Function of individual X. citri transcription activator like effectors (TALEs): – Xanthomonas citri strain 2090 from Florida does not contain a typical 17.5 RVD TALE essential for typical pustule formation, but does contain pthA3 15.5 RVD. We cloned this gene and expressed in Xanthomonas citri subsp. citri 306 ‘ pthA1-4 and tested transiently with the 14 EBE promoter:GUS construct in grapefruit leaves. Activity was very low for this TALE, suggesting it may play a marginal role. – Xanthomonas citri strain #93 carries two TALEs – pthC 14.5 RVD and pthC2 17.5 RVD. These were tested in the transient assay and found to weakly activate the 14 EBE promoter:GUS construct in grapefruit leaves. We used an in silico tool – Talvez (http://bioinfo.mpl.ird.fr/cgi-bin/talvez/talvez.cgi, P’rez-Quintero et al., 2013) to scan citrus genomes for TAL effector binding sites, which suggests that PthC2 has differential ability to activate host genes compared to PthC. Transformation: Whereas we observed the expected behavior of gene constructs in transient assays, we have been unable to isolate stable transgenic citrus lines with functional gene constructs. We continue to explore multiple approaches to overcome this technical issue: 1. We tested Carrizo for transformation with the 4 EBE promoter:avrGf1 and challenged with Xanthomonas citri subsp. citri strain 306 and 306 transconjugant carrying avrGf1 into young leaves. The carrizo genotype gave significant resistance for transconjugant Xcc 306: avrGf1 with pathogenicity test infiltrated at 10-3 cfu/ml bacterial suspension. 2. The 14EBE promoter construct efficiency is being tested in tomato; binary vector was engineered with NosT: ProBs314EBE :avrBs4: NosT in T-DNA. AvrBs4 when expressed in tomato results in hypersensitive reaction. The Binary construct in agrobacterium strain Agl-1 was used for transforming tomato Bonny Best and large Red Cherry varieties. Transformant screening is in progress for assessing resistance to bacterial spot disease. 3. We sent two of our constructs for testing in parallel at a contract transformation lab The facility at UC Davis compared the 14 EBE:GUS and 14 EBE:AvrG1 constructs with their standard control plasmid in both Carrizo and tobacco. They found that both of our constructs gave a far lower transformation efficiency than their standard in both tobacco and citrus, and transformants with GUS recovered so far have not showed GUS activity. These results indicate that a likely source of the difficulty we have encountered is the vector. 4. To date we have prepared four promoter constructs in another vector that we use commonly and have good success with. The first set of these transformants are in soil, and we will be able to test the integrity of the inserts by PCR in a few weeks. Preliminary histochemical screening for successful transformation in putatively transgenic shoots of grapefruit, sweet orange and Carrizo showed that transformation has been successful and that transformation was significantly higher in Carrizo than in grapefruit or sweet orange.
In the previous quarter report, the initial development of formulations for two naturally occurring terpenoid essential oils A and B was presented. Oil-in-water microemulsions of the two essential oils (EO) were developed with droplet sizes varying between approx. 5 to 30 nm and upto 14 % (w/w) oils loading. The formulation development was continued in this reporting period and several formulations were prepared from two different oils (EO A and EO B) using agriculturally approved surfactants. Various co-surfactants were employed to increase the loading of the EOs in the formulations. Stable microemulsions were developed for EO B with upto 35% (w/w) oil loading with the aid of co-surfactants. The droplet size distributions for the developed formulations were found to be within the 5 nm to 30 nm range as measured by Dynamic Light Scattering. Initial experiments were also performed to change the droplet charge in the formulations by the addition of anionic / cationic surfactants co-surfactants while maintaining microemulsion stability. The biocidal efficacy of formulations against the surrogate bacteria, Liberibacter crescens were tested in Dr. Triplett’s labs (Microbiology and Cell Science Department). Formulations of both EO A and EO B and corresponding control samples were prepared for testing antibacterial efficacy. Select formulations were tested for the biocidal assay against L. crescens with appropriate antibiotic and media controls. Of the formulations tested, 11 showed bacterial inhibition of over 80% and two showed more than 90% inhibition ( one each for EO A and EO B). Further experiments are being planned to test more formulations and increase biocidal inhibition. Select formulations were tested for phytotoxicity and leaf/ tissue damage in Dr. Albrigo’s labs (Department of Horticulture, Lake Alfred). The two EOs, when applied undiluted, were very phytotoxic. Most of the soft nanoparticle formulations showed low phytotoxicity at 20 to 1 dilutions. Further experiments are being planned for minimizing the leaf/ tissue damage by varying the surfactants/co-surfactants/EO in the formulations. For the application of water soluble antimicrobial agents, preliminary experiments have been performed to produce double emulsions such as water-in-oil-in-water systems. Double emulsions allow for the encapsulation and delivery of water soluble antimicrobial agents for killing the bacteria. The characterization of these emulsions by optical microscopy, droplet size, stability and release of actives is under investigation. Future studies involve improving formulations to provide higher biocidal activity, investigating the uptake of the selected formulations on HLB infected citrus plants, their interaction with the bacteria, Ca Liberibacter asiaticus, as well as their phytotoxicity on the leaves and stems of citrus plants.
Current status of the research: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to develop an efficient system of screening EFR mutants for their binding to elf18-CLas, and in vitro binding system was developed which could be used to screen a mutant phage display library. We demonstrated in vitro binding of both elf18-WT and elf18-CLas to fragments of EFR ectodomain, but not to the ones of related receptor FLS2. Binding of elf18-CLas was weaker than that of elf18-WT, but it was considered that improvements in binding achieved by phage display screening may not be evident considering the binding of WT EFR to elf18-CLas. In addition, recent structural information about FLS2 binding to flg22 indicates the involvement of BAK1 as a co-receptor directly binding the ligand. As the phage display system would not account for this interaction, it may yield mutants which would perturb the binding of BAK1. An alternative system based on split ubiquitin is currently in the process of being investigated, which will hopefully overcome these issues. Additional experiments have also been performed to determine which portion of elf18-CLas is non-functional, by making chimeric elf18 peptides with WT and CLas portions. Both WT-CLas and CLas-WT peptides fail to elicit ROS, indicating there are multiple issues with the function of the elf18-CLas peptide, and thus requiring further investigation. A collaboration is currently being set up with the laboratory of Prof. Chai at Tsinghua to obtain structural information surrounding elf18-WT and elf18-CLas binding to EFR, which would enable more straight-forward testable hypotheses. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Vectors are currently being constructed in the pCAMBIA backbone, under the expression of the 35S/FMV promoter. These constructs will contain: EFR; XA21; both EFR and XA21; and EFR and XA21:EFR chimeric. Cloning of these constructs should be completed in the next few weeks and will then be passed on to the Moore laboratory for transformation in citrus.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 August. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. A total of 939 psyllids have been found on sticky cards of which greater than 60% are in no-mulch plots with the majority in plots that do not have chemical control rather than plots that receive insecticides. Thus far 35,931 young shoots have been inspected of which 2,061 were infested with ACP. Flush infested with ACP eggs and nymphs have become much more common. Infested flush predominates with 65% found on trees in no-mulch plots. Few (5%) infested flush were seen in plots receiving insecticides with only 0.2% (5 shoots) of total infested shoots found in trees on metalized mulch and treated with soil applied insecticides. Thus, reflective mulch is providing an important added degree of protection against ACP. The third set of leaf samples for PCR analysis was collected 9 July 2013. A single HLB positive tree was detected which had been receiving receiving foliar nutrition but no insecticide in a bare ground plot. The next leaf sample for HLB is scheduled to be collected early January 2014. The third round of tree growth measurements were taken on 12 July. Two trunk diameters are measured as well as canopy height. These measurements revealed larger trunk cross sectional areas in all mulched treatments compared to their no mulch counter parts. Trees receiving insecticide treatments were larger than trees without. No consistent treatment effects differences in tree height were observed. Foliar nutrition is applied during the first week of every month and will continue until the dormant season begins. However, no response in tree growth has been detected from these treatments have thus far. Leaf samples for nutrient analysis have been collected for the fourth and final time this year. Normal grove care operations continued. These include one herbicide application on July 24 of glyphosate, Kocide was sprayed on August 2 for control of canker, and one application of Intrepid for leaf miner control on August 30.
Within the last year and-a-half, researchers trying to find solution to HLB through production of transgenic plants have used every possible approach that offered some prospect for production of tolerant/resistant citrus plant. As a result, the Core Citrus Transformation Facility (CCTF) has indeed become the platform for testing the effect of different DNA sequences (‘genes’) on transgenic plants that could result in possible alteration of their ability to sustain pathogen attack. Since most of this work in its nature is theoretical, it is not well known how the introduction of certain gene into Citrus will affect production of transgenic plants. Some genes may be easily introduced into model organisms or may cause weak effect on their phenotype. However, very often situation is rather different in Citrus. From one client, CCTF has received a group of orders that all have common DNA sequence combined with other, different DNA sequences. Those orders consisted of six vectors that were supposed to be introduced into three different citrus cultivars in certain combinations. Since the time when these orders were placed and 9/20/2013, altogether 70 co-incubation experiments were performed with more than 38000 explants. Despite all this work, no transgenic plants were produced. From another research group came an order to produce plants with the gene that severely affected the phenotype of transgenic plants. Transgenic shoots were stunted and extremely bushy, making our efforts to graft them impractical. Work on this order stopped after about 30 PCR-positive shoots were produced at which time the agreement was reached with the client who will try to place new order where this gene will be controlled by an inducible promoter. The unintended consequence of the efforts of researchers to find the ‘gene’ that may render Citrus plants tolerant/resistant to HLB by using CCTF as a testing platform is low number of transgenic plants produced in the CCTF. In the last quarter, only 20 plants were produced. They belong to the following orders: pHGJ2 vector-one plant, pHGJ3 vector-seven plants, pHGJ4-three plants, pN5-five plants, pBI121-one plant, pN7-one plant, pW14 one plant, and pMED14 one plant. The second (out of ordinary) reason that contributed to low productivity within this quarter was the massive contamination in one of the common growth rooms where CCTF also keeps germinated plants. This contamination took place in June and July and wiped out 18% of our cultures from that period. Out of three orders for transgenic plants placed in the previous quarter, two were withdrawn. Within this quarter, six orders were placed. Work with two vectors is to proceed only to the early step in shoots production. For additional three vectors, the goal is to establish possible effect of introduced genes on genome activation/transformation success rate. Because of the presence of GFP as a reporter gene in these vectors, the work on these orders may also be completed at the phase of transformation when fully developed shoots are harvested from explants.
Seasonal root sampling continues in two field sites with a third site identified. Sampling has already revealed seasonal variation in root infections and shifts in the root flush cycle caused by Liberibacter. Root cages to monitor new root growth in healthy and HLB affected trees have been installed in one site to capture the fall root flush. Do to unforeseen complications, the additional sites will have root cages installed at the next flush cycle. Sampling at a rootstock trial site is underway with 9 months of data on the effects of HLB on these new experimental rootstocks. This has already begun to demonstrate how these new rootstock lines respond to Liberibacter infection. Meanwhile seedlings of the most promising of these rootstocks along with conventional and recently released rootstocks are approaching graftable size, so Liberibacter inoculation will begin soon.
The Mature Citrus Biotechnology Facility (MCBF) continues to increase our capacity for genetic transformation of mature scion budded onto immature rootstock. To this end, we have double budded immature rootstocks with mature scion. The number of explants will be significantly increased if this double budding scheme works well. Thus far, the results are promising for grapefruit but we are still waiting for results in sweet orange. Sweet orange is not as vigorous as grapefruit. We are also increasing the number of transgenic events produced by transforming immature rootstock. Different transgenic/wild-type combinations of rootstock/scion will be tested in field studies. A number of clients have provided genetic constructs important to imparting tolerance to citrus canker. Genetic transformations of Valencia using two constructs obtained from Dr. Wang’s lab have been performed and putative transgenic shoots are regenerating which will be micro-grafted in the future. Transformations using a construct from Dr. Mou’s lab (originally provided by Dr. Dong at Duke, NC) have been used with Hamlin, two batches of Pineapple and Ray Ruby grapefruit. Mature shoot explants of Valencia will be transformed in the near future as explants become available. This construct has also been used with explants of Carrizo. Swingle and Macrophylla rootstocks will be transformed in the near future. Additional genetic constructs will be obtained from citrus researchers as the capacity of our lab increases. Molecular analyses of putative transgenics, transformed with marker genes, are underway, and these plants are expected to flower in the near future. The number of transgenes for each putative transgenic is being determined by qReal-time PCR or Southern blot. Additional laboratory equipment and consumables are being purchased and another employee hired to accomplish this objective. The temperature, photoperiod, and light source have been changed in Growth Room A to induce flowering in these transgenics. Dr. Pena indicated that thorns in mature citrus transgenics regenerated from tissue culture is normal, transitory, and a sign of vigor. During the last quarter, additional experiments were conducted with marker genes (Table 1). The number of positive shoots recovered in some experiments is relatively low, but still acceptable for mature citrus transformation. Table 1. Mature citrus transformation experiments showing transformation efficiencies. Cultivar Date Batch Plasmid Explants Positive Transformation Shoots Efficiencies (%) Ham 19 6/11/13 36 p2301 240 1 0.8 Ham 20 7/2/13 31 p2301 300 2 2.2 Pine 8 6/11/13 35B p2301 240 1 0.8 Pine 9 7/9/13 40 pE121 260 3 4.5 Val 20 5/21/13 30 pE121 810 28 2.1 Ray 2 7/30/13 37X pE121 640 7 6.7 Book chapter from previous lab manager: Orbovic, Shankar, Peeples and Hubbard (in press) Citrus Transformation using Mature Tissue. Edited by Kan Wang, IN Agrobacterium Protocols Vol 2
In cooperation with Bryan Belcher from Davis Citrus Management we have identified bicarbonate impacts on health and Huanglongbing (HLB) symptom expression for trees on Carrizo and Swingle rootstocks. Citrus groves have a history of dolomite liming for control of copper toxicity and microjet irrigation that concentrates fibrous roots in the wetted zone. Soil pH >6.5 and/or well water with bicarbonate (>100 ppm) are associated with >HLB expression. Groves with soil pH <6.5 and/or low bicarbonate water may be experiencing less deterioration in root health. Rootstock sensitivity to bicarbonate is Swingle > Carrizo > Volk > Cleopatra. We surveyed 41 grove locations in Highlands and Desoto Counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is related to well water pH>6.5 (r2 = 0.50 ) and soil pH>6.2 (r2 = 0.25). Yield records for the blocks surveyed revealthat those under high bicarbonate stress have declined ~20% in production over the last 3 seasons (2009-2012) Those blocks under minimal bicarbonate stress have increased 2%. Bicarbonate stress appears to be a major factor that compounds HLB root loss and impairs root function.
Candidatus Liberibacter asiaticus (Las) moves down in the phloem during periods of root growth. Las colonizes structural and fibrous roots, and moves freely and extensively in the roots because it does not incite plugging in roots. The roots act a reservoir of Las that moves in the phloem upward during periods of shoot activity. Root infection causes rapid decline of fibrous roots (27-40% less root mass density) before symptoms appear in the canopy. The role of root infection in disease development focused attention on the potential for interactions of HLB with soil-borne pathogens and pests. Concurrent with HLB spread through FL citrus groves Phytophthora spp., especially P. nicotianae (Pn), populations increased in a statewide survey conducted by Syngenta Crop Protection. Rise in soil populations was unexpected because it occurred during unfavorable environmental conditions (i.e., periods of exceptional drought). A greenhouse study demonstrated that Las initially induces an increase in Pn propagules, but the interaction is observed only up to the time before HLB drastically reduces root mass. Statewide rise and fall in Pn counts may also reflect predisposition to Pn infection which contributes to greater root damage. Increasing root loss on HLB-affected trees may account for the accelerated fruit drop which led to an unprecedented 14% reduction in the 2012-13 citrus crop. In the greenhouse study, presence of Pn at the time of inoculation causes a significant shift in initial Las colonization to the roots and is associated with delay in foliar symptoms. Presence of Pn at the time of inoculation causes a significant shift in initial Las colonization of the roots and is associated with delay in foliar symptoms. Presence of Pn and /or foliar phosphite increases the persistence of Las colonization in the roots. Each of these factors, Las infection or phosphite stimulates a root CHO allocation response. HLB reduces root biomass but neither Pn or phosphite interacts with the root biomass response. Phosphite interacts minimally with propagules in soil suggesting little effect of phosphites on Pn infection irrespective of HLB status.
he objectives are 1) apply a protocol for sampling grapefruit for streptomycin resistance of Xanthomonas citri subsp. citri (Xcc), 2) quantify the local systemic activity of streptomycin for control of Xcc inoculum in lesions of grapefruit; 3) evaluate the efficacy of mixing copper with streptomycin compared to streptomycin alone for reduction in risk of streptomycin resistance in Xcc. Treatments of streptomycin in four grove company locations as well as a trial with four Firewalls with an without copper were monitored this in September 2013 for incidence of resistance using a sampling protocol previously developed in our program. No streptomycin resistant Xcc were detected. These results will be communicated to EPA section 18 for their information/comment. In a greenhouse assay, Firewall sprayed on Pineapple sweet orange seedling produced locally systemic control of Xcc injected into the leaves.
Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. Strains of Xanthomonas citri subsp. citri (Xcc) and other Xanthomonads sense signals from the host which facilitate the location of leaf entry points that are specific for each bacterium-host association. In addition, there are differences in swimming as well as surface motility among the wide and narrow host range strains of Xcc. Swimming responses to apoplast extracts of lime, orange, Chinese cabbage compared to sterile water extracts, were assessed in competitive assays on culture plates. A greater effect of the citrus extracts on citrus strains (X. citri subsp. citri (Xcc) and X. alfalfae subsp. citrumelonis (Xac) and cabbage extracts on X. campestris pv. campestris (Xc) was confirmed. Surface motility assays of the extracts gave similar results for citrus bacterial strains and Xc except that the lime extract had no effect on surface motility. Gene expression in strains Xcc306, Xcc62, XccAw, XccIran2 was evaluated for fimA (XAC3241), fimA (Xac3240) (Pilus type IV), fliC (flagellin), fleN (flagella regulator), pilA (fimbria), fliA (flagella sigma factor). RNA was extracted from the edge (advancing front) and center (consolidated area) of bacterial colonies under conditions for observation of surface motility on PYM medium. Different gene transcription patterns were observed among the strains. Elevated transcription of fimA was detected in advancing front of colonies in contrast to low transcription of genes involved in flagella synthesis. Currently differences in twitching motility are being evaluated for the different strains. Objective 2. To investigate bifofilm formation and composition and its relationship with bacteria structures related to motility in different strains of Xcc and comparison to non-canker causing xanthomonads. To determine extracellular DNA presence in the biofilm matrix, biofilm was treated with DNAse in different stages of development in XVM2 culture medium for Xcc (Xcc306, Xcc62, XccAw, XccIran2), Xac and Xc. DNase was added at 0, 24, 48 and 72 hours culture incubation to determine the role of DNA in biofilm formation as well as on preformed biofilm. An effect of the DNAse in the initial stages of the biofilm formation (0h) confirmed the role of the DNA in biofilm formation for Xcc and Xac but not for Xc. After 24h some effect of the DNase was detected, but not after 72 h or after treatment of preformed biofilm. Gene expression of fimA (XAC3241), fimA (Xac3240) (Pilus type IV), fliC (flagelin), fleN (flagella regulator), pilA (fimbria), motA (flagella motor), rpfF and rpfB (quorum sensing signal) was analyzed in bacterial aggregates induced in liquid LB (high nutrient concentration) or XVM2 (low nutrient concentration that mimics the apoplast). Differences in gene transcription were found in LB and XVM2 as well as for A and Aw strains of Xcc. For both strains, fimA (pilus type IV) transcription was higher in XVM2 than LB. This is consistent with higher aggregation in XVM2 as well as the role of Pilus type IV in biofilm formation. In addition, differences in fimA transcription were observed between Aw and A strains and the two culture conditions. Transcription of fimA3240 was low in aggregates at the bottom of the flask and may be related to the lack of biofilm formation in LB. Transcription of genes related to flagella production (fleN, fliC and motA), and fimbria (pilA) varied among strains and between the culture media. Transcription patterns for genes involved in quorum sensing indicate the role of this mechanism in biofilm formation for both types of strain. Similar evaluations with the same strains were performed in the presence of apoplast extracts. Results are currently under analysis.
The main purpose of this project is to understand how effectors produced by the HLB-associated bacterium Candidatus Liberibacter contribute to the development of HLB. Our previous analysis identified four CLas proteins that are secreted by the pathogen into plant phloem and are highly expressed in infected trees. In this project, we designed experiments to identify the direct citrus targets of these CLas effectors, which will reveal key information of HLB pathogenesis. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the past three months, we have made significant progress on establishing the screens using the four CLas effectors as the baits. In particular, we have cloned the four effector genes into the Y2H bait vector pGBKT7 (Clontech). These four proteins are cleaved by a bacterial peptidase after being secreted from the bacterial cell. Therefore, their biologically relevant forms (i.e. the functional proteins in infected plants) are shorter than the peptided deduced from full-length genes. We identified the peptidase recognition site from each protein and amplified the corresponding DNA fragment from HLB-infected citrus tissues. These DNA fragments should encode the partial proteins, which will be used as the baits for Y2H screens. The four DNA fragments were inserted into the bait vector pGBKT7 respectively, and the resulting constructs were confirmed by sequencing. These constructs were then transformed into yeast strain AH109. Using these constructs, we have first excluded potential self activation activities of the effectors. Some proteins can activate reporter gene expression in yeast in the absence of an interaction partner. If this is the case, Y2H can not be applied to identify interacting partners of this particular protein. We transformed yeast cells with both the bait constructs and the empty prey vector pGADT7. The transformed cells were grown in selective media and checked for self activation. Our data confirmed that the effectors do not have self activation activities and therefore are appropriate for Y2H screens. We have also started to collect HLB-infected tissues for RNA extraction and cDNA library construction.
The person hired to work on the project has not arrived and therefore no significant results can be reported.
The person hired to work on the project has not arrived. The genes to be constructed were designed and the DNA sequences were verified. Also, the sources of citrus plant materials have been identified.
The main purpose of this project is to understand how effectors produced by the HLB-associated bacterium Candidatus Liberibacter contribute to the development of HLB. Our previous analysis identified four CLas proteins that are secreted by the pathogen into plant phloem and are highly expressed in infected trees. In this project, we designed experiments to identify the direct citrus targets of these CLas effectors, which will reveal important information of HLB pathogenesis. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first quarter of this project, we cloned the four CLas effector genes into the Y2H bait vector pGBKT7 (Clontech). These constructs were transformed into the yeast strain AH109 together with the empty prey vector pGADT7 to confirm that the effectors do not have self activation activities and therefore are appropriate for Y2H screens. In the second quarter of this project, our main efforts include: 1) collect HLB-infected tissues for constructing cDNA library that will be used for Y2H screening. We collected RNA samples from HLB-infected sweet orange tissues at different infection stages. These RNA samples were mixed and sent out for cDNA library construction by a company. We are now working with the company to normalize the library in order to minimize the influence from the over-representative transcripts of highly expressed, housekeeping genes, which may bias the screening later on. 2) construct clones that will express each CLas effector gene that is fused with a gene encoding the yellow fluorescence protein (YFP). These fusion proteins will be expressed in plant cells in order to determine the localization of the effectors in plants. We have completed the recombinant vector construction. We will start plant transformation and microscopy work in the near future.
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