This is a 3-year project with 2 specific aims: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, besides AtMKK7 gene, we also cloned the Arabidopsis MOD1 and NAC1 genes, which have been shown to provide resistance to bacterial pathogens in Arabidopsis. The T-DNA vectors have been transformed into Agrobacteria and plant transformation is underway. We have generated transgenic citrus plants overexpressing the Arabidopsis NPR1 gene and showed that they are more resistant to citrus canker. We are currently testing the resistance of these transgenic plants to HLB. We are also generating citrus plants that can accumulate more salicylic acid. For AtMKK7, about 1700 explants were incubated and regenerated shoots were tested with PCR. Twenty of these shoots were positive in the screen and all of them were grafted onto Carrizo. The twenty transgenic lines are being propagated. The presence of the AtMKK7 gene in the transgenic plants will be confirmed by PCR and the expression levels of AtMKK7 in each transgenic line will be analyzed using qRT-PCR. Resistance of the transgenic lines to citrus canker and greening (HLB) will be characterized when more transgenic plants are available. For objective 2, a total of 100 plates of hypocotyl cuttings (each plate with 40-50 stem pieces) was irradiated with a dosage of 30G gama ray. The irradiated stem pieces were placed on non-selective shooting medium. The plates are kept under 14 hour photoperiod. Shoots generated from the irradiated hypocotyls were transferred onto selective medium with 0.2 mM of sodium iodoacetate. Another batch of explants (90 tubes of ‘Duncan’ grapefruit seedlings) has been prepared for irradiation. For each irradiation, ten plates of hypocotyl cuttings were kept as non-irradiated controls for comparison. We will prepare more hypocotyls for irradiation.
Over the past quarter, we have continued to develop all aspects of our project. In particular we have progressed in the following areas: 1. Building and testing additional TAL effector and promoter constructs. We have synthetically assembled a number of TAL effector genes matching X. citri TAL effectors and showed that they transcriptionally activate our broad recognition or “super” promoter in a Nicotiana benthamiana system. We have also assembled promoters with individual TAL effector binding sites to test activity and specificity. 2. Testing activation of gene constructs against a diverse world wide collection of X. citri isolates. Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. Additionally, we are preparing and testing X. citri strains with single or multiple disruptions in their TAL effector complement to test the role of specific TAL effector proteins in the disease and resistance process. 3. Stable transformations. The transformed lines generated last Fall and Winter have progressed through selection, shoot formation and rooting, and are now growing in soil. These lines are tested by PCR as they reach adequate size, and positively scored lines have been subjected to pathogen testing by pin-prick assay with X. citri. We have identified several canker resistant transgenic lines. We are currently setting up additional transformations to generate more transgenic material for line testing and with new promoter constructs. 4. Manuscript preparation We are in the process of drafting a manuscript of our results.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. Here are the major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results were published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. We are currently assessing citrus genes modulated by Las infection in 1) Comparison of citrus leaves, stems and roots to Las infection (completed, paper in writing), 2)Comparison of healthy vs. infected leaf samples in citrus grove (microarray data collected and QRT-PCR is underway),3) Comparison of different citrus varieties that are different in tolerance and susceptibility (in progress). The alteration of gene expressions by Las in leaf, stem and root tissues of Valencia sweet orange was investigated using Affymetrix microarray analysis. Out of 30,279 probe sets, a total of 8667, 2795 and 1142 showed significantly altered (p< 0.05) expression in leaves, stems and roots, respectively. Using 2 fold change as cut-off value, 1008, 580 and 58 transcripts were significantly up-regulated in leaf, stem and root tissues, respectively, whereas, 1109, 350 and 58 were correspondingly down-regulated in Las infected plants. Differences were observed for genes involved in cell wall synthesis and remodeling, lipid metabolism, photosynthesis, secondary metabolism, and starch and sucrose metabolism. Biotic stress induced signaling and transcription factors, PR-proteins, heat shock proteins, hormones and genes involved in protein modification, redox reactions and secondary metabolism were affected more in leaves and stems than in roots. PR genes were mainly repressed in roots but showed both patterns in leaves and stems; JA genes were up-regulated in stems, down-regulated in roots but up- and down-regulated in leaves; Calvin cycle genes were mainly altered in roots; SA and heat shock proteins were not significantly altered in roots. Transcriptional factors with WRKY, AP2/EREBP, MYB, bZIP, bHLH and Zinc finger domains were differentially regulated in all tissues, but least in roots. Differences were shown by C2C2(Zn) DOF zinc finger family proteins, affected in leaves and stems only; homologs of MEE47, nuclear factor PBF-2 and ATRR1 were regulated in roots only while MADS box transcription factor family and several unclassified transcriptional factors were down-regulated only in leaf tissues. We are further analyzing the data to understand how Las affects leaves, stems, and roots since they have distinct roles and function and how they contribute to the HLB disease development.
The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac; (3) Inoculation of the transgenic plants with the HLB pathogen, and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. We have identified three transgenic lines overexpressing CtNPR1. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants. We also conducted growth curve analyses. Nineteen days after inoculation, the bacterial population in one of the NPR1 overexpression lines is 10,000 fold lower than that in the control plants. These results demonstrate that overexpression of CtNPR1 confers resistance to canker disease. We also graft-inoculated the NPR1 overexpression lines with greening to determine whether NPR1 is functional in greening resistance. We are in the process of monitoring Candidatus Liberibacter asiaticus populations in the inoculated plants using quantitative PCR. Finally, transformation of the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene, is in progress.
In this first quarter, we have investigated the experimental design and set up of plant materials and growth conditions for small molecule therapeutic treatments. We have conducted a bioinformatics and literature analysis of the transcriptome data obtained in our previous project. We have identified three different therapeutic strategies that will be tested to evaluate if they enhance citrus response to the disease, prolong life of HLB-infected plants and reduce the bacterial titre and counteract the detrimental effects on production. We have identified that a possible cause of the induced disorder in infected plants is related to an unbalanced hormone-mediated response and crosstalk. In young leaves, where the pathogen is usually transmitted by the psyllids, Systemic Acquired Resistance (SAR) was unexpectedly not induced while several genes involved in jasmonic acid and ethylene signaling and response were activated. The inductions of few SAR genes (i.e. PR1 and DIR1) in mature leaves might be too weak a response to counteract pathogen colonization/virulence. Interestingly several genes typically involved in plant responses to biotic stress were induced in the fruits such as WRKY transcription factors. Induction of ethylene biosynthesis and signaling is another detrimental response for fruits. Based on these results we defined a therapeutic strategy that will enhance SAR response and counteract ethylene in early infected tissues. An important aspect of the disease is related to the imbalance of the source-sink homeostasis due to the differential regulation and induced of several key genes involved in glucose intracellular transport, sucrose and starch metabolism. Based on these results, we have identified from public databases (i.e. Genevestigator) possible molecules that can be applied on leaves regulating these genes to correct the sugar gradient between sink and source tissues. A differential regulation of genes involved in photosynthetic light reactions was observed in fruit and leaf tissues in HLB infected (dowregulation in leaves and upregulation in fruits). This could possibly be related to the feedback regulation of sugar signaling response. Based on literature analysis this is related xenobiotic stress responses, we have also considered a possible strategy based on sugar signaling. We have constructed a solid experimental design that will clearly identify the effects of each small molecule applications on specific host responses to HLB infection. Plant categories were composed by four categories: HLB-infected and Control (healthy, uninfected) plants. Both types of plants were divided in treated and untreated groups to dissect treatment effects due to the direct enhancement of the host responses to pathogen infection and those that can have indirect effects and involved in other plant functions. The following parameters were defined: methods of HLB-infection through grafting, physiological conditions, plant age and growth conditions, timing of small molecule applications, molecular and morphological analysis, environmental conditions in greenhouse. Plants will be grown in pots and infections have yet to be started. Infected bark pieces will be obtained from citrus infected trees from commercial orchards near Lake Alfred (Florida) and confirmed positive for pathogen presence using qPCR. Plants will be randomly arranged in the greenhouse and kept under natural light conditions at 17’25 ‘C.
As previously reported Severinia buxifolia (box orange) has been determined to be a host for the Ca. L. asiaticus and transmission to citrus was obtained. Transmissions of Candidatus Liberibacter asiaticus were successfully done from S. buxifolia to both healthy sweet orange and to healthy S. buxifolia in two separate tests and a third test was performed but not yet sampled. To date we have a 50% transmission rate from S. buxifolia to sweet orange and a 72% rate from S. buxifolia to S. buxifolia. As earlier reported some plants that tested positive using real time PCR (qPCR) later tested negative. These plants were tested each month during this period and continued to test negative. This may be similar to results found with Murraya paniculata (orange jasamine) that the bacterium may be transmitted to the plants but doesn’t survive if not reinoculated. We have developed a method to quantify live and dead bacteria in plants and are using it to determine the population of live Candidatus Liberibacter asiaticus in citrus and S. buxifolia. As reported the various rutaceae (Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica) were subjected to psyllid inoculation with Candidatus Liberibacter asiaticus since all be found to be graft incompatible with citrus. As reported all of these hosts were new ones for the psyllid and in transmission experiments the psyllid did survive and in some instances layed eggs on these hosts. To date only Choisya ternata again was found positive for Liberibacter asiaticus. Positive psyllid transmission tests with Candidatus L. africanus in quarantine with Choisya ternata were obtained. Results with Choisya arizonica are still pending. Calamondin (Citrus madurensis) was infected via grafting with Candidatus L. asiaticus and psyllid transmissions were done to sweet orange with the results pending. Surveys of wooded riparian areas near orange groves that have an edge effect of what appears to be the first entrance of HLB have been sampled for the presence of alternative hosts of HLB. We have run over 100 plants of various types including citrus and to date only citrus species have been positive for Candidatus Liberibacter asiaticus and that number is very low. Sampling will again be done this spring when psyllids are active. Primers to a different region of Candidatus Liberibacter asiaticus were produced and tested. The new primer set and probe generically detects all Candidatus Liberibacter species including the potato zebra chip bacterium (Candidatus Liberibacter solanacearum). These primers and probe were tested by two other labs for confirmation and our results were confirmed. They will be sent for testing to more labs working on Liberibacter species. We have developed these primers and probe to further check the qPCR using the 16s primers. Work to further test the susceptibility or resistance of the cultivar IAPAR73 continued. Numerous propagations of this cultivar were made on four different rootstocks and were subjected to further psyllid inoculation with Candidatus Liberibacter asiaticus. IAPAR 73 plants were prepared for quarantine for testing with Candidatus Liberibacter americanus and africanus.
The project has two specific aims. We outlined below the progress made for each of them in the final quarter. Specific Aim 1: Identify sweet orange responses to Huanglongbing disease (HLB) through deep transcriptome profiling using new DNA sequencing technologies. We successfully completed the analysis of transcriptome data from immature fruits, the final developmental stage considered. We have mapped the entire deep sequencing data obtained from the four types of samples (young and mature leaves, immature and mature fruits) using a customized reference composed of NCBI unigenes from two citrus species (C. sinensis and C. clementina) and the Affymetrix sequence dataset. This analysis has increased the number of annotated HLB-regulated genes. In total, 12-36 million 85 pair-ended reads were obtained. We identified 6383-11407 genes as HLB-differentially regulated (log Fold Ratio ><0.5) and functionally categorized using Mapman knowledgebase. We grouped them based on the regulation at different stage of disease identifying those that are specifically or commonly regulated at asymptomatic and symptomatic stages. We performed a gene set enrichment analysis to identify pathways that are highly affected by the disease such as sucrose and starch metabolism, glycolysis, pentose-phosphate and phenylpropanoids. In leaves, we observed a clear down regulation of photosynthesis and the up regulation of genes involved in sucrose and starch metabolism. These altered transcriptional regulation might induce an increase in glucose and a decrease in sucrose accumulation resulting in an alteration in source-sink homeostasis. In HLB infected fruits, photosynthesis was up regulated while starch metabolism was repressed. Specific Aim 2: Define and validate gene networks and identify host (sweet orange) response biomarkers regulated by HLB at different stages of infection. The major causes of the detrimental effects of HLB disease might be related to an inappropriate hormone-mediated response which disrupts source-sink homeostasis between leaves and fruits. The innate immune system is down regulated. SAR was not induced in infected young leaves and the crosstalk between JA, SA and ethylene seems to play a key role in pathogen success. Other hormones such as gibberellins and cytokinins were differentially regulated in both tissues. We dissected the different hormone signaling and their crosstalk choosing important key genes for qRT-PCR validation. Our analysis identified heat shock proteins as major hubs in the protein-protein network and qRT-PCR confirmed transcript down regulation at all stages of the disease. In addition, five-ten early regulated transcripts were identified as good host biomarkers of asymptomatic infections in different developmental stages and tissues. These genes belong to photosynthesis, sucrose metabolism, volatile pathways, transcription factors. These genes may be used to distinguish infections before symptoms appear and as host response complement to qPCR pathogen detection.
Three new formulations of SPLAT-DMDS were developed and evaluated. The formulations release the DMDS active ingredient for up to 3 months, which is approximately 3 times longer than previous formualtions. However, results in the field have still been inconsistent. In certain field trials, were have been able to measure reduced ACP populations but in other experiments there appears to be no effect. The most promising results are obtained when the repellent is deployed after the populations of ACP are initially killed off with an insecticide. Subsequently, psyllids appear to re-colonize DMDS-treated plots slower than untreated plots. However, even these experiments have been inconsistent. An experiment was conducted during the spring and summer that tested the effect of an olfactory psyllid repellent under standard psyllid management practices. The experiment has concluded. The formulation tested was the initial SPLAT-ACP Repel. Throughout this period, three applications of the formulation were made (April 29th and May 29th and June 29th). Applications were made to 3-4 acre replicated blocks of citrus and identical adjacent blocks were used as controls. The experiment was arranged as a randomized complete block with four replicates. All blocks received the same standard psyllid management with pesticides. Psyllid populations were monitored once or twice per week March 16th through August 18th. Yellow sticky monitoring traps were used as well as the tap sampling method. Throughout the course of the experiment, psyllid populations were extremely low in the experimental plots (Below 0.25 psyllids per ten traps per block on average). We observed no significant additional reduction of psyllid populations in the plots that received the olfactory repellent as compared with control blocks without the repellent. This result was likely due to the insecticide use practiced in this grove resulting in psyllid population densities that were likely too low to allow measurement of a treatment effect. During the initial part of the season, we observed that the DMDS active ingredient was highly phyto-toxic when the SPLAT formulation was applied to peripheral (pencil-thin) tree branches. However, application to larger branches (3-5 inch diameter or greater) did not cause phyto-toxic symptoms. We have also developed a method to measure DMDS concentrations in the field and we are using this method to determine how much of the DMDS or other repellents occurs when we deploy it from SPLAT or other release devices. Our intent is to determine exactly how much DMDS we need deployed in the field to achive desirable effects, so that a more consistent release device can be developed.
The goal of this proposal has been to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to understand its role in disease spread in field. We found that Ca. Las was sexually transmitted from Ca. Las infected male psyllids to healthy females but not from infected females to healthy males or among psyllids of the same sex. The experiments were repeated this season to confirm the findings. Our findings were consistent with our previous findings and we were able to confirm that Ca. Las is sexually transmitted between the adult psyllids during routine mating. We were able to detect bacteria in ACP ovaries of recipient females with PCR. However, we were unable to detect the presence of bacteria in genital parts of male and female psyllids with scanning and transmission electron microscopy probably due to washing of bacteria during sample preparation procedures. However, we found some other unidentified bacteria like structures in ovaries of recipient females. In addition studies are in progress to determine if Ca. Las recipient females are able to new citrus plants. Our ongoing efforts focus on identification of unidentified structures and to exploit florescent in situ hybridization analysis to detect presence of bacteria in genital parts of recipient and of the donor psyllids. The procedures for this are being standardized in collaboration with microbiologists.
In the last few months, we have continued working on genetic transformation of mature material from the three sweet orange genotypes (Valencia, Hamlin and Pineapple) with the aim of improving transgenic regeneration efficiency and having a reliable mature transformation procedure for each type that could be reproduced in Florida. After some last refining, Valencia sweet orange is routinely transformed at IVIA now. The transgenic nature of the first plants acclimated to the greenhouse has been confirmed through Southern blot analysis. Hamlin is more difficult to transform but with appropriate modifications of the tissue culture media and the source material used we have been able to produce already many transgenic plants, as confirmed also by Southern blot. Pineapple is routinely transformed at IVIA since the 90’s and is being used as control. Transformation of mature Carrizo citrange was initiated later, simply because we had not enough space and personnel to work with all the genotypes at the same time. During the last quarter, we have been more focused on developing a reliable transformation system for this genotype. More than 100 mature transformants (PCR-positive shoots) have been produced so far. The key in this case is using proper source material. We have preparing new source material to attempt transformation of mature citrumelo and grapefruit in the coming months. Regarding our second objective, at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes are established in the greenhouse and we are now characterizing them in detail (genetic and phenotypically). Additionally, a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis has been synthesized and incorporated into Agrobacterium tumefaciens. It will be used to transform Carrizo citrange. In Florida, construction of the growth room has been finally initialed and according to the schedule it will be finalized before the end of February. The PI and his greenhouse manager are planning to travel to Florida next March to supervise and setting up plant growth conditions, to set up the healthy citrus mother materials, and to establish substrate, fertirrigation and phytosanitary treatments.
This is the first quarterly report from our project. Collaborators in Florida (M. Hilf, T. Gottwald) have been routinely sending a variety of citrus-greening infected citrus samples to the De La Fuente Lab, including leaves and fruits from grapefruit, pomelo, and orange. Methods have been developed for processing the samples to provide both DNA for molecular testing and bacterial suspensions for microscopic and culture-based investigations. To obtain DNA samples, a variety of tissues and tissue-disruption methods have been tested to determine which sample type and processing method produces the highest DNA yield of Liberibacter asiaticus (LAS). Leaves, pith, pulp, juice, seeds, and stems have been tested using chopping and bead-beating with various sizes and types of beads for tissue disruption. DNA was extracted from the disrupted tissue samples, and PCR was the molecular method used to determine which tissue type and disruption method yielded the highest concentrations of DNA from the bacterium. Two different PCR reactions were first compared using positive control samples and the PCR with the lower limit of detection was selected for subsequent analyses. Overall, seeds contained the highest concentrations of LAS, and the bacterium was most effectively released from the tissue using bead-beating with metal beads. Work has been conducted to implement a quantitative-PCR assay as well so the relative quantities of LAS in the samples can be measured. For microscopic and culture-based investigations of the citrus greening bacterium samples, tissue samples were initially processed in the same ways as for the DNA samples except under aseptic conditions. Samples of the bacterial suspensions produced by the tissue disruption were then plated on several kinds of low-nutrient media, some containing juice extracts from the fruits. Cultures of other bacteria (including Pseudomonas spp.) were also plated alongside some of the samples to potentially encourage growth of LAS. A collection of unknown bacterial isolates which grew from the plated bacterial suspensions from infected fruits were preserved in glycerol in the freezer for later identification and experimentation. The bacterial suspensions were also placed in sterile microfluidic chambers for observation under the microscope. Bacteria were observed in the samples in the microfluidic chambers, but it has not been confirmed if the visible bacteria were LAS. The dimensions of the bacterium observed in the chambers correspond with published data for LAS, but no growth was observed after 10 days of incubation.
Obj. 1,2. Construct cDNA libraries from (a) adult/immature psyllids, dissected gut, salivary glands and accessory salivary glands; Sequence random cDNA clones, assemble ESTs, and select unigene sets; Obj.3. Produce RNA-seq libraries and compare transcript levels in psyllids and digestive organs at key acquisition access periods (AAP) to determine the relative abundance of insect mRNAs affected by bacterial infection. NCGR assembled the Illumina reads and made available files of the UniTran sequences and expression levels for each library. We assembled the 454 UniTrans (previously assembled with PAVE) with the Illumina UniTrans. The 454 assembly has 59002 singletons and 18998 contigs, and the Illumina had 121444 contigs; these assembled into 153482 Unitrans (singletons and contigs) and 12708 has at least one 454 sequence and one Illumina sequence. The assembly has been annotated as described in the last report (i.e. with UniProt, GO and GOSlim); 18339 UniTrans have a protein hit. As the expression levels for the new super-Unitrans must come from the original assemblies, the PAVE schema and software have been modified accordingly, and we are currently modifying the interface to for correct display and query of the expression levels of this super-assembly. The Hunter ESTs have been downloaded from GenBank and are currently being split into libraries and assembled. B. cockerelli libraries analogous to the ACP cDNAs were prepared as paired end libraries during the last quarter. Libraries are expected to be submitted to the sequencing lab in January 2011. Data-mining, qPCR analysis of ACP transcripts, FISH probe design and optimization using our epifluorescence microscope equipped with a filter for Cy5-labeled probes commenced. Ten psyllid genes have been selected for initial validation by PCR, RT-PCR and FISH localization as potential targets for RNAi constructions. Additionally ten Ca. Liberibacter genes potentially involved in the infection cycle were selected from the published genome sequences. Primers have been designed and are under validation by PCR and RT-PCR. Transmission studies continue in order to determine % efficiency transmission by single B. cockerelli psyllids to tomato plants using liberi-infected and -uninfected colonies over a range of AAPs and IAPs. Key time points will be identified and results validated by qPCR for extrapolation to time point feeding for ACP and RNAseq analysis. Asian citrus psyllid colonies are successfully being reared on citrus plants, now showing 80% infection with selected plants are confirmed positive by qPCR. Time course feeding will commence when flush growth appears during Spring 2011 and psyllids will be collected for RNAseq library construction (7-8 reps each).
Obj1-DNA bar coding to establish the identity and diversity in south Florida (Stansly, Brown).PCR primers were used to obtain a ~1200 bp product; analysis underway for 50 seqs plus sequences obtained for potato psyllid (outgroup). Obj2-qPCR detection of Ca. Liberibacter in potato psyllids is optimized; in progress for the asian citrus psyllids (from etoh). Obj3-Define the gross association of Ca. Liberibacter in thick sections’develop a ‘gross anatomical road map’ of Ca. Liberibacter accumulation in key organs, tissues, and cells; Obj4-Using the resultant ‘thick section road map’, elucidate at the TEM level the specific organs, tissues and cells where Ca. Liberibacter accumulates in the vector. Adoption of the potato psyllid for study here in Arizona has proven to yield windfall savings in time, expense and manpower toward understanding the anatomical particulars of Liberibacter transmission. The most important observations came from dissection and staging of guts, from all instars, for the scanning electron microscope (SEM). As this exercise was initially very labor intensive, specimens were processed in small lots. As observations and correlations came into view, we realized how powerful they were, revised our approach to the Objectives, and proceeded to increase our sample number (‘n’) many fold in association with the skill sets gained from the experience. We are now able to process larger lots rapidly and simultaneously, and so far, have examined a total of 195 uninfected and 138 infected guts. Results from these stagings have allowed us to develop several hypotheses and conclusions on the relationship between insect and pathogen, how the pathogen is transmitted, and the cost and benefit to the vector. We are now ready to draw up a manuscript on these findings for publication. We are continuing this approach, and have redefined our ‘unit specimen’ to include salivary glands and guts of each individual in separate clusters on the SEM stub. We can now directly correlate the degree of infection in the gut with the condition of the glands of each same individual. We have attached a fluorescent label to the bacterium inside extirpated guts, and want to move to the next logical step- development of techniques to label the bacterium inside the nonextirpated gut, the latter residing in its native configuration inside the deshelled abdomen, surrounded by all the other organs and blood. We’ve developed the skills to deshell the exoskeleton from the fixed tissues inside, and are experimenting with methods to eliminate non-specific labeling. We will then commence time-course studies in association with the acquisition access studies summarized below. Lastly, two directives for transmission electron microscope studies are in continuance. The first focuses on developing the probes needed to label the bacteria with colloidal gold. The second uses the techniques of Elliott (2007 Microscopy Today 15: 30-33) to stack Z-sections of the oral box into looped animations using PowerPoint and Macromedia Flash so that access by the bacteria can be studied in 3 dimensions. Time course IAP and AAP studies are underway. Subsets of adult psyllids are allowed defined feeding times, and assayed by qPCR to confirm bacterial presence, or prepared for SEM/TEM-hybridization analyses.
For Dec 2010 Methodology The plants were put in a growth chamber (photoperiod of 16/8 hours, temperature 24/22 oC, at 60 % relative humidity) day/night, 22/6, 24/16), as described in the previous report, and monitored periodically for presence of bacteria and symptoms of HLB. After confirm the presence of both Ca. Liberibacter asiaticus (CLas) and Ca. L. americanus (CLam) the tissues of five plants were collected for RNA extraction. Since the infection of Ca. L. asiaticus progresses faster than the infection of Ca. L. americanus, sample were collected at different infection time. The tissues of symptomatic plants infected were processed as described in the previous report, and was later performed the extraction of total RNA (RNeasy Mini Kit, Qiagen). The RNA was then analyzed for integrity, quality and concentration. The next step was to send the samples to Roche NimbleGen for microarray hybridization as previously described. Time course experiments – Since the proposal experiments were with Ca. Liberibacter americanus (there was publication with Ca. L. asiaticus), and considering the importance of time course experiments for a better understanding the gene network involved in the response to infection, we decide to set up a time course experiments with both bacteria. Results The first round of hybridizations were performed by Roche Nimblegen with a customized 385K chip containing 32,000 unigenes of C. sinensis. The raw data were pre-processed using the Robust Multiarray Average (RMA) by the NimbleScan software. Normalized data were imported to R package (Bioconductor) where statistical analyses were performed. A Bayesian moderated t-test statistic was calculated and p-values were adjusted for multiple comparisons by the false discovery rate correction. A total of 634 genes with P- values ‘0.05, fold change |logFC| ‘ 2.0 and odds probability ‘ 0.95 were considered differentially expressed at a statistically significant level. From those, 419 were up- and 215 down-regulated in symptomatic plants. In order to identify the most representative metabolic pathways regulated in infected plants we applied a gene set enrichment analysis approach by using the information of Gene Ontology and KEGG annotation. We found 45 different biological processes, including those involved in response to jasmonic acid stimulus, signaling and metabolism. There is partial agreement between our results with Ca. L. americanus and Albrecht & Bowman (2008) results. They have found 279 to 515 differentially expressed genes according with the infection time. Although the statistical approaches are similar (R package, Bioconductor, RMA, fold change, and False Discovery Rate), our results are based in wide DNA chips with only sequences of sweet orange. But the overall tendency is pointed out that the interaction of sweet orange with Ca. Liberibacter americanus is quite similar to the interactions with Ca. L. asiaticus.
Production of transgenic Citrus plants in the Core Citrus Transformation Facility (CREC) continues to be at the rate of about 100 plants per three months. Orders are being serviced for clients based both in Gainesville and in Lake Alfred. The demand for transgenic material is holding steady. Additional four orders were taken to produce transgenic grapefruit carrying genes harbored in following vectors: pWG22-1; pWG24-13; and pWG25-13, and pWG27-3. However, most of the activities of the facility are directed towards completion of previously placed orders. New orders are being serviced according to the order they were placed. The list of transgenic plants that were delivered within the last quarter includes those concerned with resistance to both bacterial diseases and CTV. Canker and HLB: 1) N1* gene: one Duncan plants; 2) NPR1: three Flame plants and superNPR1-six Flame plants; 3) AS7 gene: two Duncan plants and A13* gene: four Duncan plants; 4) pMKK7 vector: 20 Duncan plants; 5) pMOD1 vector: seven Duncan plants; 6) pNAC1 vector: one Duncan plants; 7) pSuc-NPR1 vector: three Duncan plants. CTV: 1) Gene in p33 vector: 26 Mexican limes, 16 C. macrophylla, and five Hamlin plants. CCTF also produced and delivered eight more Mexican lime plants for the pHK vector order. Change in genotype of these plants is not involved in response to plant pathogens. There are about twenty soil-adapted plants that will be tested by the PCR to confirm the presence of gene of interest in their tissue. Publication supported by this grant: Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266
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