The overall goal of this project is to determine how the xylem and phloem transport systems in citrus are affected by HLB. As of this reporting date, the majority of the field data are collected or in the process of being collected. Specifically, we have measured the vulnerability to drought induced cavitation in trees grown under new advanced citrus production systems compared to conventional practices. These data are now being processed but the incoming data agree with our preliminary investigations in that citrus trees are able to adapt to a range of water availability scenarios. The overall trend is that the ACPS grown trees are more susceptible to drought, which increases the need to carefully maintain positive water balance and correct operation of the irrigation systems. Currently we are in the process of determining the anatomical and physiological differences between the groups to better understand how the trees adapt to the different irrigation practices using traditional light microscopy, transmission electron microscopy, and scanning electron microscopy. A supplement was made to this project to start experimental plant growth regulator trials to determine if plant growth regulators could be used to mitigate the effects of HLB, specifically preharvest fruit drop. We have established a 4 acre trial in Lake Alfred on Hamlin trees affected by HLB. The trial is set up in a 4×4 Latin Square experimental design and we are comparing fruit drop, leaf physiology, and leaf anatomy in sub-plots treated with a plant growth regulator (2,4-D), a commonly used surfactant, the PGR and the surfactant together, and a control block. To date we have measured fruit drop in 2 week intervals for the last three months. Despite high variability we are seeing promising results from the PGR and the surfactant individually, but at this point the data are not fully analyzed and corrected for tree health. Fruit drop counts will continue until harvest some time in November or December, and we will then analyze the remaining fruit for juice quality.
The overall goal of this project is to understand the organization of the phloem network in citrus and the potential pathways for the spread of CLas through the citrus tree. Since the last report we have established approximately 100 trees in the retrofitted greenhouse with an automated irrigation system. The trees have been girdled or grafted and we are waiting for the grafts in heal and set. In about 50% of the grafted trees new flush from the HLB affected tissue is now developing but the leaves have yet to harden off. We expect the remainder of the grafted tissue to produce new flush within the next 2-4 weeks. Prior to grafting we collected leaves for PCR analysis, and we will repeat this process at 12 week intervals for the remainder of the project. Once the grafting and girdling has healed/set we will wait for approximately 8-12 weeks and then begin destructive harvesting of the tissue in plants from each experimental group and perform the anatomical analysis. We currently have time scheduled on the microCT instrument in Berkeley, CA (mid-Nov 2013) to generate high resolution 3D images of the phloem network in healthy citrus wood which will help us better understand the potential pathways for CLas movement. Once the tissue is scanned we will begin analyzing the datasets to study the phloem anatomy. We are also developing a phloem clearing technique that will allow us to perform similar analysis using traditional light microscopy on healthy and HLB affected plant material which can be performed at the UF CREC.
Using the heating tunnel prototype built last year, which incorporates two infrared radiant heaters and four fans, 36 trees were heat treated at three different temperatures. Within each treatment temperature, three different duration times were also tested. Each of these nine combinations were repeated four times and randomly assigned to HLB infected trees. During these experiments a third heater was added to the prototype for use during the higher temperature tests. It was necessary for the temperature to increase more rapidly during these tests. The third heater also helped maintain the higher temperatures more efficiently. Two more trees were randomly selected for testing an extremely high temperature over a very short period of time. For this experiment, the third heater radiant heater was added. Reflective panels were also placed on the ground below the tree to aid in maintaining such high temperatures. This test was added to evaluate the maximum heat treatment that can be applied before permanent damage to the tree occurs. A heat transfer test was conducted to quantify and model the amount of heat the tree will absorb during each test and to measure the heat distribution throughout the tree canopy and within the tree. Small wholes were drilled at varying parts of the tree and thermocouples were placed inside. Surface temperatures and air temperatures were also collected at these sites. This test was done using the same heating tunnel prototype with some minor adjustments. The third heater was added and reflective panels were placed on the ground to limit the heat absorbed by the soil. This test will later be used as a tool to design a model for the transfer of heat into the tree during each test. To further quantify the results for tree health, physiological measurements were added to the study. These measurements include porometer readings, pressure readings, and leaf anatomy samples. For each time-temperature combination, two trees were randomly selected for these tests. Samples were taken before treatment, one week after treatment, one month after treatment, and will continue to be tested throughout the remainder of the experiment. Trees tested at lower temperatures show less signs of physical change having almost no defoliation or fruit drop. Trees tested at the higher temperatures however, were almost completely defoliated and have started to produce new growth. The prediction is that these trees will show the greatest response to the treatment. Even the trees tested with an extremely high temperature are showing signs of new growth which shows that the trees may be able to withstand even higher temperature treatments.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. Currently we have cloned 10 full-length genes in these categories in the entry vector pJET. Five of the genes were further cloned to the binary vector pBIN19plusARS and transferred to Agrobacteria. The Agro strains were sent to our collaborator Dr. Bowman’s lab to initiate citrus transformation. In the mean time, we started the process of transforming Arabidopsis to overexpress these genes and to test their defense function. T0 transformed seeds have been harvested for some constructs and will be screened for transgenic plants soon. In addition, we did extensive bioinformatics analysis to identify potential new citrus defense genes to clone and to test for their defense functions. A list of 15 genes has been generated and primers to amplify these genes have been designed. The cloning of these genes will be initiated shortly. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated.
Activities for this quarter included sensory panel and chemical analyses of fruit harvested from Huanglongbing (HLB) or greening-infected trees in winter (December Hamlin), 2012 (actually prior to start of project), winter (January, Hamlin) and 2013 spring (April and June, Valencia) herin referred to as ‘season 1′ (2012-2013) of the two seasons scheduled in the grant (2012-2013, 2013-2014). For season 1, trained sensory panels were completed and analyzed for all harvests and both varieties, while difference-from-control tests are still ongoing. Chemical analyses of sugars, acids, aroma volatiles, vitamin C, limonoids and flavonoids are completed, but the data not yet completely analyzed except for sugars, acids and vitamin C for the two Hamlin harvests. The molecular (qPCR) analysis of the titer of the pathogen (Liberibacter asiaticus) DNA in the juice has been completed and analyzed for all Hamlin and Valencia harvests of season 1. For season 1 to answer the question whether the nutritional spray programs reverse the color, size, shape and sensory flavor defects of HLB disease, the nutritional programs studied so far have shown no effect on color, size, shape or flavor, although one of the treatments may make the fruit taste somewhat sweeter. For season 1, to determine if the nutritional spray programs reverse the chemical composition of orange juice normally caused by HLB compared to conventional spray programs and juice from healthy trees, sugars, acids and vitamin C were all lower in all HLB treatments, regardless of nutritional treatments, resulting in similar solids/acid ratios for all treatments (Hamlin harvests). Not all the season 1 harvests’ data have been analyzed for limonoids and flavonoids, but so far the bitter limonoids and other flavonoids are higher in HLB-infected juice, regardless of nutritional treatment, and especially in symptomatic fruit or fruit from severely infected trees. Therefore, so far the nutritional treatments have not reduced the chemical off-flavor components or increased desirable flavor components like sugars to any significant degree. To determine any relationship between nutritional foliar spray programs and pathogen (Liberibacter asiaticus) titer in the juice using real time qPCR, compared to conventional spray programs, the nutritional spray programs did not reduced the pathogen titer in the . Therefore the nutritional treatments did not lower the bacterial pathogen titer in the juice for either variety for season 1.
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: ‘ Baseline sensitivity of Alternaria alternata population to boscalid experiments were finalized and analyzed. ‘ Statistical analysis of QoI-fitness and baseline sensitivity to boscalid was finished. ‘ A subset of 15 isolates was selected to molecularly characterize the structure of the iron-sulfur (SDHB) and membrane-anchored (SDHC and SDHD) subunits of the succinate dehydrogenase (SDH) of A. alternata. ‘ DNA extraction was performed using the 15 isolates previously selected. ‘ Nine pairs of primers were developed to amplify different segments of SDH’s gene. ‘ Three pairs of primers: AASDHBF2/AASDHBR3, AASDHCF1/AASDHCR1 and AASDHDF1/AASDHDR1 were selected for further analysis. ‘ DNA cloning and sequence for 15 isolates using three pairs of primers were performed. ‘ DNA assembling for individual isolates within corresponding SDH gene was done. ‘ Using the sequence information, the molecular characterization of SDHB, SDHC and SDHC subunits were established. ‘ The paper ‘Stability and fitness of Alternaria alternata tangerine pathotype’ is on-going. ‘ For the project: ‘Detection of resistance in the non-pathogenic Alternaria population in tangerine groves’, a new set of primers were designed (three pair of primers). ‘ The primers CytbF1/CytbR1 were selected to clone and sequence the partial segment of cytochrome b using six selected isolates. ‘ RFLP-PCR analyses were performed in a subset of 20 isolates of non-pathogenic A. alternata to identify the point mutation G143A.
Based on previous results (see previous reports), we designed a model to test the effectiveness of the selected chemicals. Shoots were collected from a single HLB-symptomatic Valencia Orange (C. sinensis) tree, infected with ‘Ca. L. asiaticus’. Previous studies have reported greater numbers of viable ‘Ca. L. asiaticus’ cells in the sieve elements of young, asymptomatic leaves, collected from new flush. All leaves used for this study were collected from new flushes on highly symptomatic branches. Nine leaves were collected for each treatment and control group. Samples were then incubated for 6 or 24 h (with or without chemical). We followed the transcriptional activity of the 16S RNA gene and the L10 ribosomal protein (encoded by the rplJ gene) as viability parameters. The amplification values were normalized to the plant gene cox2 and are expressed relative to the control (incubated without chemical) samples. After 24 h of incubation, significant differences were observed in samples treated with small molecules. Expression of the 16S RNA gene was repressed in the treated samples. The effect of the selected chemicals on the expression of the specific genes ldtR and ldtP was then determined in the infected leaves. The expression values are calculated relative to the 16S RNA gene, to assess the specificity of the chemicals to target genes. Compound A showed a strong effect (9.0 .fold decrease) on the expression of ldtR after 6 h of incubation, while B displayed similar repression values after 6 or 24 h (5.1 .and 5.2 fold, respectively. The expression of ldtP showed constant and incremental repression values over time. Compounds B and C reached maximal values of 16 and 17, respectively, while A showed a maximal repression of 7.0 fold. These results indicate that the small molecules tested act specifically on the ldtR activator. We hypothesize that in ‘Ca. L asiaticus’, expression of LdtP is increased in response to biotic stress, allowing persistence of the bacteria within the phloem of the tree. As such, the regulation of ldtP expression through inactivation of LdtR with small molecules represents a direct means of influencing stress tolerance, and survival of ‘Ca. L asiaticus’ within the host. These results demonstrate that the rational selection of chemicals to inhibit specific targets is effective to design new treatments to diminish Liberibacter asiaticus viability on infected trees. The next logical step of our research is the treatment of infected trees directly on the field to evaluate its long term effectiveness.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the LasA1 gene, the N-terminal region (LasA1-A), two version for the repeat region with different number of the repeat sequences (LasA1-B0 and LasA1-B1), the C-terminal region (LasA1-C), and the full LasA1 gene. We are analyzing these constructs for their transient expression in Nicotiana benthamana and stable expression in transgenic Arabidopsis thaliana. The transgenic lines were obtained by floral-dip transformation of Arabidopsis Col-0 plants and we are currently verifying the gene insertion and mRNA expression level on T2 Arabidopsis. Three transgenic T3 lines expressing the gene are selected for analyzing phenotypes and protein localization using GFP pGWB2 vector. We are testing the expression level of the gene constructs that were transiently expressed in N. benthamiana with 35S, PFLAG and GFP pGWB2, 6 and 12 vectors. Different constructs of LasA1 and LasA2 proteins using GFP vectors for localization, and using the PFLAG vector for protein-protein interactions were made, respectively. pGWB-PFLAG c-terminal and n-terminal Autransporter plasmids: LasA1, LasA2 and LasA1-a/b/c were over-expressed in Nicotiana benthamiana under 35S promoter via agrobacterium-mediated transient expression. Inoculated leaf samples were collected after 3 and 6 days post inoculation (dpi) and stored at -80 C for further analysis. Immunoprecipitation and elution of FLAG-tagged autotransporters, as well as ATP quantification of the challenged samples are underway.
This project is to assess how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. Our main accomplishments on this project are listed below. 1) Electron microscopy examination of the sites on the leaves of citrus plants where HLB-positive psyllids fed for 7 days demonstrated that even at early stages of infection (starting from 3-4 weeks after the beginning of the experiment) the bacteria could be already visualized in the initial sites of introduction. 2) In order to characterize inoculum sources of the bacterium available for psyllids within an infected tree, we examined the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes, young growing or matured symptomatic ones. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. We also conducted a similar experiment that was slightly modified in a way that psyllids fed on old and young leaves that were detached from plants and kept in 50 ml tubes (‘detached leaf experiment’). Some differences in the bacterium acquisition were obtained from these two experiment series. On average 48.33% of psyllids fed on old symptomatic flushes tested positive and 58.33% of psyllids fed on young pre-symptomatic flushes were positive. In the ‘detached leaf’ experiment, an average acquisition from young pre-symptomatic tissue was significantly higher than from old symptomatic flushes: with average of 64.26% and 23.9%, respectively. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. 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 (52% of Duncan grapefruit plants and 53% of Madam Vinous sweet orange plants) and less proportion of receptor plants inoculated with psyllids that fed on old mature flushes got infected (19 and 33% of the same varieties, respectively). 3) In order to assess 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 (“no young flush” plants). According to our data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is significantly lower. 4) Overall, our results support the initial observation of young flushes being more likely crucial for the disease spread at both steps of the pathogen transmission, either acquisition and inoculation are higher when young flush are present. Nonetheless, transmission associated with old tissues, which occurs at a reduced level, should not be ignored also. 5) To examine psyllid transmission rates to different citrus genotypes, we analyzed psyllid inoculation of 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. The first four varieties showed the highest infection rates (80-100% infection), while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. Poncirus and poncirus hybrids have been shown to have much greater tolerance to HLB. The fact that they are also more tolerant to psyllid inoculation with the bacterium suggests that developing hybrids of such varieties that in addition to being more tolerant would also have acceptable horticultural characteristics and would produce fruit and juice of a sufficient quality could be a solution to battle HLB epidemics before more sustainable approaches are in place.
To determine the effect of azelaic acid in the defense response of citrus we pre-treated susceptible ‘Duncan’ grapefruit plants with this chemical via leaf infiltration and subsequently inoculated the infiltrated portion with Xanthomonas citri pv citri (Xcc). Because this pathogen can be infiltrated at a specific time, location and concentration, compared to the graft inoculations necessary to transmit Candidatus Liberibacter asiaticus, we thought it would be a quicker way to determine if this compound has any effect on resistance. After Xcc inculcation the population growth was determined using colony forming units (cfu) using a standard procedure. There was no effect of azelaic acid in the Xcc growth when compared to water control. In a separate set of experiments we have initiated an Illumina NextGen DNA sequencing project comparing ‘Sun Chu Sha’ mandarin (HLB tolerant) and ‘Duncan’ grapefruit (HLB sensitive) infiltrated with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) peptide and water control. The idea is to identify genes that may be part of the PAMP-triggered immunity (PTI) and differentially expressed in the distinct genotypes and treatments. This is underway and we expect to be analyzing the data in the next quarter.
A new FT construct,FMVcDNA27, containing an FT3 cDNA insert in the pCAMBIA 2201 vector has been made with a constitutive FMV promoter that proves to be as effective at transforming citrus and tobacco as the corresponding genomic construct that has been previously used. The FMVcDNA27 construct will be used to develop a chemically inducible system for the expression of this transgene. The inducible promoter systems from the Danforth Foundation mentioned in the previous quarterly report update was not used due to unforeseen issues; therefore the work will be continued using a transcription activator-like (TAL) effector system inducible by methoxyfenozide that will activate the naturally present FT3 gene in citrus. This promoter will utilize chemical-inducible ecdysone receptor-based expression. Research has also been conducted that looks into other endogenous plant chemically inducible promoters that are not turned on by chemicals in the media used to transform plants for use in controlling the FT3 gene expression. A manuscript comparing the behavior of the three genomic clones from citrus when overexpressed in tobacco has been completed and is undergoing further review before publication. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype has been completed and gene expression levels have been compared in a month-to-month basis using the comparative CT method from qualitative Real Time PCR. The results show a promising patter that could potentially clarify the flowering pathway and the physiological effects of these three genes as it relates to the induction of flowering. The data is being currently analyzed for statistical significance and will be cross-examined using a higher concentration of DNA to verify the results. SDS pages and western blots have been done with the synthesized FT3 protein in order to identify if the HA tag and antibody will be an effective method to test for the presence of the protein when applied to plant tissue. The synthesized protein showed high specificity the HA-antibody and therefore this method will be used to assess the presence of the FT3 synthetic protein in further studies. The same procedure was performed with the Arabidopsis FT antibody and endogenous FT in citrus, tobacco and Arabidopsis as to determine if this will be an appropriate assay for the detection of FT3 synthesized protein. Another experiment involving FT3 transgenic tobacco and the effect of plant hormones Ethylene and Gibberellin (GA) as well as a GA pathway inhibitor chemical Paclobutrazol is underway to determine an effective way of preventing precautious flowering of citrus FT3 transgenic plants in tissue culture stages.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the LasA1 and LasA2 genes. We are analyzing these constructs for their transient expression in Nicotiana benthamiana and stable expression in transgenic Arabidopsis thaliana. We have obtained transgenic lines with these constructs via floral-dip transformation. these transgenic lines were verified by PCR and RT-PCR and their segregation in T2 were analyzed. Intriguingly, no obvious abnormal phenotypes were observed in the transgenic T2 lines that over-expressed the LasA1 and LasA2 genes when the LasA1 and LasA2 were fused with GFP at C-terminal. However, transgenic Arabidopsis T2 lines expressing LasA1 or LasA2 with PFLAG showed abnormal phenotypes. Arabidopsis expressing LasA1-PFLAG showed a retarded growth and overgrowth of their roots. Moreover, the leaves displayed different shape with white-silver dechlorophyllation compared to the the wild type, while Arabidopsis lines expressing LasA2-PFLAG showed similar abnormal phenotype with less severity but normal root growth. These results indicated the LasA1-GFP fusion may not function, which may be resulted from the fusion with GFP. We also expressed LasA1 protein using The Champion’ pET Expression System containing a polyhistidine (6xHis) tag in E coli. Purified LasA1 protein will be used for antibody production and crystallization study. In addition, we have made several constructs for development of transgenic citrus via Agrobacterium-mediated transformation. Immunoprecipitation and elution of FLAG-tagged autotransporters, as well as ATP quantification of the challenged samples are underway.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, 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 are being 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. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/16th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository were planted this quarter, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants with citGRP1 are growing and will soon be tested. Forty of them were test by PCR and twenty two of them are transgenic plants with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. More than thirty kan resistant shoots were obtained from citGRP1 transformed Hamilin. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil and are ready for PCR test. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. Seven transgenic Hamlin with MADS6 were planted in soil. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kanamycin resistant transformants were generated on the selective media. About twenty kanamycin resistant shoots are rooted in-vitro and one Hamlin transformant is in soil. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. The construct pBinARSplus:nbFLS2 was used to transform Hamlin and Carrizo. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted in rooting medium and eighty Carrizo and ten Hamlin transformants were plant in soil. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
In this project we are working to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector as a tool to be used in the field to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. Several sets of experiments in which we are examining how preexisting infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees are ongoing. We are also examining the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We first graft-inoculated sweet orange trees with the T36,T30 or T68 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field (FS series isolates). We are using isolates that contain only single strains and isolates that contain mixtures of strains for primary inoculations. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Additionally, real time PCR quantification is also being employed to these tests. Additionally, various rootstock/scion combinations are prepared to be tested in order to find combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the foreign protein of interest from this vector. These combinations include trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. The plants will be used for the experiments similar to the experiments described above.
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