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.
The first task in this proposal is to find citrus version of the two proteins that make up the functional design of a chimeric antimicrobial protein previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have completed the discovery of the replacement of the first component the human neutrophil elastase (HNE) the surface binding component. Since HNE is a serine protease with elastasin as a substrate and whose crystal structure has been determined, we used 3D shape criteria and electrostatic properties to search the PDB data base for a plant protein with the identical active site structure. Focusing on a set of 288 non-redundant plant derived proteins from the entire PDB database, we used CLASP to search for a match using the electrostatic and structural features of amino acids that make up the active site. This was successful and the tomato PR14a was identified as an exact match. Using the tomato amino acid sequences we then searched for a similar citrus protein by searching citrus genome information in Phytosome (http://www.phytozome.net). This was successful and we have identified a single protein that is identical in the Citrus sinensis (Cs) and clementina (Cc) genomes. We are focusing on the 165 amino acid protein from Cs which we call CsP14a. We have analyzed this sequence and have determined that it is a secreted protein and contains a 25 amino acid signal sequence. We are focusing on the 137 aa mature protein and begun the construction of two genes that encode this protein. The first is a CaMV35S expression cassette that will be used to express just the mature form of the CsP14a protein, however, we have included a signal peptide (22aa) that we have used before and know works really well at secreting proteins to the plant apoplast and xylem. This signal peptide has been added at the N-terminal of this protein and we have added a Flag Tag also at the N-terminal so that the protein can be easily detected and purified. The Flag tag is part of the secreted protein after cleavage of the signal peptide. The second version is like our CAP protein and has Cecropin B (CecB) at the C-terminal linked via a flexible linker as we have described earlier. This protein is designated CsP14a-CecB. These genes will be used for in planta expression of the proteins CsP14a and CsP14a-CecB that will be isolated and used to test their efficacy against the bacteria as we described in our last report.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project are being used to support the construction of a new greenhouse at the IRREC. UF-IFAS Facilities Planning & Operations (FP&O) conducted a competitive bidding process and identified a vendor capable of constructing FDACS-DPI-approved propagation structures. Funds were transferred to IFAS FP&O to initiate construction of a new greenhouse to support the goals of this project. Currently, site preparation at the IRREC is being performed to ready the location, and building will commence as soon as possible. – The initial cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation have been identified. They are currently emerging from the ‘HLB gauntlet’ and will soon be ready for en masse propagation.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. A total of 3,805 transgenic plants have passed through inoculation process. A total of 76,160 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects.
The Texas Citrus Budwood Certification Program has completely migrated into insect-resistant structures, as well as rebuilding the Foundation and Increase tree collection. The progress made will allow the Budwood program to continue supplying pathogen-free, high quality, true-to-type citrus budwood for the Texas citrus industry. PROJECTS – In October, 2012, construction was completed on the Phase I-II and III projects, enclosing the existing Increase tree collection inside an insect-resistant screen structure. The enclosed screen structures have new irrigation drip lines pumps and injector system. – Foundation Screenhouses 1 & 2:A new irrigation system was installed in Foundation Screenhouses 1 & 2. Foundation trees were moved from Screenhouse 5, the Stephenville greenhouse, and newly propagated trees from CCPP bud sources, and planted into SH-1 and 2. – A new irrigation system was installed in Screenhouse 5. New trees that were propagated from CCPP bud sources in October, 2012 were added to replace the Foundation trees moved to SH-1 and 2. There are currently 200 containerized trees in Screenhouse 5, and another 50 will be added this fall to bring it to capacity of 250. – The ‘TajMahal’ building that has served as a research and biological indexing greenhouse and screenhouse began renovation in May, 2013. The rooms will be made insect resistant, for housing additional Foundation trees. – All Foundation and Increase trees at the Citrus Center and Stephenville greenhouse were tested in the fall and spring for HLB and in the spring for CTV. All trees tested negative. – The Foundation collection began rebuilding in September, 2012. 34 new varieties were budded with buds from the California Citrus Clonal Protection Program (CCPP). In June, 2013 another 26 varieties were budded from buds from the CCPP. Currently there are 81 Foundation trees planted in-ground in Screenhouses 1 and 2. There are 200 Foundation trees located in Screenhouse 5. There are 83 Foundation trees located at the Stephenville greenhouse. Budwood from the Stephenville trees is cut 4 times per year and brought back to the Citrus Center to propagate new Increase trees for SH 3 and 4. – Establishment of new Increase trees began in Screenhouse 3 in September, 2012. There are over 1,000 active Increase trees in pots in Screenhouse 3, with another 1,200 trees budded to be transplanted. All Increase trees source budwood came from Foundation Screenhouse 5 and the Stephenville greenhouse. The first budwood cutting of trees in Screenhouse 3 was in April, 2013, about 4 months ahead of schedule. As soon as the capacity is reached in Screenhouse 3, then Increase trees will be started in Screenhouse 4.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we have performed the designed microarray experiment to identify genes that are induced by NAD+ in citrus. The microarray data is currently under analysis. For objective 2, in last quarter we cloned two non-host resistance genes against citrus canker into the T-DNA vector pBI1.4T and mobilized the plasmids into Agrobacterium. In this quarter we have started genetic transformation of citrus ‘Duncan’ grapefruit with the Agrobacteria. Several putative transformants have been identified.
Obj 1A: All components of the user-friendly web versions of the single (sTCW) and multi-(mTCW) psyllid transcriptome databases have been finalized in preparation for the public release which will allow researchers to quickly identify targets, not only based on expressional differences but in combination with predicted functions. Recent data obtained from the sTCW databases containing salivary glands and midgut tissue transcripts show that the salivary glands contain more transcripts that are differentially expressed in response to Liberibacter than midgut tissues. Also there are drastically more ‘biological process’ Gene Ontology categories that contain a significant number of differentially expressed transcripts in the salivary glands compared to the midgut. These data highlight the potential importance of tissue specificity, specifically the role of the salivary glands in Liberibacter transmission. Previously, it was reported that members from 3 OrthoMCL-defined clusters (mTCW) were validated in both POP and ACP. To date, the expression of transcript members from an additional cluster predicted to be important for bacterial nutrition has been validated in both ACP and POP by RT-PCR using primers designed from conserved regions. Obj 1B: Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions. Previously it was reported that a total of 9 CLas candidate genes from a list of 19, had been moved into the Yeast 2 Hybrid (Y2H) mating experiments using the ACP gut and salivary gland libraries. To date 12 gut library matings and 12 salivary gland library matings have been performed. Data analysis has been completed for 20 of those experiments with the remaining being in various stages (PCR, cloning, sequencing, etc.) moving towards completion. Through those 20 experiments we have thus far discovered roughly 44 ACP gene products that have high levels of interest making them good candidates for RNAi and will be moved into that phase of the project if they fit the criteria specific for RNAi. Previously we reported the findings from the first 3 mating projects (1 midgut and 2 salivary glands). Since the last update, we have discovered more interesting candidate effectors that will be moved into the RNAi phase of the project many of which are predicted to be important in Liberibacter adherence to host tissues. One interesting candidate is an endopeptidase putatively involved in the dissolution of extracellular matrices. The overexpression of this gene could greatly interfere with CLas transmission. An ACP gene product similar to enzymes involved in signaling cascades important for bacterial communication and subsequent biofilm formation was also identified. A CLas ‘bait’ with protease function, identified an ACP gene important for bacterial nutrition and is made available to the bacteria after processing. An interesting finding came from using the CLas ‘bait’ FlgL protein. No interactors were discovered from gut matings, only from salivary gland mating experiments which suggest the motility of Liberibacter is most important in this tissue. To date, one candidate ACP gene putatively involved in bacterial adhesion has been mated against the CLas prey library interacting gene products (‘prey’ inserts) identified are currently being analyzed. Obj 2: RNA interference (RNAi) studies are underway to functionally validate candidate effectors. To date, good quality dsRNA has been synthesized for five psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport, or transcytosis. Previously we reported on the impact of the knockdown of one of the cytoskeleton-related gene on Liberibacter transmission using the oral delivery method. Recently, both microinjection and feeding studies have been conducted for actin, the other cytoskeleton-related gene whose knockdown is expected to result in high psyllid mortality and is used as a control to optimize our studies. Mortality results from oral delivery of dsActin show that 37% more psyllids died after feeding on dsActin-treated diet in comparison to the control. Mortality results from microinjection delivery of dsActin show that 15% more psyllids died from dsActin injections in comparison to control injections. These preliminary data suggest that the method of dsRNA delivery is crucial, and should be optimized accordingly.
The target for therapeutics that we focused on this quarter of funding is CLIBASIA_01810 (renamed LdtR). Over the last year we have found that this protein is a novel transcriptional regulator that modulates the expression of proteins involved in cell wall synthesis. Since we have proved that inhibitory molecules bind specifically to LdtR to reduce the transcriptional activity of ‘Ca. L. asiaticus’ in infected plants, our goal was to identify where and how small molecules affect the protein stability and activity. Using structural modeling we selected residues that may be involved in the binding of small molecules and performed site directed mutagenesis. The consequences of mutations in the LdtR protein was tested on the binding to its cognate DNA binding sequence as well as on the ability to bind small molecules. We were able to identify three residues involved in ligand binding. Currently, molecular dinamics experiments are being performed in collaboration with IBM with the end goal of optimizing chemicals for treatment of infected trees. A manuscript with the results obtained in the LdtR studies has been submitted for publication to a peer reviewed high impact journal.
Processing of the agreement for this project was delayed and funds were only recently received by the PI. Nevertheless Hamlin orange fruit samples were harvested from neighboring groves with normal cultivation and fertilization treatment and those receiving one or two different nutritional foliar sprays from healthy and infected trees (PCR tested), in December, 2013 and January, 2013. Similarly, Valencia orange samples were harvested in April and May of 2013. Size and color measurements were conducted on the fruit prior to processing. Fruit were then washed and extracted using JBT extractors, finished, pasteurized and frozen for future analyses. Sensory tests include difference from control tests (DFC) and some trained panel analysis, while chemical analyses include measurement of sugars, acids, aroma compounds, limonoids and flavonoids which are ongoing. Fruit from HLB treatments were smaller and more green than fruit harvested from healthy trees.
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