Under Objective 1: In order to confirm biofilm formation is a bacterial survival strategy and not just a physical aggregation, biofilm was induced in vitro bacterial suspensions of the wide host range strain of Xanthomonas citri susbp. Citri (Xcc). RNA extractions were performed from bacteria from the culture suspension (planktonic state) as well as those deposited on a glass slide surface (biofilm state). Real Time RT-PCR using GumD primers previously demonstrated as valid for viability determination of Xcc was used to assay the same concentration of RNA from planktonic or biofilm bacteria. Higher level of viability was detected for those bacteria in aggregated (biofilm) form compared to those in planktonic form. Greater viability of the bacterium in aggregates was also confirmed using the labile GFP probe to detect viable cells with confocal microscopy, and the use of a bacterial respiration assay. Differences in viability between the aggregated and planktonic bacterial states increased through the time based on GumD RNA detection. No viable cells were detected in the planktonic form after 72 hours while viability was maintained in the biofilm form. RNA detection of Xcc viability was also tested on plant material. To do so RNA extraction was performed from canker lesions after Xcc inoculation of sweet orange leaves. Positive results were obtained from lesions after no treatment but not after a bactericidal treatment of the extract by heating. Under Objective 2: In preliminary assays of bacterial viability for aggregates treated with bactericide treatments was evaluated by the methodology described above. Complete elimination of viable bacteria was not achieved with any of the bactericide treatments evaluated. New assays are currently in course with different bactericide concentrations.
The antibody developer, Creative Biolabs, Inc., now has the purchase order from Penn State, which was required to initiate the project. The target peptide for NodT is now being synthesized. Screening of antibody libraries for high-quality antibodies is expected to take approximately 6 weeks, and was initiated on April 24, 2012. They plan to isolate about 5-20 antibodies with ability to bind the NodT protein.
For the Asian citrus psyllid (ACP), we constructed and sequenced two Illumina Paired-end libraries for ACP uninfected/infected salivary glands (SGs) and generated 818 M reads, totaling 83 Gb raw data. We are presently combining the ACP SG with the alimentary canal (guts) data generated previously to make the assembly DcGs. In addition, combining the data that we generated from two 454 Titanium libraries and six Illumina Paired-end libraries of Asian citrus psyllid (ACP) and the Sanger sequence data from Genbank for this species, we have made a hybrid assembly ‘ACPisp’ to yield 98,265 Unitrans from 140 M Illumina reads, 664 K 454 reads and 19 K Sanger reads, respectively. Of the 98,265 total unitrans, 22,543 unitrans (23%) were annotated with an average unitrans length of 1,848 bp. This dataset will provide deeper insights into the whole trancriptome of ACP. To translate peptide sequences from transcript (coding) sequences: (1) the ESTScan program was installed, (2) the hemiptera sequences were downloaded from Genbank, (3) these were used to train the ESTScan matrix, and (4) ESTScan was used with the hemiptera matrix to extract the peptide sequences from the ACPisp transcript sequences. An additional matrix was created using all the Genbank insecta sequences, and a second set of peptides were generated using the insecta sequences. The hemiptera matrix performed better than the insecta matrix, creating slightly more and longer sequences. ESTScan was also used to extract peptides from our WFB assembly using the same matrix. The two peptide files were used as input into orthoMCL, and the resulting clusters were used as input into cmpPAVE in order to investigate functionally similar transcripts between these two genomes. Secondly, we have made an extensive comparative transcriptome analysis of ACP and whitefly (also important agricultural sucking pest and invasive species), the potato psyllid, and pea aphid. Results indicated the presence of a suite of immunity and stress-related genes. Thirdly, for potato psyllid, we constructed and sequenced a suite of Illumina RNA-seq libraries to investigate infected and uninfected potato psyllid gene expression changes reared at different temperatures. We have identified a branch of genes that were differentially expressed under different temperatures in the Ca. Liberibacter-infected and uninfected potato psyllid. This work also will provide a good reference for the study of temperature affects for ACP in the future. We continue work on several manuscripts that are expected to be submitted during May-July.
The final step in completing the general transmission pathway is determination of the origin of Lib individuals that are able to access the stylets (adult, larval, or both) for reinoculation. Once acquired by adult PoP, the potential for dispersal of Lib by flight relies on its ability to exit the adult, or pass transovarially to the next generation larvae. Exit from either is, potentially, multidirectional. Our methodology is therefore scenario driven. Scenario #1) [Inoculum comes from primarily from larvae]. Our, and other lab’s, feeding experiments show that adults can transmit Lib during feeding, but our TEM shows no transepithelial, nor transcuticular, movement (n hot=20, n cold = 10), which would explain the origin of their midgut biofilms. If transmission by the adult is very inefficient, it must be transovarially transmitted to the next generation larvae after flight. SEM of all instars (n=333, 195) shows that 3rd and older larvae have biofilms on the midgut exterior also, but they have yet to be examined by TEM. It may be that larvae are the primary source of inoculum under field conditions. FISH of dissected guts assisted in identification of Lib, but does not distinguish between internal and external bacteria as needed for this scenario (pub. #1). #2) [Transepithelial movement to the adult midgut exterior may occur at point loci missed in our sampling.] FISH with thick paraffin sections of infra-hour time course AAP is required to see if Lib leaks thru the epithelium at point loci to establish blood-side biofilms. #3) [Access to the adult midgut exterior by chitinolysis through foregut/hindgut intima.] Alternatively, Lib may chitinolyse through weak points in the intima, and sprawl to the midgut exterior. #4) [Adult midgut biofilms as a source of inoculum.] Although the origin of adult midgut biofilms is unknown, we demonstrated that they sprawl anteriorly, proliferate in the oral region, burrow into the salivary glands, and reach their core. We have not found them inside the ducts yet, but this anatomy may get clogged or disabled by the biofilm’s intrusive movements. Lib are ca. half the diameter of the duct lumen. Alternatively, Lib may also access the mouthparts by chitinolysis through the oral region’s endoskeleton (purported for sharpshooters, Brlansky et al. 1983). Our study of the oral region (pubs #2, 3) elucidated its anatomy, but did not show any compromises of cuticle with Lib exiting through them. Views (n = 5) of the oral lumina, available in our TEM libraries, do not show fastidious bacteria, as occurs with sharpshooters (Alves et al. 2008). Our time-course experiments are underway, and when minimum AAP for adults is determined, FISH and colloidal gold may reveal where leakage into the interior occurs. Publications. We are preparing a third manuscript on the topic of: Stylet biogenesis and installation into their functional positions during the pharate adult stage of the potato psyllid, Bactericera cockerelli (Sulc). J.M. Cicero and J.K. Brown.
At this stage, we have completed Aim 1 (Identify genes positively regulating SA-mediated defense in citrus) and most work described in Aim 2 (Complement Arabidopsis SA mutants with corresponding citrus homologs). We have so far cloned more than ten citrus SA genes, all of which are at various stages of gene transformation and analysis of transgenic plants. We are actively working on Aim 3 (Assess the roles of SA regulators in controlling disease resistance in citrus) to make citrus transgenic plants over-expressing the SA genes and to assay the plants for resistance to HLB and citrus canker diseases. So far we have confirmed transgenic citrus expressing ctNDR1, ctPAD4, and ctEDS5. Additional constructs are in the pipeline of citrus transformation. While the cloned citrus SA genes are at various stages of analysis, the most advancement that has been made so far is with ctNDR1. We have obtained data to support that manipulating the level of ctNDR1 could lead to enhanced disease resistance. The main results on ctNDR1 are summarized below. We are in the middle of preparing a manuscript for publication. 1. Overexpression of CsNDR1 could complement the Arabidopsis mutant ndr1-1 for its disease susceptibility to and the lack of hypersensitive response to Pseudomonas syrinage avrRpt2 infection. ctNDR1 conferred resistance is largely dependent on the expression of ctNDR1 (dosage dependency) in Arabidopsis 2. The Arabidopsis NDR1 was previously shown to act downstream of a subset of resistance genes (i.e. RPS2 that recognizes avrRpt2) but not required by other resistance genes, such as RPS4. However, we found that ctNDR1 overexpression increases resistance to both P. syringae strains expressing avrRpt2 or avrRps4, suggesting the activation of general defense mechanism in the ctNDR1 overexpression plants. 3. Consistent with enhanced disease resistance to different pathogens, we found that higher expression of ctNDR1 also led to increased accumulation of salicylic acid, a key signaling molecule that activates broad disease resistance. 4. We performed quantitative RT-PCR analysis of NDR1 in mock-inoculated and Ca. L. asiaticus-inoculated ‘Cleopatra’ mandarin seedlings. We found that expression of ctNDR1 was inducible by HLB in two independent experiments (experiment 1 with 32 week-old seedlings and experiment 2 with 30 week-old seedlings). These results suggest a potential role of ctNDR1 in HLB resistance. 5. We have so far obtained 29 independently transformed transgenic citrus plants carrying ctNDR1 overexpressing construct. The presence of the transgene was confirmed by transgene-specific primers. We should begin to test these plants for resistance to citrus canker disease in the next few months.
In this research, we have been developing a repellent formulation for Asian citrus psyllid (ACP). A DMDS-based SPLAT formulation has been developed. It is currently formulated and produced by ISCA technologies. Our recent results have produced inconsistent results; however, there have been cases where the formulation has effectively reduced psyllid populations both in Florida and in trials in another state. In the final year of the project, we have tested new active ingredients that may be more effective than DMDS for several reasons. First, these newly identified active ingredients are less noxious than DMDS and simply do not smell as bad to humans. This makes them much easier to work with. Also, these chemicals are less phytotoxic and easier to formulate. Therefore, these new formulations may not have the drawbacks that are associated with DMDS-SPLAT. In this previous quarter, we have been working to evaluate these new formulations and we have determined that two of the newly identified active ingredients are effective in repelling psyllids when deployed in SPLAT. We are currently finishing up these tests to hopefully determine whether a more practical SPLAT-based repellent formulation can be developed than our current DMDS-based standard.
Our objective was to determine how psyllid behavior is affected by Las-infection of citrus. In previous experiments we have determined that ACP adults initially prefer to settle on Las-infected plants. This attraction to Las-infected plants appears to be dependent upon the plant release of the volatile chemical, methyl salicylate. Based on published data, it is known that Las-infected plants have nutrient deficiencies. Therefore, we hypothesized that while the Las-infected plants are initially attractive to psyllids, after prolonged feeding the psyllids experience imbalanced nutrition and choose to seek a better host. To examine this hypothesis we planned to determine how psyllids settle on plants with known nutrient deficiencies. Our previous experimental design was limited to settling behavior of psyllids on nutrient deficient and healthy plants. To obtain more robust conclusions, we have modified our experimental design to incorporate HLB-infected plants. In these experiments we plan to test psyllid settling preference in several scenarios: 1. Healthy vs. Nutrient Deficient Citrus 2. Healthy vs. HLB infected Citrus 3. Healthy vs. Nutrient Deficient Citrus + ‘HLB infected plant odor’ (HLB odor) 4. Healthy vs. Healthy + HLB odor If psyllids initially settle on HLB infected plants because of attractive odors and subsequently move due to the poor nutrient quality of the host, we expect the following results: In scenario 1, psyllids should settle and stay on healthy plants. In scenario 2, psyllids should initially settle on HLB-infected plants and then move to healthy plants (this has been previously shown in experiments). In scenario 3, psyllids should settle on nutrient deficient plants with HLB odor and then move to healthy plants. In the last scenario psyllids should settle and stay on healthy plants with HLB odor. In the last quarter, we have been successful in making nutrient deficient uninfected plants to use in these experiments. We sent leaf tissue samples from ten healthy, ten unfertilized (Nutrient Deficient), and ten HLB-infected Valencia plants to a private lab for nutrient evaluation. The results show that our unfertilized plants contain significantly less nitrogen, potassium, magnesium, calcium, boron, zinc, manganese, and iron when compared to Healthy plants. There were no significant differences in sulfur, phosphorus, or copper content between unfertilized and control plants. The HLB-infected plants have been fertilized regularly and are contain levels of nutrients that are not significantly less than healthy plants with the exception of zinc. We are in the process of assessing the volatile profile of the three plant types: Healthy, Nutrient Deficient, and HLB infected citrus. In preliminary trials we found that our existing volatile collection bell jars were too small to use on the plants selected, resulting in physical damage to the plants during handling. We have since ordered larger bell jars and are prepping for volatile collections. Upon analysis of Healthy, Nutrient Deficient and HLB infected plants, we will determine the characteristic components of HLB infected plant odor. These odors will then be applied to the plants according to the design outlined above.
Our progress for the current quarter is as follows: 1. Transgenic Duncan grapefruit lines expressing avrGf1 driven by the Bs3- PIP14 box promoter are currently being tested for resistance by spray inoculating X. citri subsp citri 306 at 10^8 cfu per ml suspended in sterile tap water on young citrus leaves. Resistance confirmed by transgenic lines will be assessed by evaluating canker lesions between transgenic and non-transgenic control plants. 2. Compared to AvrGf1, the type 3 effector AvrGf2, identified from X. fuscans subsp aurantifolii strain C, seems to provoke a faster hypersensitive reaction on Duncan grapefruit. The avrGf2 coding sequence was fused with the Bs3- PIP14 box promoter into the pK7FWG2 binary vector and transformed into A. tumefaciens strain Agl-1. In planta transient expression in Duncan grapefruit was assessed for induction by X. citri subsp citri 306 TALEs using the pathogen inducible promoter (Bs3- PIP14 box):avrGf2 construct for citrus canker resistance. Upon activation of the construct by Xcc TALEs, the expressed AvrGf2 elicited a resistance response that was faster and more effective than that of a Bs3-PIP14 promoter construct driving AvrGf1. 3. Transgenic Duncan grapefruit carrying the AvrGf2 construct are in the development pipeline to produce stable transformants. Transfomation was performed on 2,049 grapefruit epicotyl segments. 4. Germination and epicotyl transformation experiments with Duncan grapefruit are continuing. Molecular characterization involving PCR is ongoing and used to confirm transgenic lines from the putative transgenic plants regenerated. To date, a total of 190 putative transgenic plants have been screened and 23%, 20% and 6% of the plants tested positive for the Bs3 promoter, nptII and the avrGf1 genes, respectively.
Cytoplasmic citrus leprosis infected samples previously sent from our cooperator in Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. continued to be negative in PCR and antibody tests for cytoplasmic citrus leprosis virus. Additional sets of samples were sent during this funding period. All samples were prepared for electron microscopy to verify the PCR results. Viral particles similar to those previously published for cytoplasmic citrus leprosis were discovered in all the samples. Sizes and locations of the virions were verified. Sequencing was done and a new virus was identified by our lab in conjunction with another funded project. New primers are now available for detection and we plan to perform mites transmissions on both types of the virus at the USDA, ARS, FDWSRU in Fort Detrick. Mr.Leon in Colombia as earlier reported did mite transmission experiments with the previously PCR negative isolates from Colombia. With our new primers we have found that he has transmitted this new virus with Brevipalpus mites from Colombia. Symptoms of the new virus are identical to citrus leprosis.
All data analyses and writing for this research was completed, and a publication entitled “Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium” has been submitted to a peer-reviewed journal and is currently under review. Data finalized and discussed in this publication include: qPCR data from total and viable LAS cells over time in different culture media and next-generation sequencing data of the microbial communities associated with LAS in cultures, among several other aspects of the research conducted for the purpose of this grant. This data was also presented at the 3rd International Conference on Huanglongbing, and comments from other researchers in this field were incorporated into the manuscript. While this publication does not provide a defined method for the culture of LAS, it does provide many useful clues for prolonging LAS viability which we expect to be instrumental in future development of a culture medium via collaborations among the scientific community.
Cytoplasmic citrus leprosis infected samples were sent from our cooperator in Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. Five sets of samples were sent during this funding period and again samples did not arrive in very good condition due to delays in shipment. PCR was done on all samples and all samples were prepared for electron microscopy to verify the PCR results. Viral particles as previously published for cytoplasmic citrus leprosis were discovered in all the samples. However the PCR was not positive for cytoplasmic citrus leprosis. Since the samples were not positive samples for mite transmissions were not sent to the quarantine lab at the USDA, ARS, FDWSRU in Ft. Detrick for endemic mite transmission tests. Mr.Leon in Colombia as previously reported did transmission experiments with the mites and these PCR negative isolates from Colombia. Funding for Mr. Leon’s work was received and he continues to do transmission tests. PCR positive samples were successfully shipped from Mexico and Panama to quarantine in Maryland as controls for PCR tests. In January the first experiments were planned with with the endemic mites from Florida and PCR positive leprosis samples from Colombia. However none of the samples were PCR positive. We began sequencing funded by another grant to find out why the samples were not PCR positive for cytoplasmic leprosis compared to samples from Mexico and Panama.
This is a 4-year project with 2 main objectives: (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 the transgenic approach proposed in objective 1, besides transforming the Arabidopsis MKK7 (AtMKK7) gene into citrus, we are making transgenic citrus plants overexpressing SA biosynthesis genes. We expect that citrus transgenic plants overproducing SA would have increased resistance to citrus canker. Although exogenous application of SA does not increase resistance to citrus greening, increasing endogenous SA levels may have different effect. Transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene are currently under canker resistance test. We have propagated these plants for citrus greening test. We are trying to generate citrus transgenic plants overexpressing several other Arabidopsis disease resistance genes including ELP3 and ELP4. The mutant screen proposed in objective 2 has been continued. More gamma ray-irradiated Ray Ruby grapefruit seeds have been gamma ray irradiated. Part of the seeds will be plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Some shoots formed on these gamma irradiated seeds have been screened again on the selective medium. Part of the seeds will be directly sown into soil, and seedlings from these seeds will be test for greening resistance. We would like to test whether a direct genetic screen could work for identifying citrus greening-resistant varieties. We will continue to germinate gamma ray-irradiated Ray Ruby grapefruit seeds in soil and inoculate the seedlings with psyllids carrying greening bacteria. We have been watching the development of greening symptoms on the seedlings.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We searched the citrus genome database (http://www.phytozome.org/citrus.php) with BLAST and identified nine genes similar to AtNPR1 or its Arabidopsis homologs. Among them, CtNPR1 (also named CtNH1) is the most closely-related to AtNPR1 based on phylogenetic analysis, supporting an orthologous relationship. The Figwort mosaic virus (FMV) promoter was used to overexpress CtNH1 in citrus. Previous studies in soybean showed that the FMV promoter is significantly stronger than the Cauliflower mosaic virus (CaMV) 35S promoter for gene expression (MPMI 21: 1027). Three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts have been identified. When inoculated with X. citri subsp. citri (Xcc), they all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xcc in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. CtNH1 plants have been propagated by grafting and are inoculating with Candidatus Liberibacter asiaticus (Las) in two laboratories. A microarray experiment was conducted using CtNH1 and non-transgenic Duncan grapefruit inoculated with Xcc. A needleless syringe was used to infiltrate the leaves with the bacterial culture (OD600 to 0.3). Three time points were used for this study. For each time point, three replications were used. Data analysis indicates that at p value <0.01, a total of 451, 725, and 2144 genes were differentially expressed at 6, 48, and 120 hours post inoculation (HPI), respectively. Using the visualization tool Mapman 3.5.1, the differentially regulated genes (Log FC ' 1 and Log FC ' -1) were mapped to give an overview of the pathways affected. Interestingly, at 120 HPI, a large number of genes involved in protein degradation and post-translational modification were differentially regulated. Furthermore, numerous genes involved in signaling also showed differential expression at this time. The results indicate that a large number of genes involved in the regulation of transcription were up-regulated in the transgenic plants at 120 HPI, and also at 48 HPI, although to a lesser extent. The photosynthetic pathway was affected to a larger extent at 48 HPI, which is signified by a large number of genes involved in photosynthesis being up-regulated in the transgenic plant when compared to the non-transgenic citrus. We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into 'Duncan' grapefruit. To date, ten transgenic lines have been obtained. They are ready for Las inoculation.
We now have citrus transformants in the greenhouse at UF for all of our constructs. PCR confirmations for all plants have been completed and indicate that from 17 to 88% of the surviving plants are positive for the R gene constructs. The least successful was the snc1 constitutive mutant expressed using the AtSUC2 promoter (2 positives out of 12 total). In addition, we have 11 (out of 15) citrus transformants containing the GusPlus reporter expressed using the AtPAD4 promoter, which we have recently found to be inducible by psyllid feeding. Our working hypothesis is that restricting expression of the R proteins to the phloem will lessen the negative impact that these proteins may have on normal growth and development. Transgenic citrus plants expressing either SNC1 or SSI4 wild type proteins using the phloem-specific AtSUC2 promoter appeared to exhibit a normal growth and leaf phenotype. This result was predicted since the respective R proteins should not have been activated in the absence of pathogens, and hence, would not trigger the hypersensitive response. In contrast, however, expression of the constitutively-active ssi4 protein in a phloem-specific manner resulted in approximately 60% of the plants with an obvious negative phenotype consisting of yellowish leaves, wavy-edged leaves, reduced internodal lengths, general stunting and leaf drop. This stunted phenotype resulted in death for 6 out of the 15 original transformants (40%). Although phloem-specific expression of the ssi4 mutant protein often (60% of the plants) resulted in either death or a negative growth phenotype, phloem expression of the snc1 mutant protein produced no abnormal phenotype. However, only 2 out of 12 plants were confirmed transgenics (AtSUC2/snc1). A similar tendency for the ssi4 mutant protein constructs to show an abnormal phenotype was also seen when expressed using the wound-inducible AtPAD4 promoter. The AtPAD4 promoter-snc1 construct showed no unusual phenotype. There are two questions that remain unresolved: 1) Why are some transformants showing a negative growth phenotype and others not, and 2) Do any of the R protein constructs confer resistance to Liberibacter? We are planning experiments that will evaluate the survival of Liberibacter in our transformed citrus lines. In addition, psyllid feeding tracks are being characterized histochemically to determine the relationship between AtPAD4/GusPlus reporter expression and the location of stylet sheaths and callose induction.
Analysis of transgenic citrus lines: We confirmed the presence of transgenes in transformed citrus plants using standard PCR techniques. In one line, SUC2-snc1 mutant (20-7), it was unusually difficult to establish that the desired construct was present. It required serial dilutions of genomic templates to reduce interference of the inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with the presence of this specific construct. It is possible that constitutively expressed snc1 mutant may affect the production of phenolic or other interfering compounds. In order to evaluate the survival of Liberibacter asiaticus (Las) in our transformed citrus lines, we first focused on the development of an assay to detect the presence of the bacteria in heavily infected, symptomatic citrus leaves. These were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. Citrus leaves were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials were in the range of 40 mg. The primers used for PCR detection of the Liberibacter asiaticus were based on 16S ribosomal DNA, and as plant controls, the cytochrome oxidase COX gene was used (Pelz-Stelinski et al., 2010, J. Econ. Entomol. 103, 1531-1541). We were able to detect Las and Cox amplicons in genomic DNA isolates from these relatively small quantities of transgenic citrus leaf material: either in green (asymptomatic) or yellow symptomatic Las-infected leaves. Setting up a calibrated curve for real time quantitative PCR: Next, we generated Las and Cox PCR amplicons to be used in specific standard curves in the real-time PCR reactions to determine copy numbers of respective genes. The Wingless (Wg) gene that serves as the psyllid control was obtained from the genomic DNA isolated from 10 uninfected psyllids. Real-time PCR reactions required testing multiple variables in order to fine-tune the Las-detection assay, some being the primer and amplicon concentrations. We tested a range of amplicon concentrations from 10 ng to 1 pg (=12,190,283 copies), and in later experiments, down to 12 copies of Las, 14 copies of Cox and Wg. Also, lower concentrations of PCR primers were more optimal. As a starting point, we tested the expression of AtPAD4-GUSplus transgenic plants responsive to wounding to correlate the wounding event itself with the actual psyllid feeding. Preliminary citrus wounding experiments by slit-cutting, or needle-puncturing determined that the AtPAD4 promoter was very specifically induced by wounding. We performed numerous histochemical studies, including aniline blue, acid fuchsin, toluidine blue, Evans blue as individual and with combined staining, and fluorescence techniques to detect psyllid stylet sheaths. These were performed on cross-sections identified by GUS staining spots generated in response to psyllid-feeding (=wounding). After numerous attempts we were unable to establish this technology as a useful tool to meet our overall goal of the early Liberibacter detection in citrus plants.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
