Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Mutagenesis of Arabidopsis EFR Random mutagenesis was performed on the extracellular domain of EFR, and a library containing approximately 10^6 clones with an average mutation frequency of 0.4% was produced. From this library 13,000 clones were screened for ROS induction in response to elf18CLas. Unfortunately, No elf18CLas responsive clones were found. It was observed that there was a high frequency of non-functional clones in this library (as assessed by ROS production induced by wild-type elf18), so a further library was produced with a lower mutation rate (0.1%). A further 6,000 clones were screened from this library without isolating any elf18CLas responsive clones. Given the lack of positive results arising from these screens it has been decided to use different approaches to engineer elf18CLas responsiveness to EFR. Firstly, target mutations were produced in EFR at sites which are known to be important for elf18 binding and responsiveness. However none of these produced a response to elf18CLas. Secondly, we have shown that elf18CLas fails to compete well with elf18, in ROS and growth inhibition assays, suggesting that binding of elf18CLas EFR is not occurring. Therefore, a first step toward engineering an EFR variant capable of responding to elf18CLas is to evolve an EFR variant that gains binding to elf18CLas. In order to engineer EFR capable of binding elf18CLas, experiments have been initiated to determine the feasibility of performing a phage display screen to identify mutants of EFR. Initial data indicates that fragments of the EFR extracellular domain can be expressed in E. coli and can bind to biotin-labeled wild-type elf18. Further experiments are underway to determine the minimal region of EFR necessary for binding and the specificity of binding, which will later enable mutagenesis of this region. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Transgenic Arabidopsis plants are being produced with XA21 or XA21-EFR to assess their resistance to Xanthomonas campestris pv. campestris 8004. This work is required to test unambiguously the functionality of XA21 in conferring anti-bacterial disease resistance in dicots. In addition, tomato plants are being transformed with XA21 to determine functionality in this species. These plants will also be crossed with tomato EFR lines to determine the effectiveness of the presence of both genes in bacterial resistance.
This study addresses two general questions: 1) Will our constructs disrupt normal growth and development in citrus, and 2) Will these constructs confer a degree of resistance to infection by Liberibacter asiaticus? We have answers for the first question and seek a one-year extension to address the second. Objective 1. Express R proteins in a phloem-specific manner in Arabidopsis and citrus. It was evident very early from the results of our experiments, and of others, that the Arabidopsis SUC2 promoter was phloem-specific in citrus and, thus, efforts were directed towards the generation of transformed citrus containing wild type and constitutive mutants of the two R genes, SSI4 and SNC1. Our rationale was that by restricting expression to phloem tissues (or to the wounding response) potential negative effects on growth and development would be minimized. From 30-60 transformants of each R gene variant were obtained in Arabidopsis and at least 10 in citrus (Duncan grapefruit). In Arabidopsis, some stunting was observed when transformed with the constitutive ssi4, but not with wild type SSI4, or with wild type or mutant SNC1. A similar result was obtained with citrus; however, the stunted growth (or seedling death) phenotype was much more pronounced. However, as with Arabidopsis, no abnormal phenotype was observed with either variant of SNC1. e triggered by psyllid feeding. This objective is a variation of the first, except the restriction in expression of the potentially harmful R genes was imposed by the wound-inducible PAD4 promoter, a promoter known to be activated by aphid feeding in Arabidopsis. Our rationale was that in case AtSUC2-directed expression resulted in a stunted growth phenotype, the use of an inducible promoter, such as PAD4, would provide a way to evaluate the effectiveness of R protein expression in inhibiting Liberibacter infection. Our expectation was that the Pad4 promoter would not be active, except upon deliberate wounding under controlled conditions. As with the AtSUC2 promoter, the expression pattern of PAD4 was more variable in Arabidopsis as determined using a GUS reporter; however, PAD4/GUS expression in transformed citrus appeared to be strictly wound-inducible. Expression of the R gene variants using the PAD4 promoter gave a result similar to that obtained in Arabidopsis: expression of the SSI4 constitutive mutant was sometimes harmful to the plant; whereas, expression of the constitutive mutant of SNC1 was not. These experiments can be summarized as follows: 1-Restricted expression of the wild type SSI4 and SNC1 genes using either the AtSUC2 or AtPAD4 promoter had no negative impact on growth and development in citrus. 2-Similarily, expression of the constitutive mutant of SNC1 had minimal effect on growth and development. In contrast, expression of the constitutive mutant of SSI4 is sometimes harmful to normal growth and development in both Arabidopsis and in citrus. Additionally, preliminary tests indicate that none of the constructs effected psyllid feeding preferences. In preparation for assessing disease resistance of the transformed citrus, a single leaf assay to monitor the early events in the transfer of Liberibacter from the psyllid to the plant has been developed as outlined in the proposal pending with the CRDF (Nov 2012). In brief, the real time PCR protocol was refined by developing calibration curves for the Las and various plant and psyllid control amplicons so that detection is now reproducible down to 12 copies. In addition, significant improvements have been made in single-leaf cage design that will enable feeding to be restricted to a 6 mm area of leaf.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This new greenhouse is compliant with DPI regulations for propagation facilities. – The initial cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation have been identified and are currently being propagated.
For the last three months of 2012, the Mature Tissue Transformation Laboratory (MTTL) continued to operate in the ‘maintenance’ capacity mode. Level of operation was determined by the amount of plant material available and its quality. The process of increasing the number of rootstock plants is slow and it has been hindered by low germination rate of seeds that are old. Although new seeds were ordered in December, they will not be available until late January/mid February when Swingle citrumelo and C. macrophylla fruit are available. One of the batches of Hamlin buds grafted in early October had low percentage ‘take/success’ rate. The outside provider of grafting services claimed that the buds coming from mother plants were not of the highest quality. In the meantime, this person has left the business and the facility contracted other provider. In couple of experiments, a high percentage of explants that were used in co-incubations with Agrobacterium got contaminated. We are investigating whether those incidences were the result of human error in the steps of transformation taking place in the laboratory, or if plants that served as starting material for explants were infected while in the growth chamber. During these three months, six co-incubation experiments were performed. Four of those experiments were done with Valencia explants and two with Hamlin explants. For the Valencia experiments, we cut 2170 explants and 1030 explants were cut for Hamlin experiments. In order to be able to assess the ability of the lab to process different orders at the same time, two additional Agrobacterium strains were used for co-incubation experiments. One of those harbored a binary vector with the gene for green fluorescent protein (GFP) as a reporter gene. In one of the Hamlin experiments, out of 16 shoots inspected for GFP fluorescence two were positive. Those two shoots were micro-grafted on Carrizo rootstock plants. Some GUS assays were done on shoots obtained from experiments done earlier. Out of 19 shoots, one was positive. Two additional Ray Ruby plants were cleaned of microorganisms and are ready to become source of budding material. One more Hamlin plant was also cleaned.
The four most promising anti-NodT scFv antibodies have been selected for further development. Anti-NodT antibody #1 has been successfully expressed in E. coli. This means that we can generate as much of the antibody as needed. The antibodies are being augmented with two 6xHis epitope tags – one at the amino terminus, and one at the carboxy terminus. The protein can be detedcted with anti-His antibodies. The anti-NodT scFv antibody is soluble, and should be usable for protein immunoblotting and other applications. We experienced some delays in cloning the scFv antibody DNA into the appropriate citrus transformation vector. However, these difficulties have now been solved and we now expect to have the scFv citrus transformation construct completed within a few weeks, and we will commence transformation immediately.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance.A construct containing CEMA gene stacked with the NPR1 gene has been constructed. Also, another vector containing a AttacinE gene stacked with the NPR1 gene is also under construction. Correlation of transgene expression with disease resistance response: More than 150 transgenic lines with different genes have been analyzed using ELISA by either C-myc or LIMA antibody (which also works for CEME) to measure transgene expression. As expected, significant differences were observed in our transgenic plants. Correlations between the data obtained from ELISA and other molecular data with HLB challenge response data are underway. Transgenic lines examined by ELISA include 40 lines with NPR-1, 50 lines with LIMA, and 9 lines with CEME. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): We have finished construction of several parts of the T-DNA region of a pCAMBIA0390 derived binary vector for cre-lox based marker-free selection. A fusion codA-hptII gene driven by the d35S promoter have been constructed and a cre gene driven by a glucocorticoid-responsive elements promoter have also been constructed and cloned into a pUC based vector. We are experiencing problems cloning the glucocorticoid receptor gene driven by a constitutive mirabilis mosaic virus promoter as all sequenced clones have mutations and/or deletions in them. Work is underway to rectify this.
Monthly monitoring of all the alternative host plants continued this quarter. Transmission rate data were the same as found in last quarter on the 6 citrus relatives tested. Severinia buxifolia (Sb), Calamondin (Cal), Zanthoxylum fagara (Zf), Citropsis gilletiana (Cg), Choisya spps (Ch), and Esenbeckia runyonii (Er), were all found to be alternative hosts of Candidatus Liberibacter asiaticus. Bacterial populations in Zanthozylum fagara, Citropsis gillentiana, Choisa spp. and Esenbeckia runyonii maintained for a relatively short period of time, or maintained at a low population level (Cq value around 35). This followed the same trend found with Murraya paniculata. Las persistency in the plant, percentage of psyllids with Las and Cq values in the psyllids was summarized. Monthly monitoring of the live bacterial population dynamics in Severinia buxifolia and Valencia sweet orange by PMA-qPCR ended in April 2012. The data of the last month showed that Severinia and Valencia both contained high levels of live bacteria around 10^7, and 3×10^7 bacteria/gram tissue, respectively. The total population was similar to the these levels in both Severinia and Valencia.
Three different psyllid transmission tests using all of the alternative host plants were completed except for tests using Amyris since all transmission attempts from HLB infected citrus to Amyris was negative (no PCR positives or symptomatic plants) Monthly monitoring of the bacterial population dynamics in Severinia buxifolia by PMA-qPCR showed that the live bacterial population level dropped in January 2012 to about 10^6 bacteria/gram tissue, and then came back to the high level in February and March (about 10^7 bacteria/gram tissue), while total population fluctuated from 10^7 to 1.2×10^8 bacteria/gram tissue. Monthly monitoring of bacterial population dynamics in Valencia sweet orange showed that the live bacterial population also dropped in January 2012 to about 10^7 bacteria/gram tissue, and then came back to the high level in February and March (about 4×10^7 bacteria/gram tissue). The total population fluctuated from 10^7 to 10^8 bacteria/gram tissue.
In alternative hosts tested in psyllid transmission experiments, positive PCR transmission results from citrus to alternative host were obtained for the following species: Severinia buxifolia (Sb), Calamondin (Cal), Zanthoxylum fagara (Zf), Citropsis gilletiana (Cg), Choisya spps (Ch), and Esenbeckia runyonii (Er). No eggs or nymphs were found on any of these alternative hosts (except for Sb and Cal), and further transmission tests (three reps were done for each) are still under investigation. The psyllid feeding behavior and living status on those plants was recorded since the psyllids seemed not to readily feed on some of the plants even when force fed. Monthly monitoring of the live bacterial population dynamics in Severinia buxifolia by PMA-qPCR showed that the live bacterial population remained high (about 10^7 bacteria/gram tissue) through this time period, while total population fluctuated from 10^7 to 10^8 bacteria/gram tissue. Monthly monitoring of live bacterial population dynamics in Valencia sweet orange by PMA-qPCR showed that the live bacterial population remained on the high level (about 3×10^7 bacteria/gram tissue) through these three months, while total population was about the same as live ones in these three months, 3.2×10^7 bacteria/gram tissue.
HLB in Florida is associated with bacterial pathogen Candidatus Liberibacter asiaticus (Las) and transmitted by Asian citrus psyllid (Diaphorina citri) to citrus. Some plant species have been listed as hosts of the psyllid and/or the associated bacterium based on field samples, but their status as alternative hosts has never been throughly studied. In this work the following rutaceous plants were investigated: Murraya paniculata (orange jasamine), Murraya (Bergera) koenegii, Murraya exotica, Severinia buxifolia, Citrofortunella microcarpa (Calomondin), Citropsis gilletiana, Esenbeckia runyonii, Zanthoxylum fagara, Choisya aztec ‘Pearl’, Choisya ternata ‘Sundance’, Helietta parvifolia, and Amyris texana, were studied to investigate their alternative host status. Possible transmission pathways for each plant were tested with repeated psyllid transmission experiments as well as grafting where compatible. After inoculation, plants were monitored for symptom development and tested by real-time PCR (qPCR). At the USDA, ARS, FDWSRU, Ft. Detrick, MD, psyllid transmission tests, Asian citrus psyllids transmitted Las to M. paniculata and M. exotica, but not M. koneigii. Disease symptoms did not develop in Murraya plants, and positive infections were determined by PCR. Back-inoculations from M. paniculata to sweet orange was successful however there was some variability in infection rates, titer, and the Las bacterium did not persist in M. paniculata (published). At the Texas A&M Citrus Center, Weslaco a collection of eight rutaceous species were established and included two Amyris species (torch wood), Zanthozylum fagara (lime pricklyash), Helietta parvifolia (baretta), Esenbeckia berlandia, Casimiroa tetrameria and two Choisya species (Mexican orange) and were tested as hosts for the psyllid. Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica all were found to be feeding hosts for the psyllid. Egg laying was found on torchwood and egg laying and nymphal development were found on C. ternata. At CREC we experimentally demonstrated the alternative host status of several plant species, of which 6 plants (Calomondin, C. gilletiana, E. runyonii, Z. fagara, C. aztec and C. ternata) were 1st time reported as hosts of Las. S. buxifolia, Calomondin, C. gilletiana, E. runyonii, Z. fagara, C. aztec and C. ternata were infected by Las although the transmissibility varied and was based on bacterial persistency and psyllid activities. At the USDA, ARS, Beltsville quarantine work with dodder as an alternative host to study the plant infection process and for its use with plants that are not graft compatible with citrus was done and published. qPCR has limitations since it does not differentiate live bacterial genomes (LBG)and dead bacterial cells. Propidium monoazide (PMA), a novel DNA-binding dye, has been used with many bacterial pathogens to effectively remove DNA from dead cells, but no applications on uncultured bacteria like Las have been reported. In our work we devised PMA-qPCR protocols and optmized them to work with plant and psyllid materials. They were then used to determine LBG in various studies, such as establishing correlation between LBG and microscopic counting, checking the reactions of different citrus plants to Las infection, and checking the connection between LBG and leaf symptom expression. Lastly, the LBG dynamics inside HLB positive citrus and non-citrus hosts was monitored monthly through a 20-month period, and a seasonal development pattern was observed in both hosts. The optimized PMA-qPCR developed provides an accurate way to determine LBG in plant hosts of Las, which should benefit various HLB research and serve as a crucial component in HLB management. This work has been accepted for publication.
Work has been continuing on the development of a construct using the FT3 cDNA insert and an FMV promoter. This construct will eventually be used to test the efficacy of the FT3 cDNA as compared to the genomic DNA construct currently being used. Over the past several months, extensive testing was conducted to establish a more effective disinfestation technique for use on seeds and other explant tissue. This technique should allow for the continuous use of seed for transformation, even many months after their initial collection. Transformation of Carrizo has picked up following the most recent harvest of seed. These transformants will be used in the experiments examining the effects of GA and day length on FT phenotype. This is month 7 of the in vivo tracking of FT1, FT2, and FT3 and samples are continuing to be collected and processed. These data will be evaluated at the end of the year-long trial to compare month-to-month variations in gene expression. The FT3 protein that was commercially synthesized has finally arrived and experiments with direct application of the protein will be commencing shortly.
In the previous period ‘Duncan’ grapefruit (considered susceptible to HLB) and ‘Sun Chu Sha’ mandarin (considered moderately tolerant to HLB) were inoculated with Flagellin 22 (flg22). Tissue samples were collected before inoculation (time 0) and at 6, 24, 72 and 120 hours post infiltration with flg22. Total RNA was extracted from all the samples (3 replicates of each) and we determined and analyzed gene expression using comparative Ct real time PCR. Genes associated with SAR and PAMP-triggered immunity (PTI) as well as genes in the salicylic acid and jasmonic acid biosynthetic pathways were studied. So far during this period we have studied the expression of 11 genes (EDS1, EDS5, ICS1, NDR1, NPR1, NPR3, PAL1, PR1, R13032, RAR1, SGT1). The results have been run through a Q-test for outliers and are in the process of being statistically analyzed using JMP Genomics. Expression levels of additional genes will also be added to the study.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 60 different peptides for activity against HLB. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids.
The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ Finished 2012 survey of 19 groves, encompassing 32 blocks sampled. The total area sampled corresponded to 850 acres in 7 counties of Florida. ‘ During 2012, 855 isolates were collected. The number of cultivars were Lee, Minneola, Murcott, Orlando and Sunburst. ‘ Out of 855 isolates, 336 were tested for pathogenicity, and 274 were confirmed as pathogenic (82%). For each pathogenic isolate, monoconidial cultures were done. ‘ Using the RZ-based microtiter assay, 163 isolates were screened for fungicide sensitivity to azoxystrobin and pyraclostrobin. Forty percent of the isolates tested in 2012 were found to be resistant to strobilurins. ‘ DNA extraction of 100 isolates was done in order to identify the point mutation that corresponds with the resistant phenotype. ‘ A greenhouse experiment was conducted to test the ability of resistant and sensitive isolates to infect tangerine plants previously applied with a full rate (15 fl oz) of Abound (azoxystrobin). During this experiment 10 different isolates were used (5 sensitive and 5 resistant). After 5 days of inoculation, the numbers of lessions were counted on individual leaves per treatment-isolate combination. A significant difference (p<0.001) was found between sensitive and resistant isolates, means that resistant isolates were able to infect plants previously covered with the full rate of fungicide.
We are examining how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. The first objective of this project is to examine initiation of HLB infection after psyllid inoculation to investigate how introduction of the pathogenic bacterium into different types of flushes of a tree affects establishment of infection. We have conducted electron microscopy examination of the sites on the leaves of citrus plants where psyllids carrying the HLB bacteria fed. Psyllids were allowed to feed on the plants for 7 days and after one more week the sites where they have fed were used for microscopy observations. Our results demonstrated that even at so early stages of infection the bacteria could be already visualized in the initial sites of introduction. To examine what types of flushes are more susceptible to psyllid inoculation with the HLB bacteria, we exposed sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes to HLB-infected psyllids. The leaves on which the psyllids fed were analyzed by PCR to see if the HLB bacterium could be detected soon after the exposure of leaves to infected psyllids. As a result in these experiments, we were able to detect presence of the bacterium fairly early after the initial exposure, approximately between two weeks and one month (similar to our results from electron microscopy studies). Plants exposed to infected psyllids have been transferred to greenhouse and further monitored for the development of infection. We have repeated this experiment several times and now are analyzing and comparing infection rates of plants with young flushes versus plants with only matured flushes. According to our data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is significantly lower. To characterize inoculum sources of the bacterium available for psyllids within an infected tree, we are evaluating the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes during infection development and their ability to transmit infection to new trees. 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 using small traps made up of mesh material and after 21 days 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 were monitored for the development of infection. Analysis of numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes revealed that more receptor plants that were inoculated by psyllids kept on young flushes became infected and less proportion of receptor plants inoculated with psyllids kept on old flushes became infected with HLB. The next objective is to examine psyllid transmission rates from and to citrus varieties that are highly tolerant to HLB. We have propagated 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. Currently these plants are being exposed to HLB-infected psyllids. After 1-month exposure, plants were moved to greenhouse and monitored for the development of HLB infection. The first four varieties showed the highest infection rates, while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. The results obtained in this project are now being prepared for a publication.
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