We are still working to obtain stably transformed citrus containing the BS3 promoter with added TAL effector binding elements (4 or 14 EBE) fused to a defense response inducing gene. We have obtained three transformants with a 14 EBE construct driving either the AvrGf1 or AvrGf2 Xanthomonas effector gene in Carrizo citrange, a citrus variety more amenable to transformation. PCR-based analysis of gene expression demonstrated that these constructs were induced as expected upon infection with the virulent strain X. citri strain Xcc306, validating that the promoter works in stably-transformed citrus. Whereas expression of AvrGf1 or 2 genes in orange and grapefruit triggers a hypersensitive defense response, this reaction doesn’t occur in Carrizo and we are not able to assess resistance to X. citri in these plants. In Duncan grapefruit, our efforts to transform constructs with AvrGf1 and AvrGf2 transgenes, where an inducible hypersensitive response is expected, have led to the isolation of seven putative transformants. These were sequenced to determine whether the transgene construct was intact. We found that all seven had deletions in the area of the transgene comprising parts of the promoter region and Avr gene. We believe our difficulty in obtaining transgenic grapefruit is arising either because the construct may have a tendency to recombine in Agrobacterium or during the transformation process, or the promoter may be leaky at some point during the transformation process, even though we have shown that it is tightly regulated in transient assays in leaves. Therefore, we are taking further steps to assess the stability and background expression of the construct. To investigate construct stability, transgenic tobacco plants (Nicotiana tabacum) resulting from the constructs pCAMBIA2201, pCAMBIA2201:NosT:Bs3super::avrGF2, and pCAMBIA2201:NosT:Bs34box::avrGF2 have been generated to determine whether the constructs are stable in another system. If construct sequence optimization is necessary, it will be easier in the tobacco system. We are also carefully assessing the potential for background expression of the construct. Four Carrizo transgenics containing the 14EBE construct fused to GUS were obtained, and these will be verified for the presence of the intact transgene by PCR, followed by GUS staining to determine whether there is any unexpected expression in any tissues other than leaves. In addition, N. tabacum and N. benthamiana transgenic plants carrying the 14 EBE construct fused to GUS will be stained to determine whether GUS is expressed anywhere in the plant. If one of the added EBEs produces unwanted background expression, we will be better able to determine which one is problematic in this system. We will also attempt to transform Duncan grapefruit with a construct containing the Bs3 promoter without any added EBEs, fused to an Avr gene or to GUS, to assess whether the unaltered promoter produces any background expression. Work also continues in the tomato model system, where one transgenic line carrying the disease resistance construct showed a reduction in symptoms in initial tests. T2 plants are being generated for further study.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to perform screening on complex EFR mutant libraries required to discover mutants which respond to elf18-CLas ,we have been developing a FACS-based screen. To this end we have generated a number of reporter lines (using GFP) in both suspension cultures and transgenic Arabidopsis plants. The reporter lines are driven by the FRK1, WRKY30 and PER4 promoters. We have tested two PER4p:GFP cell suspension lines for responsiveness to elf18, and both of these give clear induction of the reporter gene following treatment. However, when protoplasts were produced of these lines, elf18 responsiveness was no longer observed. We are currently retesting these lines to ensure the buffer conditions and EFR expression is correct. In addition, plant and cell suspension lines transformed with FRK1p:GFP and WRKY30p:GFP are also in the process of being tested. In addition to the mutagenesis approach, we screened the Nordborg collection of Arabidopsis ecotypes for sensitivity to elf18-CLas or reciprocal chimeric peptides of elf18-Ecoli and elf18-CLas. Of this collection, none show ROS in response to either elf18-CLas or the chimeric peptides. We did observed one line (Se-0) which had enhanced response to the CLas-Ecoli-elf18 chimera. This chimera has some activity in Col-0, but only at high concentrations. Further testing of this ecotype revealed that it also enhanced ROS to elf18-Ecoli and to flg22, indicating that it was not a variant of EFR which was causing the enhanced ROS. Indeed the sequence of EFR from Se-0 contains no non-synonymous SNPs. We have been also investigating the possibility of targeting other PAMPs. To this end we conducted a bioinformatic comparison of known PAMPs with those in C. Liberibacter asiaticus. From these search we identified CSP22 (Felix & Boller, JBC 2003, 278:6201) as a potential candidate, since it is conserved in the sequence required for recognition. We are currently waiting for delivery of the CLas-CSP22 peptide to test. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. These constructs have been constructed and tested and a manuscript is under revision. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Transformation experiments are ongoing; to date, a total of 5,781 ‘Duncan’ grapefruit and 956 sweet orange segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). A total of 580 and 219 grapefruit and sweet orange shoots, respectively, were transferred to rooting media. These shoots were first analyzed histochemically for GUS expression. The results show that collectively 6 grapefruit shoots were GUS positive with the constructs EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 and 1 sweet orange shoot GUS positive with the construct EFR-XA21-EFRchim. Other grapefruit shoots (47) collectively stained partially (chimeric) for GUS with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201, while 5 sweet orange shoots stained chimeric for GUS with the constructs EFR, EFR-XA21 and EFR-XA21-EFRchim. Chimeric shoots were those segments with less than 85% of blue staining.
The measurement of petiole phloem area for all treatment was completed by December 2014. Data from all heat treated trees with dry air are being compiled. Physiological measurements including leaf water potential, stomatal conductance, and phloem area are being analyzed to evaluate possible effects of heat treatment on HLB-infected citrus trees. Chlorophyll fluorescence, fruit set, fruit diameter, leaf area, and PCR data are being analyzed to assess the effects of heat treatment on overall tree health and symptom reduction. According to the preliminary analysis of the physiological measurements, there is no evidence that heat treatment had an effect on these markers. Since the physiological measurements can heavily depend on weather, they might not be the best indicators of heat treatment performance. Chlorophyll fluorescence measurements were not significantly different among heat treated and control trees but further studies on this parameter need to be done in future experiments. Preliminary data analysis shows that trees heated to 55’C had significantly more and larger fruit when compared to control trees. Starting in January 2015, fruit drop measurements on trees heated with dry air will be collected. Fruit from under heated and control trees is counted on a weekly basis. This data will also be used to evaluate the effect of the heat treatment on symptom reduction. Fruit quality will also be analyzed. Fruit quality analysis was done on trees heated with steam in August 2014. Total acid, total brix, acid/brix ratio, and pounds solids per box were evaluated. There was no significant difference in total acid between heated and control trees. However, total brix, acid/brix ratio, and pounds solid per box were all significantly higher in heat treated trees when compared to untreated trees.
During summer 2014, 9- and 13-month post treatment physiological tests on stomatal conductance, water potential, and leaf anatomy samples were continued. Also, fruit set data, fruit diameter data, average leaf area data, and average leaf area index (LAI) was collected and analyzed. The results of the fruit set analysis show there is a significant difference between the amount of fruit on the trees that were heated to 50’C and 55’C as compared to the sick control trees. The fruit set on the trees heated to 55’C and 50’C are significantly higher compared to untreated trees. The fruit diameter data shows there is a significant difference between the average fruit diameter on the 55’C treated trees as compared to the control trees. There is also a significant difference between the 45’C treated trees and the 55’C treated trees. The fruit on the 55’C trees is also significantly larger than the fruit on the 50’C trees. There was no noticeable difference in the average leaf size for any heat-treated or control trees. However, there were some differences in the average LAI. This measurement was taken to help measure the leaf density of the tree canopy. The trees heated to 55’C and 45’C had a noticeably higher average LAI than the control trees. The trees heated to 55’C had a significantly higher LAI than the trees heated to 50’C. The trees heated to 50’C dropped more leaves than the trees heated to 45’C directly after treatment. Leaf anatomy samples were processed and analyzed. The leaf petioles from 7 days after treatment and 1, 3, and 6 months after treatment were cross-sectioned and stained to measure phloem area. Since the disease is phloem-limited, the phloem was analyzed for any changes due to heat treatment. Ideally, in heat-treated trees that are recovering, the phloem area should increase once the bacterium is no longer present. The results suggest there was an increase in phloem area directly after treatment in all treated trees, and it continued until 1 month after treatment. Then, the phloem area began to decrease again.
The work in the Core Citrus Transformation Facility (CCTF) continued without interruption. Co-incubation experiments using different type of explants and Agrobacterium strains are still being done on a weekly basis resulting in production of more transgenic plants. Three new orders were received during the last three months although most of facility’s productivity came as result of work on older orders. Significant effort was invested into production of rootstock plants carrying the NPR1 gene requested by the CRDF. About 850 shoots were tested in the primary PCR with 135 of them being positive. Forty eight of these shoots died either before or after grafting, and 27 were negative in the second PCR. The other 60 positive shoots were grafted. Out of those, 27 were moved to pots. Presently, there are 29 plants in the greenhouse that are 6-10 inches tall. In about six weeks many of those plants will reach the size when they could be cut into nodal explants for propagation. In the period covered by this report, CCTF produced plants for the following orders: pNPR1-30 plants, pNPR1-G-five plants, pX4- two plants, pELP3-G-one plant, pELP4-G- one plant, pMG105- eight plants, pPR2-one plant, pHGJ2- one plant, pHGJ8-one plant, pW14- two plants, pMED16-four plants, pN7-three plants, pN18- four plants. Out of total of 63 transgenic plants, 29 were Carrizo citrange, three were Swingle citrumelo, one was C. macrophylla, one was Mexican lime, one was Valencia orange, and 28 were Duncan grapefruit. Within previous year the efficiency of transformation and the ability of explants to regenerate shoots are little lower than they were in previous years. The reason for this is probably the low quality of seedling explants which are starting material in our experiments. Seeds used for production of Duncan grapefruit, Valencia orange, and Hamlin orange seedlings that are cut into explants are obtained from fruit harvested on CREC property where HLB is widespread. Fruit have unhealthy appearance; seeds are smaller and have altered color. Seed vivipary which occurs in older fruit of Duncan appears four months earlier than it used to. This is a strong indicator of disrupted hormonal balance within the fruit which may contribute to changes we noticed. For this reason, we intend to change the way fruit are acquired. Duncan grapefruit will be picked from CREC property only between September and January. Depending on the quality of fruit this season CCTF may start getting Duncan fruit from the outside source even before January. Fruits of sweet oranges will be acquired from the outside sources throughout the whole season and CCTF will require assistance for this.
The main accomplishments during this quarter: 1) We were continuing to infect and transform mature tissues of of citrus using Agrobacterium with the shoot enhancing genes we constructed. The explants used were greenhouse grown Washington Navel, Pineapple and Valencia. More calli formed than with the regular vectors. However, because the numbers of calli produced were relatively small, rates of shoot regeneration between the control vector and transformation enhancing vectors had not been compared. We were preparing a large number of adult explants for future infection experiments. We also started to use a number of techniques to reduce the contamination problems and a large number of explants of adult tissues for infection. 2) We were characterizing the enhancement of transformation efficiency of juvenile tissues of citrus using our regenreation enhancing genes in detail and also verified some of the results obtained previously. 3) Verification experiment for the role of an endogenous plant hormone in citrus regeneration from juvenile tissues upon transformation was performed and some progress had been made. However, more time is needed to generate significant results. We hope this study could shed some lights on the role of that particular hormone in adult tissue generation after infection. If so, the experimental results may guide designs of additional gene constructs for enhancing adult tissue transformation.
The main accomplishments during this quarter: 1) We improved a sterilization technique used for greenhouse-grown mature/adult shoot tissues and the contamination problems have been significantly reduced. 2) We have infected mature/adult tissues of Valencia and Washington orange using our transformation enhancing genes (K and I genes). Our preliminary results show that the use of the K and I genes we developed lead to drastic increases in transformation efficiency of mature tissues when compared to the conventional Ti-plasmid vector containing no K or I gene. 3) We have observed that the transport of an endogenous plant hormone in explants plays an important role in shoot regeneration efficiency. We also observed that manipulating the transport of that hormone improves shoot regeneration and genetic transformation efficiency of juvenile citrus explants. We are currently testing the effects of the same manipulation on adult tissues of citrus. We hope that manipulation can further enhance transformation efficiency of adult citrus tissues. 4) We are writing one manuscript that reports the drastic enhancement of citrus transformation efficiency of juvenile tissues of citrus. We will work on the second one, the effects of the transport and its manipulation for an endogenous plant hormones in explant tissues, once the first one is submitted.
We have concluded our phase 1 search employing our recently developed bioinformatics tools PAGAL and SCALPEL that led to the identification of 3 potential citrus candidate proteins that could serve as replacements for the CecB lytic peptide domain of our previously described chimeric antimicrobial protein (CAP; Dandekar et al., 2012 PNAS 109(10): 3721-3725). Using the same tools we have further refined our search within these particular proteins to identify a smaller segment that was tested for antimicrobial activity after chemical synthesis of the protein candidates. The following citrus proteins were chemically synthesized a 22 aa version of HAT (CsHAT22; a 52 aa segment of this protein was previously identified) a 15 aa segment of ISS (CsISS15 ‘ a negative control) and 20 aa segment of PPC (CsPPC20). These proteins were used to test the efficacy of their antimicrobial activity using the following bacteria, Xanthomonas, Xylella, E.coli and Agrobacterium. Using the same search criteria we identified a 22 aa N-terminal segment of the 34 aa Cecropin B (CBNT22) protein and a 12 aa segment of cathaylecitin (CATH15), representing protein with known antimicrobial activity that could serve a positive control for our bioassays. CsHAT22 and CsPPC20 were able to inhibit bacterial growth at levels comparable to CBNT22 and CATH15, however, CsISS15 displayed no detectable antimicrobial activity (as expected). We have recently obtained Liberibacter crescens and will soon test the antimicrobial activity with this bacteria as a surrogate for CaLas the causative agent of HLB. We have designed 3 constructs 1) CsP14a with a secretion sequence and Flag tag, 2) CsP14a ‘ CecB (this is construct 1 expressed as a CAP with the original CecB and 3) CsP14a-CsHAT52 (the 52 aa version of the CsHAT protein from Citrus) for testing in CTV vectors system and in transgenic plants (tobacco and citrus rootstock). These three constructs have been successfully incorporated into CTV vectors and are being infected to develop plant materials that can be used for challenge with HLB. All three of the above constructs have been introduced into binary vectors and then incorporated into Agrobacterium strains and these are being used to transform tobacco and Carrizo rootstocks. The plant transformation process in underway and will culminate in the isolation of transgenic plants that can be tested for disease resistance efficacy.
The main accomplishments during this quarter: 1) We did three Agrobacterium infections using adult tissues of Washington Navel, Pineapple and Valencia from greenhouse-grown plants. With the K and I genes, we observed more calli formed than with the regular vectors, which is a positive sign. However, the numbers of calli produced are relatively small comparing to those of the juvenile explants. We started the step to regenerate shoots from calli we have already produced but no significant results can be reported at this time. 2) We have observed endogenous concentrations of a hormone may play a role in citrus regeneration efficiency from juvenile tissues. We have started additional experiments to verify that observation. If that is true, we will modify the gene cassettes we originally designed accordingly.
OVERVIEW The Texas Citrus Budwood Certification Program continues to expand and evolve as the source of certified pathogen free, true-to-type citrus budwood for the Texas citrus industry. The program evolved significantly, becoming Texas Department of Agriculture (TDA) certified in January, 2014, and U.S. Department of Agriculture (USDA) certified in June, 2014. In addition several projects were completed to upgrade the program and facilities. – All Foundation and Increase screenhouses were certified by TDA in January and by USDA-APHIS in June. – All Foundation trees were tested for HLB and CTV in the spring-2014 and were all negative. – All Increase trees in the Screen Structures I-II-III were root tested for HLB in the fall, 2013 and tissue tested in the spring, 2014 for CTV and HLB. All were negative. – All Increase trees in the screenhouses 3 and 4 were tested in the spring for CTV and HLB. All were negative. – Screenhouse 5 received a new roof in the spring-2014. – New tables were added to Screenhouse 4 in the spring-2014. – All Foundation and Increase screenhouses will be at full capacity by fall-winter 2014. – The “TajMahal” Foundation greenhouse/screenhouse renovation will be complete this fall. The structure will be certified by TDA and USDA-APHIS to house containerized Foundation trees. – The Stephenville remote location greenhouse has 85 containerized Foundation trees, with a capacity of 100. The greenhouse will be at capacity by this winter. – Budwood sales for the year were 195,960. Rio Red Grapefruit buds totaled 155,401.
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 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. We now are examining the possibility of treating infected plants with antimicrobial peptides to allow them to recover from an HLB infection. We are beginning to work with a couple of teams of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. We are screening a large number of transgenic plants for other labs. We have promising transgenic plants that are being rescreened to ensure efficacy against HLB. We have had a collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We have found that three different lines appear to be giving strong tolerance against HLB. We are propagating the plants for more extensive analysis.
Obj 1A: To identify target psyllid effectors, the mining capabilities for the single (sTCW) and multi-(mTCW) [C. Soderlund, W. Nelson, M. Willer and D. Gang. (2013) TCW: Transcriptome Computational Workbench; PLOS ONE: 8(7), e69401] psyllid transcriptome databases have been finalized and released on line. Thus far over 500 candidates are available for hypothesis-testing using the database tools available at the website, including Gene Ontology and differential expression (GOSeq, EdgeR) profiles. Two manuscripts have been submitted. Obj 1B: Yeast-2 hybrid studies to capitalize on in vitro protein-protein interactions important in psyllid-Liberibacter interactions are ongoing. Previously, we reported 17 CLas candidate genes from a list of 25 putative candidates (identified in silico using the db, obj. 1A), were cloned into the Yeast 2 Hybrid (Y2H) mating experiments using the ACP gut and salivary gland libraries. To date 24-gut and 24-salivary gland library matings have been performed. From these experiments we discovered a number of potentially lucrative ACP gene products (‘prey’), and have moved them into the dsRNA station in the pipeline. Based on predicted functions, including some shared in common with other host-pathogen/parasite systems, it is becoming possible to hypothesize a pathway of CLas invasion into ACP tissues and organs (circulative). Among the promising candidate effectors are an ACP protein that comprises domains annotated as having antibacterial defense and quorum sensing functions. The knockdown of genes involved in quorum sensing processes could interfere with biofilm formation or signaling pathways that are essential for CLas establishment and survival. To identify potential CLas factors involved in lucrative points in the circular, propagative transmission pathway, ACP genes are used as ‘bait’ in mating studies with the CLas library. We last reported results from 14 ACP candidates mated against the CLas prey library. Thus far, 25 ACP gene candidates have entered the Y-2H station to test for interactions using the CLas prey library. Data analysis has been completed for 19 of these experiments, with the remaining being in various stages (PCR, cloning, sequencing, etc.) and moving towards completion. More than one third of mating experiments have produced potentially, biologically relevant prey products. The most interesting thus far are ACP bait genes with annotations to endocytosis/phagocytosis pathways, with one in particular that interacts with a CLas receptor-like protein known to be required for CLas invasion. Further biochemical analysis (in silico) of this ‘prey’ protein has confirmed it to be the aqueously soluble domain/portion of the protein. A second interesting candidate is also an endocytosis-related ACP gene that interacts with a CLas prophage protein. The CLas prophage/CLas/ACP interaction highlights another possible avenue for abating vector transmission. A third candidate has been identified, which may function as a regulatory protein that interacts with a microtubule attachment protein. This suggests it may have a role in Liberibacter adhesion and invasion of the psyllid host. Confirmation of Y-2H interacting proteins, and identification of additional interactors using immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) are underway. Two CLasY-2H candidates predicted to be involved in adhesion and quorum sensing were used to optimize IP and Co-IP assays owing to the abundance of these ACP interactors identified in Y-2H assays. About 50% of the adhesion-related candidate was found to be expressed in the soluble protein fraction. Optimization of pulldown assays is underway. Obj 2: RNAi studies are underway to functionally validate candidate effectors. To this end, good quality dsRNA has been synthesized for thirteen psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport or transcytosis, and nutrition. Knockdown experiments for 10 candidates have been completed using oral delivery and microinjection, with knockdown frequencies ranging from 50-100%. Five genes showed reduced transmission frequencies ranging from 18-25%. Confirmation of knockdown (qPCR), followed by transmission bioassay of additional candidates identified in silico (transcriptome) and those identified using in-vitro Y-2H studies, is ongoing.
We received an e-mail last week of April from the Citrus Research Development Foundation requesting revisions to be made to the original proposal #909. Revisions were sent that week. Research agreement funding CATP13 proposal #909 was forwarded to UF for execution first week June. We were notified of the available funding until July 7, 2014.
The goal of this project (#894) is to supplement project #707, specifically to determine the efficacy of plant growth regulators (PGRs) as a tool to mitigate declines in citrus tree root and canopy growth resulting from HLB. This project will extend our current work to include detailed greenhouse trials designed to help inform field applications of PGRs on established Hamlin and grapefruit trees in Lake Alfred and the Indian River region. The greenhouse studies will enable us to control environmental variables (soil type, tree age, secondary infections, etc.) that are not possible in the field and develop a fundamental understanding of how PGRs (e.g. 2,4-D, cytokinins, GAs) affect HLB-affected trees compared to healthy trees. Progress in the first quarter included procurement of sixty clean ‘Valencia’ / Kuharske nursery trees and inoculating half of them with HLB by grafting them with shoots from PCR positive trees. At the same time we also non-destructively estimated the root system size of every tree with an electrical procedure that measures root resistance and capacitance. This measurement will serve as the baseline for the current healthy root system status before deterioration from HLB infection begins.
The bacterium Candidatus Liberibacter asiaticus (CLas) is closely associated with the development of HLB and is transmitted into the citrus phloem via the psyllid insect. Using the fully sequenced CLas genome, we have identified 27 proteins that are predicted to be secreted outside the bacterial cell. These bacterial proteins are called effectors. In other bacteria, effectors are required for pathogen virulence enabling nutrient acquisition, insect feeding, and suppression of defense responses. The goal of the funded research will be to investigate the expression patterns and citrus targets of four different CLas effectors that are expressed in HLB-infected citrus trees. We hypothesize that these effectors are important for bacterial survival or HLB symptom development by targeting important citrus proteins to manipulate their host. A detailed understanding of these effectors and their citrus targets will facilitate HLB detection strategies as well as provide pathogen targets that can be manipulated to enhance plant tolerance and resistance. We have made significant progress in developing tools to assess CLas effector expression. When a gene is expressed, it is first transcribed into RNA and then translated into protein. In order to assess effector expression at the protein level, the Ma lab has previously generated antibodies that can recognize each of the four CLas effectors. During the funding period, we have purified each effector and used this to affinity purify each antibody. This has resulted in significantly enhanced detection of effector proteins with no interfering background detected in citrus. These purified antibodies can now be used for effector detection as well as effector targets in the funded work. In the Contained Research Facility at UC Davis, we have started a time-course experiment to investigate the detection of PCR positives as well as the expression of each effector in navel and mandarin orange over time after graft inoculation with CLas. We have validated that the infected material is PCR positive for CLas, contains the four effectors, and expresses the effectors at the protein level. Leaf tissue from plants at time zero (before inoculation) and up to six months after inoculation has been collected and has been processed to isolate DNA, RNA, and proteins. PCR positive results were obtained after three months and protein positives (based on effector expression) were clearly obtained after four months. One tree exhibited a putative protein positive at the three month time point that was subsequently validated at month four. We found that feeder roots displayed PCR positives first, followed by young leaves. In collaboration with Siddarame Gowda at the University of Florida, citrus expressing each effector is being generated using the CTV-based expression system to determine if effector expression results in any obvious morphological changes, enhances susceptibility to CLas, or promotes psyllid feeding. We have generated three expression lines for three effectors that have been validated using ELISA. The fourth expression line is in progress.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
