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.
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.
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