The objectives of this research are 1) investigate the effect of heat stress on Las; 2) monitor healthy and HLB-affected citrus genome response to heat; and 3) optimize field thermotherapy. To reach our goals in objective 1, we have exposed both HLB-affected and healthy periwinkle (40 C) and citrus (42 C) to heat stress. DNA (to study phage gene copy number) and RNA (to study gene expression) has been extracted. Changes in gene copy number for genes located in the phage region have been studied. Additionally, expression levels for Las genes were studied with RNA-seq and several genes of interested were identified. Reverse-transcription PCR has confirmed the results of the RNA-seq analysis and has suggested a possible mode of action behind the successful elimination of Las seen in thermotherapy. For objective 2, we have conducted a comparison study between field heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the citrus trees treated with heating. Additionally, potted Las-positive and negative citrus were exposed to 4 hours for 4 days of 40 C, 85 % relative humidity (similar to heat exposure in field setting) in a controlled greenhouse. RNA-Seq data (from new flush present before heat treatment and the new flush that developed after heat) were analyzed. There were 3,722 differently expressed genes (DE) between Las-negative and positive trees not exposed to heat. Flush that appeared after heat treatment on the positive plants had 294 DE as compared to flush on unheated positive plants and 1308 DE as compare to flush on healthy trees. Some heat shock and oxidative proteins were identified in the DE lists. Additionally, potted Las-positive and negative citrus were exposed to 30 hours of 40 C, 85 % relative humidity and sampled during the exposure. RNA has been extracted from these samples. Time 0 before treatment and time 30 hours (of continuous heat) are being monitored for changes in gene expression for heat shock genes and other identified stress genes using RT-qPCR. As for the objective 3, over 3 years of prior data (tree Las Ct values, treatment procedures, and temperature logs from one location) have been summarized. Extensive analysis of temperature and humidity data using KS nonparametric test, ANOVA, Tukey’s HSD, and other measures have shown that each HLB-affected trees respond uniquely to heat treatment. The greatest decrease in Las titer and overall duration of this decrease varies for each tree (6-18 months) and is not solely dependent on heat but most likely affected by the biology of the tree. When comparing 7 versus 9 days of treatment, the longer treatment did not increase titer reduction. Also, six days was not more effective than a 4 day treatment. Regarding temperature, the greatest effect was present at 40, 41, and 42 C for 5 to 7 hours for 3 out of 4 test plots. A detailed correlation chart indicates other combinations of temperature and durations can also be effective at reducing Las. All statistical analyses shows that the response to heat stress is unique for each Las-infected tree. Although fruit drop did decrease for many of the treated trees, due to the large variation in data, the decrease was not statistically significant. Fruit was harvested and juice made. Volatile production analysis of juice from commercial grove 1 has been finished. A total of 63 aromatic volatile compounds were detected by HS-SPME-GC-MS. Discriminant analysis separated the “no heat” juice from the “heat” juice. Juice made from the product of the heat-treated trees had “fruity” and “pineapple fruit” top-notes. Juice is still being analyzed for sugar/acid, secondary metabolites, and Las titer. Juice quality taste panels are complete and panelists could distinguished between heat treated and no treatment juice sampled juice from the two commercial HLB positive Valencia groves (p<0.05). USDA Picos farm juice was not significantly different (p=0.052). Almost all panelists who differentiated between juice correctly said that heat-treated HLB juice was sweeter, less acidic, less bitter, and had more flavor and body than the juice produced from unheated trees.
Seed decontamination 1. Develop guidelines for seed propagation that prevent contamination of seed by citrus canker. Surface disinfection of seeds. Objective is to develop economical and easy-to-implement seed cleaning methods for removing Xcc bacteria without loss of seed viability. Progress: Citrus seeds obtained from a local grower were free of any X. citri infection. Therefore citrus seeds were artificially inoculated with X. citri . Seeds were soaked in a freshly prepared X.citri culture (OD 0.002) in a beaker for 1 hour. Contaminate seeds were then placed on sterile filter papers and dried at room temperature for 24 hrs. These were then treated with PPM and Chlorox according to the plan of the project. Seeds were rinsed in sterile water and serial dilutions of the rinsed water were plated for bacterial count. Similarly intact seeds were placed on LB for determining the efficiency of decontamination protocols. Some data has been collected, with the rest being collected from the pictures of LB plates. After completion of data collection, data will be analyzed. The same experiment will be repeated once more. Evaluating the 2006 CHRP decontamination procedure. Progress: These experiments are in progress along with experiments using sodium hypochlorite and PPM for decontamination. Development of rapid and sensitive molecular diagnostic methods for on-site detection of Citrus canker. Progress: Initial standardization experiments are going on. The PI met with our collaborators at UF to obtain additional isolates. Cutting and tissue cultivation On 7 July, 4 schedules of acclimation of tissue cultured citrus rootstocks were applied to C-35, C-54 sour orange, US-812, SW-13 and KC-13 root stocks. Any roots were removed and stems transplanted into a peat:perlite blend in seedling trays. Relative humidity (RH) started at 90% under layers of 30% and 50% shade cloth, overlain with reflective R-3 insulation board (from inside out). RH was decreased step-wise every 2, 3, 4 or 5 days until the end of days at RH = 50%. Covers were similarly removed. Success was very high for most varieties at the fastest acclimatization rate, at over 90%; except sour orange. A second set was started in early October. Cutting propagation began on May 6th and have generally been initiated monthly since. Initially cuttings with 1, 2 or 3 nodes were used. No differences in rooting were noted but multiple nodes required pruning to a single node. Thereafter single node cutting have been used. Different auxin concentrations and carriers were also evaluated. Best results were 4000 and 8000 ppm. Both concentrations have been carried forward. Success has been >90% to date using common rootstocks of Kuharski and C-35.
The last quarter of 2015 was dedicated to microscopy with additional time allotted to starting the second year experiments and to write manuscripts. There was a great deal of time dedicated to data analysis and field plus greenhouse preparation. In November, we began with the planned treatments both in the greenhouse and field. We followed the proposed plan of work with the following experiments. I: Effect of drenching application of SL on HLB-infected trees. Soil in potted Valencia trees was drenched with SL at the predetermined concentration. Tree characteristics were noted and changes recorded on a bi-weekly basis. II: Effect of spray application of SL on HLB-infected trees (Repeat experiment). This is a repeat of the experiments involving spraying SL on Valencia potted trees in the greenhouse. Tree characteristics were noted and changes recorded on a bi-weekly basis. III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. This treatment has been postponed. IV: Effect of spray application of SL on other varieties of citrus in groves. ‘Midsweet’ was selected as a second variety to be tested for SL effect on tree health. Tree physical characteristics and fruit drop are being monitored. V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic compounds, successfully used in other crops, are being tested for the potential effectiveness in citrus trees. So far, diluted sucrose solutions have been effective in enhancing new flush in trees with advanced stages of HLB. For all trees, data is being collected on the appearance of new growth, flowering, fruit drop and vegetative growth in general.
In the initial phase of this research project, development of field work protocol and fertilizer solution chemistries for the greenhouse portion have been the primary focus. Grove test site selection is still underway and once all of the grove sites are identified, the bulk of the field data collection work will begin. Of the four primary objectives, one and two have been the primary focus. Objective 1: Leaf nutrient thresholds We are working to identify and select groves that will be suitable for this phase of the research. One of the groves identified on the CREC campus was used to test the protocol of the work to be done on a regular basis. LAI, tree canopy volume, PCR, soil samples, and leaf tissue nutrition were all measured. Leaves for nutrition were measured for chlorophyll index (SPAD), leaf area, dry mass, and scanned using a flatbed scanner for color as well as leaf circumference and leaf area. Objective 2: Determine soil conditions that favor root health, including root hair and VAM proliferation i. While groves are still being selected for this research, the preliminary work, as mentioned in Objective 1 above, has been done and includes the soil sampling at the 0-6 inch and 6-12 inch depth. ii. Cuttings were taken from both healthy non-infected (HLB-) trees as well as HLB+ infected trees. These cuttings were rooted using plastic seed germinating domes to limit transpiration. After the cuttings rooted, they were switched to aeroponics chambers for further root development using standard fertilizer solutions. In the meantime, solution chemistries were developed to contain the correct mix of nutrients for the target phosphate concentration using soluble phosphorus, tricalcium phosphate (TCP) and rock phosphate (RP). Initial groups of HLB suspect cuttings were placed into three aeroponics chambers with specific nutrient chemistries. Those chambers are, Chamber 1, complete fertilizer minus soluble phosphate and using TCP; Chamber 2, complete fertilizer minus soluble phosphorus and using RP; and Chamber 3, complete fertilizer solution with soluble phosphorus. The purpose behind the use of solid TCP and RP is to establish stable solid-liquid equilibria that regulate concentrations of P and Ca in solution for optimizing root growth and health. We succeeded in establishing stable chemical equilibria with both PR and TCP in the slightly alkaline pH range, and the trees are now being grown in aeroponics to monitor and measure root proliferation.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Trangenic citrus plants expressing Las AI also display growth retardation. Meanwhile, to further study the function of LasAI in citrus, transgenic citrus were generated to express LasAI, LasAI N-terminal, LasAI C-terminal, LasAI repeat region, LasAII and GFP control, respectively. We have obtained transgenic citrus plants transformed with different domains of LasAI, Interestingly, transgenic plants show different degree of growth retardation, in particular the full length LasA1 and LasA1 C-terminal shows slower growth compared to the other constructs. Using RNAseq and RT-qPCR, we were able to identify the up- and down- regulations of some important genes involved in host-pathogen interactions and biosynthesis of secondary metabolites in these transgenic plants. Transient expression of LasAI and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Because of high level expression of these effector proteins, we developed a novel in vitro screening system that can evaluate small molecules against these Las effectors. The library consists of more than 30 million compounds obtained from the small molecule libraries of the TPIMS (Torrey Pines Institute for Molecular Studies). Interestingly, a few groups of compounds showed interference activity against the mitochondrial localization of LasAI. Meanwhile, to concert this screening, we developed another in vitro screening system in conjunction with the culture screening using Liberibacter cresence (Lcr). From these screening of 65 scaffold chemicals, we identified a number of chemical groups that disrupted the interaction between LasA1 and mitochondria and inhibit both Las and Lcr growth. We are narrowing down to individual compounds that inhibit the function of the Las AI effector or kill Las bacteria via other pathways, and measured the dosage effect of these potential candidate. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this transgene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector revealed its unique sub-cellular localizations. Understanding the molecular mechanism of how the effector induces HLB-like symptom is underway. We also analyzed the expression of LasA1 in Las-infected citrus plants. RT-PCR results indicate that LasA1 expression is correlated with severity of HLB symptoms. In particular, LasA1 was expressed more in the yellow leaves or the yellow spots than green spots of the symptomatic leaves with blotchy mottle. We successfully optimized the production of LasA1 polyclonal antibody. The new antibody increased the detection sensitivity and specificity. The new LasA1 antibody can detect LasA1 protein in Las infected periwinkle plants using western blot. Moreover, we detected LasA1 in Las infected citrus leaves in histological localization experiments. From the tissue print results, we identified the presence of a strong diffuse signal in the vascular bundle, indicating that LasA1 is abundant in las infected tissue and that diffuses in the phloem, which indicates that LasA1 was secreted outside of Las bacteria and move in the phloem system.
The objectives of this research are 1) to develop cost effective thermotherapy protocols for field application by optimizing temperature and relative humidity conditions in the tent; 2) to develop a mathematical model derived from our data and grower s data which will be used to determine the best treatment duration in future applications; and 3) to study gene expression of HLB-affected citrus plants that received heat treatment, and identify critical citrus genes that may be induced by heat stress for the benefit of suppressing HLB. To reach our goals in objective 1, we have exposed both HLB-affected and healthy periwinkle (40 C) and citrus (42 C) to heat stress. DNA has been extracted and amplified for Las 16S rRNA and certain phage genes. A standard curve for a normalization gene has been established and data is being analyzed using the delta Ct method. Additionally, changes in expression levels for these genes are being monitored. Reverse-transcription PCR is currently being used to confirm the results of gene expression data. As that Las cannot be cultured, a kill curve of Liberibacter crescens was determined. A dramatic decrease in viability was shown after L. crescens was exposed to 10 minutes of 46 C. For objective 2, we have conducted a comparison study between field heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the citrus trees treated with heating. Additionally, potted Las-positive and negative citrus was exposed to 4 hours for 4 days of 40 C, 85 % relative humidity (similar to heat exposure in field setting) in a controlled greenhouse. RNA-Seq data was analyzed using DESeq2 with a FRD of 0.5 and fold change above 2. Using new tender flush as a sample, there were 3,722 differently expressed genes (DE) between Las-negative and positive trees not exposed to heat. Flush that appeared after heat treatment on the positive plants had 294 DE as compared to flush on unheated positive plants and 1308 DE as compare to flush on healthy trees. Some heat shock and oxidative proteins were identified in the DE lists. Analysis and confirmation are ongoing. As for the third objective, over 3 years of prior data (tree Las Ct values, treatment procedures, and temperature logs from one location) have been summarized and are currently being used to determine an algorithm that relates environmental conditions with decreases in Las titer. Extensive analysis of temperature and humidity data using KS nonparametric test, paired T Test, and other measures have shown that each HLB-affected tree responds uniquely to heat treatment. The greatest decrease in Las titer and overall duration of this decrease varies for each tree (6-18 months) and is not solely dependent on heat but most likely affected by the biology of the tree. When comparing 7 versus 9 days of treatment, the longer treatment did not increase titer reduction. Also, six days was not more effective than a 4 day treatment. Regarding temperature, the greatest effect was present at 40, 41, and 42 C for 5 to 7 hours for 3 out of 4 test plots. A detailed correlation chart indicates other combinations of temperature and durations can also be effective at reducing Las. All statistical analysis shows that the response to heat stress is unique for each Las-infected tree. Although fruit drop did decrease for many of the treated trees, due to the large variation in data, the decrease was not statistically significant. Juice quality taste panels have just been completed. Panelists could tell the difference between juice from commercial Valencia trees that were heated and not heated with a 95-99% confidence interval. Further analysis of taste panel data is ongoing. Volatile production analysis for one group of data (out of three) has been finished. A total of 63 aromatic volatile compounds were detected by HS-SPME-GC-MS. Discriminant analysis separated the “no heat” juice from the “heat” juice. Juice made from the product of the heat-treated trees had “fruity” and “pineapple fruit” top-notes.
The program research objectives are to develop an effective and sustainable bacteriophage (phage)-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. Our approach has been to develop a bank of virulent (lytic) phages and/or antibacterial particles called tailocins , which are derived from phages. We have identified seven tailocins with activity against Xac and developed a large bank of virulent phages representative of the Caudovirales (tailed phages). Tailocins XT-1 and XT-4 exhibit broad host activity, killing 13/13 Xac isolates in vitro and have shown efficacy in greenhouse studies. A tailocin cocktail composed of XT-1 and XT-4 reduced lesion formation by an average 51% , as compared to non-tailocin challenge plants inoculated only with Xac. We have previously reported on the identification of the cassette encoding for tailocin XT-1.We now report that the tailocin cassette is 16,880 bps and encodes for 22 ORFs. We have identified genes encoding for the tail tube, tail sheath, tail fiber, baseplate assembly and tail tape measure proteins. Further analysis of the XT-1 cassette will identify all genes and allow for an initial bioinformatic comparison of tailocins XT-1, XT-4 and XT-7. The newly identified XT-7 tailocin shows activity against 10/13 Xac isolates tested including the Xac 306 strain. The type IV pilus dependency of Xac phages CCP504 (podophage), CCP513 (siphophage) and CCP519 (myophage) was confirmed using a pilA gene deletion Xac mutant. The deletion mutant exhibited no twitching motility and was resistant to all three phages, whereas the in trans complement exhibited twitching motility and was sensitive to the three phages.
The focus of the project is to develop a bacteriophage (phage) and/or phage components (tailocins) system with activity against Liberibacter crescens strain BT-1 that will be a model for Candidatus Liberibacter asiaticus . Strain BT-1 harbors two prophages (LC1 and LC2) that we have determined are not inducible when the organism is exposed to UV or oxidative stress, which may indicate that the prophages are defective. Both of the phages are predicted to be podophages (i.e., no separate tail structure). Multiple constructs are being produced to determine the correct fusion that will produce a mature and active tailocin with activity against L. crescens. The assay system we have developed for L. crescens will be used for testing activity. Our goal is to construct tailocins with specific targeting to L. crescens by changing the C-terminal portion of the tail fiber. As a method to determine validity of the approach, we have constructed an unmarked and in-frame deletion mutant of the tail fibers of tailocin Bcep0425 and complemented in-trans with the homologous tail fiber and chaperone genes. We then constructed a series N-terminal tail fiber region derivatives (i.e. 153aa, 183aa and 243aa) of tailocin Bcep0425. When the N-terminal regions were joined with the heterologous C-terminal tail fiber regions of a known prophage we observed no activity against the new target. This is not surprising since the tail fiber structure is an elongated homo-trimers with a fibrous morphology and .-sheet topologies with unusual repetitive folds such as a triple .-helix, so it is necessary to identify the correct fusion. We will continue to make new constructs to identify the correct fusion. We are also continuing to search for naturally occurring phages of L. crescens. Rhizobium spp., Agrobacterium spp. and L. crescens BT-1 were used as hosts for enrichment with weed extracts, water and soil samples. No phages were detected for BT-1, however Rhizobium phages were isolated but showed no activity against L. crescens.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. During the grant period (October 1, 2012 to September 30, 2015), funding was used to provide commercial grade citrus plantings for researchers described below at the Mid Florida Citrus Foundation A.H. Krezdorn Research Grove: UF/IFAS Researchers o Plant Improvement Team (Drs. Grosser, Gmitter & Castle) . New cultivars . HLB resistant rootstocks . Deciduous crops (Dr. Castle) o Entomology (Drs. Rogers & Stelizinski) . HLB vector management . Management of other insects and mites o Plant Pathology (Dr. Dewdney) . Alternaria resistance o Horticulture (Drs. Futch, Wang, Albrigo & Vashisth) . Weed Management . Anti-microbials . Foliar nutrition USDA o Citrus Rootstock Improvement (Dr. Bowman) . HLB resistant rootstocks Private Organizations (Citrus Projects, unless otherwise noted) o New Varieties Development & Management Corporation . Evaluation of promising cultivars o Florida Agricultural Research o Syntech o Eurofins o Massey Research Company o Ag Consulting o Evans Properties . Pecan
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). Our previous study indicate that Las contains a functional SA hydroxylase that degrades SA and its derivatives. SA and its derivatives play important roles in plant defenses. Las employs SA hydroxylase to suppress plant defenses. Our central hypothesis is that we can improve HLB management by counteracting SA hydroxylase. We will focus on counteracting SA hydroxylase using inhibitors based on structure based design. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design will be tested for their inhibitory effect against SA hydroxyalse.
The goal of the proposed study is to characterize the effect of using endophytic microbes in controlling HLB. Our hypothesis is the outcome of the interaction among Las, psyllid and citrus is affected by the citrus phytobiome. In order to achieve the goal of this study, the following objectives will be conducted: Objective 1. To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees. In this objective, we will investigate the phytobiomes and endophytes of survivor trees and HLB diseased trees. Metagenomic approaches will be used to investigate the microbiomes of survivor trees and HLB diseased trees. Metatranscriptomic approach will be employed to characterize the expression profile of the microbiomes of survivor trees and HLB diseased trees. We will culture the most representative or beneficial microbes based on metagenomics information. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. In this objective, we will test whether the endophytic microbes affect citrus attractiveness to psyllids, psyllid feeding, and Las establishment in planta. The endophyte application will be through grafting roots or branches of survivor trees or using cultured representative or beneficial microbes from survivor trees. Grafting allows endophytes in survivor trees to be transmitted to testing trees directly without the culturing step and without risking the loss of microbes that are difficult to cultivate. We have extracted RNA and DNA from the rhizosphere, rhizoplane, and endosphere from healthy and HLB diseased trees. The samples have been sent out for sequencing analysis. We will have a better picture about the microbiome of citrus after analysis of the data.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. Three bacteria showing high antimicrobial activities have been sequenced to help us understand the mechanism. Currently, the genome sequencing was finished and we are analyzing the results. Four bacterial strains: two Burkholderia, one Pseudomonas geniculata, one Rhodococcus strains have been tested for their activity in and all showed induced plant defenses and against infection by Xanthomonas citri. To further study the antimicrobial producing bacteria, tow Burkholderia strains have been labeled with GFP tag. Seven other strains are being labeled with GFP or RFP tag. We also investigated the antibiotic genes in nine antimicrobial producing bacteria that we isolated previously. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. For the field trial, we have selected the grove and conduct survey on HLB disease severity. We are comparing the different delivery methods to improve the efficacy of beneficial bacteria. A root drench delivery method has been established. This delivery method will apply bacteria close to the roots and reduce the loss during surface application. We continue to isolate and test the antimicrobial producing bacteria from Florida citrus groves. To understand the beneficial traits, we have sequenced multiple bacteria. The data is under analysis.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. TALEN targeting the promoter of CsLOB1 is also being done using citrus protoplast. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been accepted for publication by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. Currently, we are constructing different sgRNA sequences to target CsLOB1. We are continuing to generate more transgenic lines to get biallelic mutations in the EBE region of the CsLOB1 gene. We have sequenced the EBE regions of several commercial varieties to design appropriate sgRNA for gene editing. We are improving the gene editing efficacy by improving the Cas9/sgRNA constructs. In addition, we are studying the function of the susceptibility gene CsLOB1 and its downstream genes.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). Our previous study indicate that Las contains a functional SA hydroxylase that degrades SA and its derivatives. SA and its derivatives play important roles in plant defenses. Las employs SA hydroxylase to suppress plant defenses. Our central hypothesis is that we can improve HLB management by counteracting SA hydroxylase. We will focus on counteracting SA hydroxylase using inhibitors based on structure based design. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design will be tested for their inhibitory effect against SA hydroxyalse.
The goal of the proposed study is to characterize the effect of using endophytic microbes in controlling HLB. Our hypothesis is the outcome of the interaction among Las, psyllid and citrus is affected by the citrus phytobiome. In order to achieve the goal of this study, the following objectives will be conducted: Objective 1. To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees. In this objective, we will investigate the phytobiomes and endophytes of survivor trees and HLB diseased trees. Metagenomic approaches will be used to investigate the microbiomes of survivor trees and HLB diseased trees. Metatranscriptomic approach will be employed to characterize the expression profile of the microbiomes of survivor trees and HLB diseased trees. We will culture the most representative or beneficial microbes based on metagenomics information. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. In this objective, we will test whether the endophytic microbes affect citrus attractiveness to psyllids, psyllid feeding, and Las establishment in planta. The endophyte application will be through grafting roots or branches of survivor trees or using cultured representative or beneficial microbes from survivor trees. Grafting allows endophytes in survivor trees to be transmitted to testing trees directly without the culturing step and without risking the loss of microbes that are difficult to cultivate. We have extracted RNA and DNA from the rhizosphere, rhizoplane, and endosphere from healthy and HLB diseased trees. The samples have been sent out for sequencing analysis. We will have a better picture about the microbiome of citrus after analysis of the data.
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