oxygen species, and then their elimination is a complicated one, and replicates an immune response process found in many other diseases. Citrus varieties that are able to maintain a fine balance between callose and reactive oxygen species generation and then elimination without either one gaining "control" may be more inclined to continue to produce fruit over many years. We also conducted proteomic and transcriptomic analyses of the seed coat vasculature. The results support this 'back and forth' immune arms race, where the host tries to accumulate callose and ROS, while CLas tries to inhibit them. We identified specific genes and proteins that play a role in this process, and these can become targets for generating tolerance Objective 2 (Identify novel CLas-phloem interactions in HLB progression):During this reporting period, we continued our research as proposed in Objective 2 (Identify novel CLas phloem interactions in HLB progression) and made significant progress. In particular, we completed the transcriptome analysis of CLas in seed coat using vascular tissue-enriched tissues. We were able to detect most genes in the genome (93-99%), which is much improved from the previous dataset using the midrib tissues. PCA analysis confirmed an overall change in transcriptomics of CLas when the bacterium colonized midrib vs seed coat. Further analysis revealed that, although 68 CLas genes were differentially expressed in both midrib and seed coat, 164 showed differential expression only in seed coat. These results are consistent with our observation that CLas in midrib and seed coat may perform different functions. GO term analysis of the 164 seed coat-specific differentially expressed genes (DEGs) suggests that they are involved in primary metabolism, indicating more active anabolism. Intriguingly, we found two Sec-dependent effectors (SDEs) and a peroxidase that showed specifically induced expression in seed coat. These genes may contribute to the difference we observed in callose deposition manipulation by CLas in seed coat vs midrib. Objective 3 (Reduce phloem plugging to reestablish sugar transport):Our focus in the past year has been on screening sweet orange seedlings infected with CTV-RNAi vectors targeting three different genes independently. We successfully screened sweet orange seedlings infected with two CTV-RNAi vectors. The two vectors failed in producing the desired tolerant HLB phenotype. The sweet orange seedlings infected with the third vector failed to get infected with HLB as determined by qPCR. Similarly, the sweet orange control seedlings either infected with CTV wild type or not infected failed to get infected with HLB indicating a problem in the ACP used for infecting the sweet orange seedlings with HLB. We are working on repeating the screening assay. Further, we had a fourth CTV RNAi construct that failed to infect sweet orange citrus seedlings upon graft inoculating from the C. macrophylla plants. We are working to repeat the sweet orange infection of the 4thCTV-RNAi construct into sweet orange. We also started testing the effect of a callose inhibitor on HLB trees. We sprayed2-deoxy-d-glucose (DDG) on HLB trees, and we are following the tree health and callose levels. These experiments are ongoing, but we already observe an improvement in tree health for the trees that were sprayed with DDG. Objective 4 (Engage stakeholders and disseminate our findings): Work from this project suggest that the phloem plugging, and other plant responses, play a bigger role than the levels of CLas bacteria inside the plant, in determining the disease severity. We were able to identify few tree characteristics that correlated with the yield of the tree, but the Ct value was not one of them. One of the values that was positively correlated with fruit yield was the percentage of photosynthetically active radiation interception in the canopy (%INT). Intercepted PAR (INT) indicates the amount of photosynthetically active radiation (PAR) caught by various canopy layers as the PAR travels through the canopy. Results from three different field trials showed that %INT (but not Ct values) can efficiently predict yield and can be used to efficiently determine the HLB disease severity. We conducted a field day for the Florida growers. We provided hand-on instruction for growers on how to measure light interception to determine the canopy density of the tree using a light meter and PAR-meter. Demonstrated measurements on two group of trees that received different nutrition.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Chiara Bernardini, Donielle Turner, Chunxia Wang, Stacy Welker, Diann Achor, Yosvanis Acanda Artiga, Robert Turgeon, Amit Levy. Candidatus Liberibacter asiaticus accumulation in the phloem inhibits callose and reactive oxygen species. Plant Physiology 28;190(2):1090-1094.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Stacy Welker, Amit Levy. Comparing Machine Learning and Binary Thresholding Methods for Quantification of Callose Deposits in the Citrus Phloem. Plants 11(5), 624
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Agustina De Francesco(p), Amelia H. Lovelace (p), Dipan Shaw, Min Qiu, Yuanchao Wang, Fatta Gurung, Veronica Ancona, Chunxia Wang, Amit Levy, Tao Jiang, Wenbo Ma. Transcriptome Profiling of Candidatus Liberibacter asiaticus In Citrus and Psyllids. Phytopathology 112(1):116-130
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Stacy Welker (G), Myrtho Pierre, James P Santiago, Manjul Dutt, Christopher Vincent, Amit Levy. Phloem Transport Limitation in Huanglongbing Affected Sweet Orange Is Dependent on Phloem-Limited Bacteria and Callose. Tree Physiology 9;42(2):379-390.
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Our project target audiances is the citrus industry stakeholders and the scientific community. We presented and discuss relevant results with citrus growers in grower's meetings and other extension activities. Grower's relevant data generated during this project was published in a citrus trade journal, and manuscripts with scientific data were submitted to research journals . Changes/Problems:The main changes from the plan occured due to the COVID pandemic, that caused adelay in recruiting the students and postdocs for the project, and let one student to leave, delaying the initiation of the work. What opportunities for training and professional development has the project provided?The work of the project team has provided training and mentoring fora graduate student, two post-docs and several technical workers. A geaduate student presented results from this work in the APS-southern devision meetin in 2021. How have the results been disseminated to communities of interest?We presented results of this work to the citrus steakholders in the 2021 Citrus Expo grower meetingin in Fort Myers, on the UF/IFAS citrus research website and in an article published in the Citrus Industry trade journal in August 2021. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The goal of our project is to increase our understanding on the Candidatus Liberibacter asiaticus (CLas)- phloem interactions, during HLB disease. These studies are challenging due to the phloem being a narrow tissue buried inside the stem, and the low and inconsistent titers of CLas in tissues studied so far. We have developed a novel system, using the citrus seed coats, which contain a extraordinary high amounts of CLas. The project will allow to identify bacterial genes that are important for CLas virulence, and citrus genes that are involved in disease progression and immune responses. Additionally, we are testing new treatments to reduce the disease progression, including downregulating genes that are involved in symptoms development and applying chemical treatments that will result in unplugging the phloem. Lastly, we will deliver our findings to the citrus stakeholders, while providing training to new student and postdocs. Manipulating the HLB related bacterial and host genes, and applying the treatments to unplug the phloem, will be used to increase HLB tolerance, and increase our capacity to control HLB by integrating strategies to keep plant healthy through genetic engineering and/or chemical treatments. Objective 1 (Phloem dynamics in CLas-infected fruits): We have collected seeds from HLB infected and uninfected Duncan Grapefruit fruits during the summers of 2020 and 2021. We have isolated the seed coat vasculature form these seeds, and confirmed the accumulation of CLas with qPCRs, Transmission electron Microscopy (TEM) and Fluorescence in-Situ Hybridization (FISH). We studied the dynamics of CLas, reactive oxygen species (ROSs) and callose phloem plugging in the healthy and infected seed coats. We have quantified the expression levels of genes related to callose and ROS in the seed coats. To further study CLas dynamics in the fruit, we also collected sweet orange fruits displaying various HLB symptoms, and isolated various fruits tissues for analysis under the TEM to compare CLas and phloem plugging in symptomatic and asymptomatic fruit. For RNA extraction from the seed coat vasculature, we tested four different RNA extraction methods (Qiagen RNeasy Plant Mini Kit, trizol extraction, phenol/chloroform extraction, and TENS-PCIextraction) and found TENS-PCI extraction method works best for citrus seed coat total RNA extraction. Eight total RNA samples (4 infected and 4 healthy) are ready to besubmitted for RNA-seq assay. We also tested two methods for total protein extraction and found that one method (Sigma Plant Total Protein Extraction Kit) works well for extracting total protein from citrus seed coat. We are waiting for the MS spec for proteomics analysis. Objective 2 (Identify novel CLas-phloem interactions in HLB progression): We have completed two CLas transcriptome analyses. Firstly, we analyzed the CLas transcriptome using bacterial cells enriched from the leaf midrib tissues. We determined the highly expressed CLas genes, which offer insight on the most important biological functions when CLas colonizes citrus leaves. We also profiled the differentially expressed CLas genes in citrus vs Asian citrus psyllids (ACP). These results represent the first comprehensive CLas gene expression profiling and have been published in the journal Phytopatholgy (De Francesco et al., 2021). Secondly, we analyzed CLas transcriptome in seed coat. Using RNA extracted from seed coat vascular tissue, we were able to detect most genes in the genome (93-99%) - this is much improved from the previous dataset using the midrib tissues, which covered 32-69% of the CLas genome (this is still much higher than previous studies). We then analyzed the differentially expressed genes (DEGs) from midrib, seed coat, and insect samples. PCA analysis suggests that the transcriptomic profiles shift based on where CLas is in the plant and insect host; and there are specific changes between midrib and seed coat samples. For example, three Sec-dependent effectors (SDEs) were found to be specifically highly expressed in seed coat but not in midrib or in ACP. Objective 3 (Reduce phloem plugging to reestablish sugar transport):We are targeting three genes to downregulate by the CTV vector. We have successfully cloned truncated sequences from the three genes into the CTV vectors as confirmed by restriction digestion and sequencing. We Agro infiltrated the three CTV vectors into N. benthamiana where they established systemic infection which enabled us to isolate virions and successfully infect C. macrophylla. In C. macrophylla we confirmed the stability of the constructs by RT-PCR. As C. macrophylla does not reveal a pronounced HLB phenotype, we grafted each construct into a set of sweet orange seedlings. Upon confirming CTV infection in the sweet orange seedlings by ELISA against the coat protein of CTV, we infected the plants with HLB via the Asian citrus psyllid vector. Currently we are monitoring the development of the HLB phenotype in the citrus seedlings. We cloned a truncated version of an additional gene into the CTV vector as confirmed by restriction digestion and sanger sequencing. We are working to get these vectors into citrus. Furthermore, collaborator Dr Wenbo Ma identified two Clas effectors that are highly upregulated in the citrus plant. We introduced these genes into the CTV vector to over express them in the citrus plant. We are working to get these genes into citrus. These two effectors are also being transformed into Citrus grapefruit for stable expression using agrobacterium. Objective 4 (Engage stakeholders and disseminate our findings): Work from this project suggest that the phloem plugging, and other plant responses, play a bigger role than the levels of CLas bacteria inside the plant, in determining the disease severity. To test this in the field , we measured different tree characteristics to see which measurements are correlated yield. We were able to identify few tree characteristics that correlated with the yield of the tree, but the Ct value was not one of them. One of the values that was positively correlated with fruit yield was the percentage of photosynthetically active radiation interception in the canopy (%INT). Intercepted PAR (INT) indicates the amount of photosynthetically active radiation (PAR) caught by various canopy layers as the PAR travels through the canopy. Results from three different field trials showed that %INT (but not Ct values) can efficiently predict yield and can be used to efficiently determine the HLB disease severity. We have presented the results of this work in the Citrus Expo grower meeting, in the UF/IFAS citrus research website and in the Citrus Industry trade journal, to educate growers to use canopy characteristics, and not Ct values,as a better way to detemine HLB disease severity.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Amit Levy, Taylor Livingston, Chunxia Wang, Diann Achor, Tripti Vashisth. Canopy health, but not Candidatus Liberibacter asiaticus Ct values, are correlated with fruit yield in Huanglongbing affected sweet orange trees.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Agustina De Francesco, Amelia H. Lovelace, Dipan Shaw, Min Qiu, Yuanchao Wang, Fatta Gurung, Veronica Ancona, Chunxia Wang, Amit Levy, Tao Jiang, Wenbo Ma. 2021. Transcriptome profiling of Candidatus Liberibacter asiaticus in citrus and psyllids. Phytopathology.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li, Q., Canton, M., Wu, H., Zhang, X., Zale, J., and Mou, Z. (2021). Efficient artificial microRNA vectors for gene silencing in citrus. Plant Cell Reports 40, 2449-2452.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Merritt, B.A., Zhang, X., Triplett, E.W., Mou, Z., and Orbovic, V. (2021). Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant. Plant Cell Reports 40, 1947-1956.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Amit Levy and Tripti Vashisth. Citrus canopy health is highly important for HLB tolerance. Citrus Industry, August 2021, 10-13.
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