On May 8th, the research team met with growers at the University of Florida’s Citrus Research and Education Center in Lake Alfred. The project and the need for grower participation were discussed. At that time, one grower had provided data on the use of enhanced nutrient programs, soil and leaf analyses, and yield. Since then, other growers have begun providing data. As data are received, they are entered into Excel spreadsheets in preparation for analysis. Preliminary exploration of the data has begun.
In this fifth quarter, and based on the low number of trees that acquired HLB infection 5 months after the first inoculation with HLB infected buds, 200 plants of Valencia orange on Kuharske Carrizo rootstocks were re-infected with one HLB infected bud per plant during the last week of September and the first week of October, 2011 using the standard inverted ‘T’ budding technique as describe by Ferguson (2007). Each plant will be inoculated with one additional HLB infected bud on March of 2012. HLB infection will be verified monthly by quantitative real-time PCR (qRT-PCR) starting on February 2012 as described in the 9/30/11 quarterly report. In brief, tree leaves per each inoculated tree will be harvested to verify HLB infection. Petioles and midribs of the citrus leaf will be used (100 mg per sample) to extract total DNA using ‘Qiagen DNeasy Plant Mini Kit’. After DNA isolation the yield and purity of the DNA will be estimated by measuring OD260 and OD260/280, respectively, with a NanoDrop Spectrophotometer ND-1000. The qRT-PCR for Candidatus.Liberibacter species will be performed using 16S rDNA primer and probe sets as described by Li et al. (2006). While we are waiting for the plants at the greenhouse get HLB diseased, we are going ahead with an alternative plan using citrus diseased trees at Florida’s orchards, which will allow us to start applying the different therapeutic strategies in 2012 to evaluate if they enhance citrus response to the disease, prolog life of HLB-infected plants and reduce bacteria titer and counteract the detrimental effect on citrus production. Literature cited: Ferguson J. 2007. Your Florida dooryard citrus guide. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Document HS 884. Li W., Hartung J.S. and Levy L. 2006. Quantitative real time PCR for detection and identification of Candidatus Liberobacter species associated with citrus huanglongbing. J. Microbiol. Methods 66: 104-115.
Dr. Pena and his greenhouse manager traveled again to Florida last October 2011 to continue evaluating the project. They started evaluating the establishment of the citrus germplasm. They checked the cultivars to guarantee that they were true to type since passing through in vitro conditions for cleaning the germplasm can cause somaclonal variation. Plants that were not true to type were discarded. They also found that the germplasm was very clean but plants were shorter than they should be for their age. They suggested changing the photoperiod in the growth room to be able to manipulate the plant growth. The suggestion was discussed with the facility coordinator and steps towards a reprogramming process to have a better control of the program have been initiated but at this date the programmer was not able to start the job. The germplasm was organized in lots that will be the material of origin of the mature in vitro experiments in the laboratory. Mother plants were also selected to be transplanted next year. The inventory was organized and I was trained in how to establish a ‘production’ schedule with the current germplasm and how to plan and manage the lots in the future. The greenhouse personnel was again trained in grafting. The personnel is taking too long to master the technique, and they are not consistent in the quality of the job they do which does not help to establish a calendar. Training, checking, and keeping the personnel on task in the growth room has been extremely difficult. Dr. Pena and his greenhouse manager also dedicated some time to check the infrastructure and growth room protocols. We found that the humidifiers are still not working. As a result of many months of waiting the solution was to bypass the filters to get enough water in the humidifiers and to place the program in manual daily. It seems like the system was not designed properly and a new set of filters will replace the current system next year. The facility coordinator is still looking for somebody to do this job. Growth room protocols were checked. The use of coconut fiber as proposed initially has been postponed until we master the current situation without major problems. Use of coconut fiber requires more trained personnel that is not available at this moment in the growth room. Once we master the current situation we will be able to move forward on this. We started doing citrus mature transformation experiments in the laboratory. We used A. tumefaciens with the pCambia 2301containing the GUS gene as a marker. The first experiment was done the first week of November with a small batch of Valencia 1-14-10 and we were able to obtain some positive plants. They are currently micro-grafted in vitro and will be ready to transfer to soil by the end of January 2012. We will evaluate efficiency of transformation after a few experiments are performed with the different cultivars.
The following has been accepted for publication in the Proceedings of the Florida State Horticultural Society: Huanglongbing (HLB) was first discovered in Florida in 2005. In response, Florida citrus nurseries began treating rootstock seed trees located outdoors with insecticide applications to reduce risk of psyllid transmission of ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent. In 2008, a survey identified two ‘Carrizo’ citrange trees with symptoms of HLB. To assess the potential for seed transmission from HLB-affected seed source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008, 1557 seedlings germinated from ‘Pineapple’ sweet orange seeds from trees in Collier Co. were assayed by quantitative polymerase chain reaction (qPCR) using 16S rRNA gene primers. Of these seedlings, a single plant was positive for (Las+), although additional tests were negative. In 2009, no Las+ plants were detected among 332 ‘Murcott’ tangor seedlings from trees in Hendry Co. From nurseries in 2008, one Las+ seedling was detected in 290 seedlings from fruit located on symptomatic branches of two ‘Carrizo’ citrange trees, but it’s Las+ status was not confirmed after repeated testing. In 2009, a single Las+ result was obtained for one of 100 Cleopatra mandarin seedlings, whereas no Las+ seedlings were detected for 125 seedlings from seeds from two trees of ‘Swingle’ citrumelo, 649 seedlings from four trees of ‘Kuharske’ citrange, or 100 seedlings from one tree of ‘Shekwasha’ mandarin. Despite the occasional Las+ qPCR tests, no plants developed HLB symptoms. The most probable explanation for these results is transient transmission of Las from seed obtained from HLB-infected trees with no subsequent disease establishment.
We have continued our work to investigate the susceptibility of various Rutaceous plant species in Florida to Candidatus Liberibacter asiaticus (Las), and the psyllid transmission from these hosts to citrus. Also work has been done to determine the performance of the bacterium in these alternative hosts and to see if passage through them affects the biology (pathogenicity) of it. We have now developed improved methodology to quantitatively detect live bacteria inside HLB hosts. This technique will soon be reported in a published manuscript and will be available to other researchers. We have found that the population of live bacteria is different from the population of the total DNA detected in the different plants infected. The qPCR method has been correlated with direct counting of bacterial rods/field of view and this has been converted to rods/.L using parameters from the microscope. The logarithmic values of the rods/.L data were plotted against Cq values from their qPCR side and a standard curve by linear regression was developed using the data. We are using this with some of our different alternative hosts and with different citrus cultivars. No significant differences were found among the different citrus cultivars that were tested. More testing was done on various alternative hosts which included Severinia buxifolia, Calomondin, Xanthoxylum fagara, Citropsis gillentiana, Chiosya spp., Esenbeckia runyonii and Amyris texana. One hundred percent transmission was accomplished with 10 psyllids per plant from citrus to S. buxifolia but only a 46% transmission was accomplished from S. buxifolia to citrus. Positive transmissions were accomplished from citrus to Calomondin, X. fagara, C. gillentiana, Choisya spp. and to E. runyonii. Transmission results back to citrus are pending. Live bacterial population data and the live bacteria ratio (LBR) over a period of 12 months were obtained for both Severinia and sweet orange.
Since funding was received cytoplasmic citrus leprosis infected samples were sent from our cooperator in Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. The samples did not arrive in good condition and that has now been fixed. 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 the samples. Drs. Schneider and Damsteegt have now received the isolation chamber at the quarantine lab at the USDA, ARS, FDWSRU in Ft. Detrick and it is now functional. The appropriate quarantine permits are now in place so that both mites from Dr. Jorge Pena, University of Florida, Homestead, FL and cytoplasmic citrus leprosis samples from Colombia can be safely brought to the facility. Mr.Leon in Colombia already has Brevipalpus colonies and has begun transmission experiments with the mites and isolates from Colombia. Funding for Mr. Leon’s work has not been received to date and this is being resolved. PCR positive samples were successfully shipped from Mexico and Panama to quarantine in Maryland. In January the first experiments are planned with endemic mites from Florida and PCR positive leprosis samples from Colombia.
We developed Xcc strains that express green fluorescent protein (GFP) in two different forms to monitor bacterial survival: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Evaluation of protein stability confirms that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal laser scanning microscopy (CLSM) was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. To confirm the role of biofilm as a survival strategy, viability of bacteria in aggregates was evaluated in vitro based on amplification of a specific length fragment from gumD gene. The amplification of the 445-bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc due to the instability of the long mRNA fragment. By this approach bacterial survival in biofilm aggregates as compared to planktonic cells was demonstrated in culture. This detection method may become a practical tool for study of survival of Xcc. Aggregation of viable bacteria in biofilms confirmed their role in survival outside of lesions and potential for protection from bactericide treatments in the field or in the packinghouse during the fruit disinfection process. Persistence of viable bacteria in biofilms explains the occasional recovery of Xcc from exposed symptomless fruit after rigorous disinfection with chlorine and sodium ortho-phenylphenate (SOPP). Aggregation and biofilm formation were confirmed for wide (Xcc A) and limited host range strains (Xcc A* and Xcc Aw). Higher aggregation and biofilm formation was demonstrated for Xcc A than Xcc Aw or Xcc A*. The higher biofilm formation of Xcc A was associated with greater motility on agar (swarming) and lower motility in liquid medium (swimming) than Xcc A* strains. Moreover, differences in biofilm structures between wide and narrow host range strains in initial stages of aggregate formation were observed by scanning electron microscopy (SEM) and CLSM. Greater flagellation and presence of swarming cells (with high ability for movement) in the Xcc A strains compared to Xcc Aw was observed by transmission electron microscopy and gene expression analysis. This suggests that action of flagella-like structures may account for the difference in biofilm formation between these strains. Differences in biofilm formation and motility among wide and limited host range strains may account in part for their difference in virulence. Based on the SEM, Xcc A is able to aggregate and form biofilm on both Mexican lime and grapefruit while Aw produces biofilm on lime but not grapefruit. An additional objective was to evaluate the effect of different bactericides on biofilm formation or removal of pre-existing aggregates. Adhesion of Xcc to borosilicate slides was measured by colorimetric assay after exposure to sublethal concentrations of NaCl, SOPP, NaClO and CuSO4. Bactericides did not inhibit biofilm formation but sometimes increased adhesion to the surface even when bacterial growth was not directly affected by the bactericide treatment.
The goal of this project is to genetically manipulate defense signaling mediated by salicylic acid (SA) to produce citrus cultivars with enhance resistance and/or tolerance to HLB and other emerging diseases that are challenging the citrus industry. Genetic engineering has been widely used to introduce disease resistance traits in crop plants, however, its application in citrus has been fallen behind due to the lack of adequate target gene information. With the recent release of citrus EST database and genome sequence, citrus researchers just begun to develop transgenic citrus with novel desirable traits. Since SA is known to play a central role in disease resistance against broad-spectrum pathogens in many plants, we chose to begin this project by focusing on genes positively regulating SA-mediated defense. We have three specific objectives in this project: Objective 1: Identify genes positively regulating SA-mediated defense in citrus Objective 2: Complement Arabidopsis SA mutants with corresponding citrus homologs Objective 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have made significant progress in the project in year 2010-2011 funding period as summarized below: 1. Bioinformatics analysis revealed that citrus and Arabidopsis share strong sequence conservation, most known Arabidopsis SA genes on our candidate gene list have homologous sequences available in the citrus sequence database. For some SA genes belonging to large gene families, we used phylogenetic analysis to identify the potential orthologs. 2. We have so far cloned ten citrus SA genes, among which six genes have been transferred to corresponding Arabidopsis mutants and are under analysis for defense responses. 3. Defense analysis indicates at least one citrus SA gene, CsNDR1, could complement disease susceptibility to Pseudomonas infection conferred by the Arabidopsis corresponding mutant, ndr1-1. CsNDR1 also rescued the HR defect of ndr1-1 in response to the avirulent strain P. syringae avrRpt2. The levels of disease resistance grossly correlated with the levels of transgene expression, suggesting dosage-dependent defense activation by CsNDR1 in Arabidopsis. A manuscript entailing function of the CsNDR1 gene in Arabidopsis is under preparation. 4. The citrus cultivars US-812, US-942, and US-802 were transformed with pBINplusARS constructs containing the citrus SA genes ctNDR1, ctEDS5, ctPAD4, and ctNPR1. Approximately 20,000 explants were transformed in 33 separate transformation groups. After micrografting regenerated shoots, transgenic plants are identified by PCR. Transformed plants are being regenerated and propagated to be used for replicated testing with HLB. It is planned to begin HLB testing transgenics with each of these SA pathway genes during the coming year. Citrus transformations will begin in the next three months with other constructs containing additional citrus defense genes ctACD1, ctJAR1, ctNHL1, and ctMOD1, and the corresponding transgenics will also be propagated and tested with HLB. Taken together, we have provided proof-of-principle data to demonstrate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes but also as a powerful tool to facilitate functional analysis of citrus genes. Several key SA regulators, when overexpressed in citrus, are expected to confer increased resistance to the greening disease and other emerging disease challenge the citrus industry.
Research on LAS in this quarter has focused on fine-tuning of experiments to estimate viable LAS concentrations over time in different culture treatments. Experiments involve replicate culture treatments inoculated with a LAS suspension obtained from seed of infected pomelo fruits, as in previous work, but in each successive experiment minor improvements/modification have been made to the experimental design. Improvements have been made in 1) sample processing and 2) data collection. The goal was to improve reproducibility/accuracy of results between experiments. Sample processing, including methods for obtaining inoculum and standardization of culture treatments, have been improved. Previously, some problems with contamination have been observed, which we suspected was from the fruit processing step. Improved methods for surface sterilization of the fruit have been implemented, and problems with contamination of the inoculum from seeds have been reduced. We have also modified the culture treatments. Results from the 25% and 50% pomelo juice culture treatments were somewhat similar within an experiment (compared to 1/3 King’s B media), but were more different between experiments. Therefore, we decided we need an additional more standardized juice solution that did not come from a new sample pomelo each time. We eliminated the 25% pomelo juice treatment and replaced it with a 50% juice solution obtained from a specific brand of store-bought grapefruit juice. Results with this new treatment seem to be more reproducible. Overall, results still show that LAS viability is maintained longer in a juice-based media than in 1/3 King’s B media. Data collection steps have been improved as well. Comparisons of standard curves between different qPCR runs showed that the plasmid standards were degrading slightly over time. This caused difficulty comparing qPCR data for unknowns between runs. To prevent this problem, more strict rules are being used for application of qPCR plasmid standards. Standards for all qPCR runs needed for an entire experiment are now being aliquoted at the same time, and each aliquot will only be used twice before disposal. This has greatly improved reproducibility of standard curves between runs. Results from experiments with LAS being conducted over the past few months show that, currently, LAS concentrations and viability in the seeds are increasing during this part of the year. We have been advised that sample quality will most likely start to decrease around December/January. Because of this, and because our experiments are improved in design, we are now working to start a larger-scale set of experiments very soon. We are considering adding one more culture treatment which is 100% grapefruit juice. We have also continued monitoring these LAS cultures in microfluidic chambers. However, we have not been able to get the LAS cells to stick to the chamber walls. We are thinking of ways to improve the stickiness of the chamber surfaces in the hope that this will help attachment.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. In addition to providing a degree of resistance to bacterial pathogens, overexpression of R proteins often results in in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: The transgenic citrus plants (Duncan grapefruit) containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well as transgenic control plants have been transported from the UF Citrus Research Facility (Lake Alfred) to our laboratory at the Microbiology and Cell Science Department. Out of the 53 transformants transported, 3 did not survive. The remaining 50 appear to be stabilized in their acclamation to our growth room environment. Currently, arabidopsis SNC1 (wt) and scn1 (constitutive mutant) transformants are being tested for resistance to Pseudomonas syringae (Psm 4326); however, since expression is largely limited to phloem cells, a more meaningful assay must include exposure to Liberibacter-infected psyllids. The design of assays and arrangement of the necessary collaborations are in progress. Our working hypothesis is that overexpression of the constitutively active mutants of the R protein genes Atsnc1 and Atssi4 will alert the endogenous innate immunity system of the plant and, thereby, provide resistance to Liberibacterium. In order to monitor the activation state in Arabidopsis lines transformed with the R protein constructs, we crossed (cross pollination) these lines with a homozygous line containing a reporter for the innate immunity response: the pathogen-inducible BGL2 (PR2) promoter driving the GUS reporter (kindly provided by Dr. Xinnian Dong, Duke University). Two homozygous AtSUC2/snc1 mutant and two homozygous AtSUC2/SNC1 wild type lines were crossed. Additionally, we crossed four other snc1, ssi4 mutant and wild type lines which had undetermined zygosity. In order to confirm that the PR2/GUS reporter line is functioning properly, activation tests are being conducted using salicylic acid, its analog INA, BHT, and pathogen P. syringae Psm4326 in induce reporter expression in the PR2/GUS reporter line. BTH (0.3 mM) and INA (0.5 mM) were the best inducers over the course of 72 hr. SA (0.5 mM ) induced GUS expression at 24 hr and plateaued for up to 72 h. Bacterial pathogen (Psm 4326) induction levels were the highest at 72 hr, but, overall, lower than those induced by other SAR agents. The use of these pathogen-inducible reporter lines will not only monitor the activation state of immune response of our R constructs, but they will also provide spatial information to confirm that phloem tissue is being activated. Two additional reporter lines to monitor the immune response are being developed to increase the sensitivity by using GUS plus (P2/GUS plus) and to monitor an additional pathogen-inducible promoter, PR5/GUS plus. Out of eight PR2/GUSplus transgenics (Arabidopsis, T1 generation), three showed constitutive expression (‘all blue’), while the remaining five showed either residual main vein expression, or no expression. From the five PR5/GUSplus transgenics, only one showed residual main vein expression, in line with published reports.
Recently we identified several sulfur chemicals from guava that repel Asian citrus psyllid (ACP) in the laboratory, but are difficult to formulate into controlled release devices for field use because of their high volatility. As we continue to work on formulating these sulfur compounds into devices that will have practical application, we have also investigated several potential “of-the-shelf” essential oils for their repellency against ACP. These were chosen based on their known repellency to many insects and based on their perceived similarity to guava in chemistry. Also, we have found that volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2-undecanone, a major constituent of rue oil repelled ACP adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of ACP when co-presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, .-caryophyllene, .-pinene, .-gurjunene and linalool did not repel ACP adults compared with clean air. Chemical analysis of the headspace components of coriander and lavender oil by gas chromatography-mass spectrometry revealed that .-pinene and linalool were the primary volatiles present in coriander oil while linalool and linalyl acetate were the primary volatiles present in lavender oil. Coriander, lavender and garlic chive oils were also highly toxic to ACP when evaluated as contact action insecticides using a topical application technique. The LC50 values for these 3 oils ranged between 0.16 to 0.25 ‘g/ACP adult while LC50 values for rose and thyme oil ranged between 2.45 to 17.26 ‘g/insect. Our current efforts are focusing on quantifying the airborne concentrations of these essential oils found to have behavioral activity against ACP that are required to induce the effect. Our current results suggest that garlic chive, lavender, and coriander essential oils should be further investigated as possible repellents or insecticides against ACP. Also, these repellents may be useful in organic citrus production, which currently has few available tools for management of ACP. We have also developed a method with which to sample and quantify the airborne concentrations of sulfur violates directly in the field. This has allowed us to precisely measure the concentrations of repellent chemicals needed in the field to affect psyllid behavior which is helping guide development of practical release devices. Our field results with DMDS released from SPLAT in 2010 were mixed. While some trials with the initially developed formulation appeared to show reductions of ACP populations, others did not. However, we have now completed analysis and processing of data from an investigation of four new (advanced) SPLAT formulations of DMDS that were designed for longer field longevity. Two of these four formulations produced very good results, suppressing psyllid populations better than the previous formulations and longer than was previously achieved. These two formulations lasted longer than four weeks, but their full potential could not be investigated because the trial was unfortunately interrupted by a pesticide application. We plan to further investigate these two formulations in 2011 and hope to replicate these results.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. ‘ A protocol for the accelerated production of transgenic plants has been published:Dutt M., Vasconcellos M., Grosser J.W. (2011) Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle). Plant Cell, Tissue and Organ Culture 107:79-89. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Seeds of precocious rootstocks (based on data from the St. Helena project) were harvested and planted for subsequent budding with transgenic precocious sweet oranges (Vernia and OLL series). Plans are underway to build a PVC-pipe scaffolding structure/rapid evaluation system (RES) in our transgenic greenhouse, similar to our successful RES in the field. This will allow horticultural manipulation of the precocious transgenic germplasm to demonstrate the reduced juvenility. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. Clonal propagation of these transgenic oranges onto the available liners of the precocious rootstocks mentioned above is underway. Transformation with early-flowering genes: ‘Duncan grapefruit plants transgenic for the poplar ft1 gene have been produced and are being tested for precocious and/or induced flowering using 2 different promoters. More experiments with the citrus ft constructs are also underway. Progeny plants of transgenic tobacco are being assayed for phenotype and transgene segregation.’ 122 transgenic Carrizo trees were generated following a co-transformation experiment using two vectors. The first containing 35S-cft1 and the second containing AtSUC2′ gus. The objective is to rapidly evaluate transgene expression in the fruit. PCR analysis revealed that 16 lines contained both cassettes. Plants have not flowered 12 months after transformation. Plants are currently being evaluated in an unheated greenhouse for cold stress in order to hopefully initiate early flowering in spring 2012.
Over the past quarter, we have made progress in the following areas: 1. We have made progress with our Nicotiana benthamiana – GUS test system to examine effector specificity for induction. We have developed improved vectors and used these to demonstrate specific promoter activation by three distinct TAL effectors thus far. We have also tested a RACE method to map which UPT boxes in our test promoters are used. This method is still in development. 2. Novel TAL effectors from additional citrus canker strains have been isolated, and sequence analysis has been initiated. 3. Transformation of Duncan grapefruit has continued. At present, we have over 330 new candidate stable transgenic lines in soil, with six different promoter-gene constructs. We are systematically characterizing these on a molecular basis to confirm presence of the specific genetic elements transformed. Pathogen testing will begin shortly, and we will seek to identify the best performing transgenic lines. New Fall seeds will be available soon for additional transformations. 4. Transformed lines of sweet orange and Ruby Red grapefruit are at the rooting stage.
Over the past quarter, we have made progress in the following areas: 1. We have analyzed the contributions of individual PthA proteins by knocking out specific pthA genes from X. citri strain 306 from Brazil and testing them with GUS reporter gene fused to our super promoter Bs3 construct containing binding sites for 17 X. citri TAL effectors. Strains in which the genes pthA 1 and 2 were disrupted activated the reporter gene at levels nearly comparable to the wild-type strain. These results suggest that there is little significant contribution of these effectors to gene regulation. Strains disrupted for (i) pthA 2 and 3 showed a 25% reduction in GUS activity. (ii) pthA 1 and 3 showed a 50% reduction, pthA 1,2 and 3 showed a 60% reduction, and deletion of all four pthA genes showed a 98% reduction in GUS activity, similar to a type three secretion deficient strain, 306.hrpG. This results suggest that pthA4 is the principle effector in the activation of gene expression, with additional smaller contributions from the other three TALEs. These results confirm that combinations of UPT boxes allows triggering of engineered resistance promoters by more than one TALE which should reduce pressure on individual TALEs to evolve to evade detection. 2. A robust transformation system using either epicotyls or cotyledons has produced a large pipeline of transformed plant material. More than 800 Duncan grapefruit lines have been transformed, made shoots, and roots, and been transferred to soil, with six different promoter-gene constructs. 153 Ruby Red grapefruit transformants and 63 pineapple sweet orange transformants are now in soil. It is slow work, and further attrition occurs following molecular characterization to identify the desired lines. 3. Once plants are transferred to 4 inch pots and reach adequate size, the final line selection process can take place by pathogen testing. We have now begun analyzing new stably transformed Duncan plants containing the super promoter:resistance construct using the avrGf1 gene. Two candidate lines showed reduced pustule formation by pin prick inoculation, relative to a non-transformed control line, and a hypersensitive reaction to infiltration inoculation, compared to a water-soaked lesion in the susceptible control. These reactions suggest that the test construct is successfully conferring canker resistance in these stable lines. We are continuing to study these plant lines and additional stable lines that are reaching adequate size for pathogen testing. Lines identified from this analysis will be candidates for grafting and filed testing. These results support our hypothesis that a resistance construct based on a promoter containing multiple citrus TALE binding sites can confer transcriptional activation and disease resistance to canker strains.
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 20 transgenic lines of Duncan grapefruit, and the transgenic plants are currently under canker resistance test. After canker resistance test, we will identify transgenic lines overexpressing AtMKK7, then chose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we are repeating the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Another two quarts of seeds have been treated with gamma-ray irradiation. All seeds were irradiated at 50 Gy, as we previously found that this dose will not significantly decrease the germination rate of the seeds. Both untreated and irradiated seeds were 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. Shoots formed on these gamma irradiated seeds will be 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.
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