Our objective was to determine how psyllid host seeking behavior is affected by Las-infection. In previous experiments, we have determined that ACP adults initially prefer to settle on Las-infected plants than non-infected counterparts. This attraction to Las-infected plants appears to be dependent upon the plant’s release of one specific volatile chemical that attracts psyllids. After settling on infected plants for a short period of time sufficient to acquire the Las pathogen, these potentially infected psyllids move from infected plants to healthy plants. Thus, the Las-pathogen is affecting the behavior of psyllids in way that promotes its own spread. 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. In the last quarter, we have worked towards creating nutrient deficient, yet uninfected plants. We have successfully achieved a low level of nutrient deficiency in some plants and conducted initial settling trials. In these trials, psyllids were initially caged on healthy citrus. Subsequently, the cage was removed and the psyllids were allowed to either stay on the healthy plant or move to a nutritionally deficient plant. In this experiment, an average of 69.5% of psyllids chose to remain on the healthy plants while on average 26.7% moved from healthy to nutrient deficient plants. In concurrent experiments, psyllids were initially caged on nutrient deficient plants and then allowed to move to healthy plants. In these experiments, an average of 72.4% of psyllids chose to move to the healthy plants, whereas an average of 24.7% of psyllids remained on the nutrient deficient plants. We have continued to restrict application of fertilizer to these nutrient deficient plants in order to achieve further deficiencies. Samples of plant tissues have been sent for nutrient analysis, and upon receiving that data, another replication of these experiments will be conducted. In future experiments we will rescue these plants with each nutrient to determine which nutrients may be involved in affecting psyllid settling behavior. These data may be useful in predicting psyllid movement between citrus groves depending on nutritional status of trees. Also, this will help explain movement of pathogen by psyllids from tree to tree as a result of pathogen-altered psyllid host seeking behavior.
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. Serial dilutions of genomic templates were employed to reduce interference of inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with this specific construct. It is possible, therefore, 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. Infected leaves were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. These were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials (leaf samples) were in the range of 40 mg. PCR primers for 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 standard curves in real-time PCR reactions for copy number determinations. 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 and 14 copies of Cox and Wg. 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 antibody labeling 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 early Liberibacter detection requirements in citrus plants.
This is the end of the second year plus a 6 month NCE for a currently funded multi-investigator, multi-institution project. Although many parts of this research were successful, it cannot be continued in its present form. The USDA group, who was receiving almost 50% of the funding, does not want to continue for a third year. The post-doc who was working on the project has left and it is difficult to get a new post-doc, particularly in Ft. Pierce, when only a year is left on the project. The group has published a paper on their successful efforts (Marutani-Hert, Mizuri, Evens, Terence,McCollum, Gregory, and Niedz, Randall. 2011. Bud emergence and shoot growth from mature citrus nodal stem segments. Plant Cell, Tissue and Organ Culture 106:81-91). The Moore laboratory is also making good progress on the use of cell penetrating peptides to get molecules into citrus without having to use Agrobacterium. In the past 6 months, we have been doing successful experiments with DNA as well as proteins. This is far from a mature technology but shows. promise.
This project sought the development of in vitro regeneration techniques for Murraya paniculata, a presumed host plant citrus relative highly favored by psyllids; these regeneration methods were then to be used to attempt first the genetic transformation of Murraya with marker genes, to optimize the transformation protocol. If successful, then insecticidal or psyllid-suppressive gene construct could be introduced. The ultimate objective was to attempt the development of a deadly trap plant for psyllids that could be deployed in citrus groves to potentially decrease psyllid populations and consequent inoculum potential. Further, such deadly trap plants could be used in the urban landscape to decrease the reservoir of uncontrolled CLas inoculum from commercial or residential areas impacting nearby citrus production areas. We were successful in developing a reasonably efficient regeneration protocol for Murraya via organogenesis, with defined levels of hormone and growth regulator supplementation as well as appropriate plant tissue management and handling techniques; a manuscript on this work is under preparation, the first ever report of in vitro regeneration of this citrus relative. We struggled, however, with the objective of achieving successful genetic transformation. One bottleneck was the unavailability of a reliable source of abundant and viable seed sources necessary to initiate the large-scale experiments that we wanted to conduct. Despite this, we explored various parameters for genetic transformation of Murraya, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments were conducted after testing various plasmids and Agrobacterium strains. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were likewise tested, and we established a standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events was pursued. Though the project has ended, we have shared our results with ctrus transformation experts, and the work is continuing in collaboration now with the Core Citrus Transformation Facility at the UF-CREC, to attempt to further refine and improve our abilities to transform Murraya, and perhaps ultimately to produce, test, and deploy the deadly trap plants we aimed to develop, to test their value and utility as part of integrated approaches to manage HLB disease in Florida citrus.
Funds for this project have now been received. The antibody service provider has provided a quote for anti-NodT antibody production. Antibody production and screening will be initiated soon and is expected to take approximately 3 months.
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 objective 1, we have generated transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene. The transgenic plants are currently under canker resistance test. We will propagate these plants for citrus greening test. We have shown that overexpressing the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker, suggesting that the salicylic acid (SA) signaling pathway plays an important role in citrus disease resistance. We recently established an Arabidopsis-Xanthomonas citri subsp. citri (Xcc) pathosystem with the support of a USDA special grant. Using the Arabidopsis-Xcc pathosystem, we found that mutants of the SA signaling pathway are more susceptible to Xcc. A manuscript about these results has been accepted by PLoS ONE. We are trying to generate citrus transgenic plants that accumulate high levels of SA. For objective 2, we are continuing the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Two quarts of seeds treated with gamma-ray irradiation at 50 Gy have been plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated were 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. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test. We are also testing whether a direct genetic screen would work for identifying citrus greening-resistant varieties. We germinated gamma ray-irradiated Ray Ruby grapefruit seeds in soil and inoculated the seedlings with psyllids carrying greening bacteria. We are watching the development of greening symptoms on the seedlings.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. Previous work focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. However, repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. Therefore during the present quarter we focused on developing a system for tomato. Small side shoots from tomato plants were placed in 50 ml culture tubes with the cut stem end immersed in water in a microfuge tube. This design was adapted from one shown on a poster by Ammar et al. at the Citrus Health Research Forum in Denver in October. Adult psyllids are placed in the culture tubes and within 7 days most tomato shoots are qPCR positive for CLps. After two weeks Ct values are typically less than 25. This system appears promising since chemicals can be introduced into the water for plant uptake. We have not yet demonstrated chemical uptake, but the system has all of the other essential characteristics necessary for a chemical genomics experiment and we plan to initiate these experiments soon. The project received a no-cost extension to Jan 31, 2012 due to delays in initial funding, so there are 5 quarterly reports this year.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. Previous work showed that the model plant Arabidopsis thaliana can be infected with CLps by potato psyllids. However, only plants growing in soil could be infected. We were not successful in inoculating plants grown in solid or liquid culture media as would be best for screening chemicals that may affect transmission or bacterial replication. Work during this quarter focused on testing methods to expose plants to chemicals before and after psyllid feeding. We tested growing plants in hydroponic culture but did not find CLps positives following psyllid inoculation. Additional testing is planned. We repeated screening of soil-grown plants of 10 Arabidopsis ecotypes for susceptibility or partial resistance using a larger number of Arabodopsis plants per ecotype to obtain better discrimination among ecotypes. qPCR assays to assess CLps levels in the plants have not yet been completed for all samples, but as in previous tests most plants are positive for CLps. Because of the difficulties we have experienced in inoculating Arabidopsis plants in culture systems that would be suitable for chemical screening, we will begin testing small tomato shoots in liquid culture.
In this project we are examining how the distribution of the HLB bacterium within infected trees affects the efficiency of HLB transmission by psyllids and what types of tree flushes provide better inoculum for psyllids to transmit the infection. During this period of funding we established and maintained a healthy-psyllid colony and an infected-psyllid colony for the use in our experiments. One portion of healthy psyllids is maintained on healthy Murraya paniculata plants in special insect cages and the other portion is exposed to HLB-infected citrus plants to generate HLB-infected psyllids that can be further used for inoculation of new citrus plants. Some of the transmission experiments were conducted using already available HLB-infected plants. In addition, we keep inoculating new sweet orange and grapefruit plants using the infected psyllids and graft-inoculation with HLB-containing buds to generate more plant material that can be used in our further experiments. One of the obstacles that we face in our research is that we have plenty of old symptomatic flushes on the infected plants, but much less often we have young, asymptomatic yet containing the bacteria tissue that grows on the infected plants. This can be explained by the fact that growth of HLB-infected plants (especially small greenhouse trees that we are working with) usually slows down. To overcome this obstacle, we try to generate more HLB-infected plants. For examination of psyllid acquisition of the bacterium from different types of flushes, we conducted several trials in which healthy psyllids were placed on either a young growing flush or an older symptomatic flush of an infected tree. Psyllids were secured on those flushes by using small traps made up of mesh material. After 21 days traps were removed and psyllids were analyzed by PCR with HLB-specific primers. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. These plants are being monitored for the development of infection. The numbers of plants that become infected upon inoculation with psyllids fed on different types of flushes will be analyzed and compared. Another observation: TEM examination of the material extracted from psyllids demonstrated that psyllids fed on different types of flushes contained different forms of Las: psyllids fed on the young flushes contained mostly rod shaped bacteria, while bacteria extracted from psyllids that fed on the old symptomatic flushes were mainly present in a round form. Our next step is to understand whether the HLB bacterium can have different forms or can be present at different stages in different types of tissues. Several approaches are being undertaken to characterize those forms. We are also examining what types of flushes are more susceptible to psyllid inoculation. In these experiments infected psyllids were placed on different types of flushes of healthy plants. Plants were grouped in two sets: first set in which only young flushes were exposed to psyllids and second set in which psyllids were placed on old flushes for a period of 21 days. The inoculated plants are being maintained and monitored for infection to analyze and compare a percentage of plants that become infected.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las is much shorter that we expected. In our population of psyllids in the containment room, the proportion of infected psyllids born on newly inserted healthy plants starts increasing after about 30 days suggesting that the receptor plants begin becoming donors at about that time. We are examining this process in more detail now. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and plants containing potential anti-psyllid genes for Kirsten Pelz-Stelinski’s lab and for Bob Shatters et al. lab in Fort Pierce. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or anti-psyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. We have about 50 new peptides that are now being screened. We are eliminating peptides that do not work and continuing to make and screen new ones. ‘ We have greatly improved our efficiency of screening. We are using small plants in order to screen faster. However, we have to balance psyllid damage with inoculation of HLB. We now are ‘pulse-inoculating’ plants by incubating them about 2-3 weeks with psyllids between intervals of no psyllids in the greenhouse. ‘ We have greatly improved the CTV vector to produce probably 100x more peptide. ‘ We have modified the vector to allow addition of a second anti-HLB gene. ‘ We have obtained permission and established a field test to determine whether the CTV vector and antimicrobial peptides can protect trees under field conditions. ‘ We continue to supply infected and healthy psyllids to the research community. ‘ We are testing numerous genes against greening or the psyllid for other labs.
Transcriptional analyses of citrus stem, root, and leaf responses to Candidatus Liberibacter asiaticus infection Candidatus Liberibacter asiaticus is known to cause Huanglongbing disease, which is currently the most destructive disease that affects citrus plants. Previous studies indicate that Ca. L. asiaticus is distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. The leaves, stems, and roots play distinct roles in the photosynthesis and transportation of water, nutrients, etc. However, the effects of Ca. L. asiaticus on gene expression in the stems and roots remain to be elucidated despite the recent progress that has been made toward understanding the transcriptome of leaves that are infected with Ca. L. asiaticus. Dramatic differences were observed in the transcriptional responses in the citrus leaves, stems, and roots to Ca. L. asiaticus infection. Overall, 1909, 884, and 111 genes were regulated in leaves, stems, and roots, respectively, by Ca. L. asiaticus infection. Only 2 genes overlapped in the leaves, stems and roots, whereas 289 genes were regulated in both the leaves and stems, 16 in the leaves and roots, and 6 in the roots and stems. The low similarities among the leaf, stem and root expression profiles indicate that Ca. L. asiaticus affects them all differently. Further analyses showed that Ca. L. asiaticus reprograms multiple cellular and metabolic processes in citrus and identified genes whose expression are regulated in organ-specific manners. Broad variations in expression levels were detected for genes that are involved in carbohydrate metabolism, cell wall biogenesis, lipid metabolism, hormone signaling, secondary metabolism, transportation, amino acid metabolism, and signaling and transcriptional regulation. Our analysis has revealed that Ca. L. asiaticus reprograms multiple metabolic and cellular processes in citrus and has identified genes whose expression are regulated in an organ-specific manner. Most of the genes were regulated in the leaves, followed by the stems and the least were observed in the roots. The genes that showed significantly altered expression between the organs were very different in each of the three organs, indicating organ specialization in response to Ca. L. asiaticus, which affects their distinct functions. In another study, host response of Rangpur lime (Citrus . limonia Osbeck) which shows tolerance to the bacteria, to Las infection, was examined using suppression subtractive hybridization (SSH). Differential expression of selected genes of Ca. L. asiaticus in cultivars of citrus Our result indicate that the Ca. L. asiaticus genes involved in the survival and virulence in the plant are up-regulated in planta compared to the insect. We tested whether these potential virulence related genes would be differentially expressed in susceptible and tolerant varieties of citrus. Genes that were highly expressed in planta were selected, their expression were evaluated in selected cultivars of citrus. The selected genes included those involved in Heme biosynthesis, ABC transport of iron and Zinc, production of an RTX type toxin (CLIBASIA_01555: hemolysin) and a metalloprotease (CLIBASIA_01345: serralysin). The citrus cultivars selected ranged from those highly susceptible to Ca. L. asiaticus infection, to the ones that are tolerant. The expression of of these genes were significantly lower in the moderately tolerant and tolerant varieties of citrus, however, most these genes were expressed at very high levels in the sensitive varieties Riored, Kinkoji and Valencia. Thus, our results indicate that there is a significant difference in the expression of Ca. L. asiaticus genes depending on host reactivity to the disease.
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 have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. 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 Xac 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. A manuscript entitled ‘Overexpression of the Citrus CtNH1 Gene Confers Resistance to Canker Disease’ is in preparation. The CtNH1 plants have been propagated by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). No conclusive results can be reported at this time. Microarray experiments were conducted using the transgenic line CtNH1-1 and non-transgenic ‘Duncan’ grapefruit inoculated with Xac306. 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. A second manuscript describing these results is in preparation. 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. We will characterize these plants by Northern blot and propagate the lines with overexpressed CtNH1 for Las inoculation.
Huanglongbing (HLB) and Citrus Bacterial Canker present serious threats to citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters are used to generate numerous transformants of rootstock and scion genotypes. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. Several events continue to grow better than all controls at 8 months after initiating the challenge, with 35% greater trunk-cross-sectional area increase than the overall experimental average and 64% greater growth than the mean of the controls, but do not show immunity to CLas development. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. D35S produces the highest level of expression but with great variability between events. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and significantly suppressed CLas growth in culture. After verification these will be used to create transgenes. Anthocyanin regulatory genes, give bright red shoots (UF Gray collaboration) and were tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Unfortunately, when antibiotics were left out of regeneration media, almost no red shoots were recovered. However, high anthocyanin apples are reported to have field resistance to bacterial fire-blight, presumably due to high levels of phenolic compounds. Red citrus transgenics will be tested for HLB, ACP, and canker resistance. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 34 months and a repeated trial has been in the field for 22 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. Comparison of field-grown and greenhouse-grown valencia following graft-inoculation show much more rapid CLas development in greenhouse-grown trees. Several new collaborations are being explored to feed new HLB-suppressing transgenes and novel strategies into the citrus transformation pipeline.
A transgenic test site has numerous experiments in place at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than seventeen months. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place: Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Discussions have been ongoing with Eliezer Louzada of Texas A&M to plant his transgenics which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Additional plantings are welcome from the research community.
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