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. This proposal seeks to investigate the role of CLas proteins that are predicted to be secreted outside the bacteria during infection. These CLas secreted proteins are called effectors and we are focusing on four effectors that can be consistently detected in infected samples. The project objectives are to: (1) characterize the expression of four CLas effectors in citrus after graft and psyllid inoculation, (2) identify and validate citrus targets of CLas effectors, and (3) characterize the role of CLas effectors in suppressing defense responses and HLB symptom development. We have made significant progress on completing all three objectives. Objective 1: In the contained research facility at UC Davis, we have completed a time course experiment investigating the detection of CLas and individual effectors after graft inoculation in Navel and Mandarin oranges. Leaf and feeder root tissues were collected each month for 9 months (April-November 2014). Stem samples were used to probe for protein level expression of effectors using a tissue imprint method. Our results indicate that feeder root samples and samples from young leaves were PCR positive before mature leaf samples. We were able to detect transcript for one effector using qPCR at month 6 on one Navel plant. Protein level expression could be detected using the tissue imprint method by four months post-graft inoculation. However, we did have high background for the tissue imprint method during later time points. These experiments will be repeated after psyllid inoculation using Navel, Mandarin, and Lisbon Lemon this year. Psyllid inoculation experiments will begin at the contained research facility in April or early May, 2015. Objective 2: Co-immunoprecipitation and mass spectrometry has been performed for three of the effectors. This technique enables the identification of the effector as well as plant targets. We have been able to identify each effector by mass spectrometry, indicating that our method is working well. We were able to identify proteins significantly associating with two of the three effectors. The third effector did not identify any statistically significant associated proteins. We also performed mass spectrometry on phloem samples and feeder roots at the completion of the graft inoculation experiment for Navel and Lisbon samples, identifying a total of 642 differentially expressed citrus proteins in diseased plants. Two citrus proteins that were differentially expressed were also identified as associated with effectors and represent promising targets for follow up. Objective 3: In collaboration with William Dawson, we have generated citrus expressing each effector using the CTV-based expression system. These plants do not exhibit any obvious morphological changes. Dr. Dawson is currently testing these plants for enhanced sensitivity to CLas or altered psyllid feeding. We have obtained material from Dr. Dawson to detect each effector using specific antibodies. If the effectors can be easily detected, we will use this material to repeat immunoprecipitation experiments in Objective 2.
We have concluded the activities of objective 1 of our proposal which have focused around finding a native citrus protein replacement for cecropin B the C-terminal component of the chimeric antimicrobial (CAP) protein. We had identified CsHAT52 using one set of bioinformatics tools and confirmed antimicrobial activity with a portion of this protein that we designated CsHAT22. Bioassay of CsHAT22 revealed a minimum inhibitory concentration (MIC) of 50 uM with Xanthomonas, 100 uM with Xylella and 300 uM with Liberibacteria crescens (Lc). Using two additional bioinformatics programs, PAGAL and SCAPEL and have successfully identified and tested 2 additional proteins, CsPPC20 and CsCHITI25 that were compared to CB and the N-terminal 21 amino acids of CB designated CBNT-21. Among the test strains used Xanthomonas was most susceptible to the peptides with CB and CBNT21 showing and MIC values 25 uM and the MIC values for CsPPC20 and CsCHITI25 were 50uM and 100uM respectively. Both Xylella and the BT-1 strain of Lc gave MIC values of 200 uM for CBNT21 against both Xylella and Lc BT-1. CsPPC20 was more active than BNT21 against Xylella giving an MIC value of150 uM and as active against Lc BT-1 giving an MIC value of 200uM. CsCHITI25 was as active as CsPPC20 against Xylella but not as active against Lc. Based on these results we have included CsPPC20 as an additional construct for testing in planta and excluded CHITI25. CTV vectors for expressing CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and evaluation and testing of the efficacy of these vectors is underway. The construction of a CTV vector to express CsP14a-CsPPC20 is underway. Binary vectors for Agrobacterium-mediated transformation of CsP14a, CsP14a-CB, CsP14a-CsHAT52 and CsP14a-CsPPC20 have been completed and the transformation process of tobacco and Charrizo tissues is underway for the isolation of transgenic plants. This is being done at the plant transformation facility at UCDavis. We obtained transgenic Carrizo citrus transformed with NE-CB as a positive control. These plants are in the CRF and last week they were exposed to infected psyllids. Once transgenic Carrizo are obtained with the 4 vectors they will be transferred to the CRF for testing with infected psyllids for resistance against HLB.
This project aims to characterize conditions for optimal deployment of a superinfecting (SI) Citrus tristeza virus (CTV)-based vector as a tool to be used in the field to prevent existing field trees from the development of the HLB disease and to treat trees that already established the disease. We are moving towards completion of the objectives of this proposal. For the Objective 1, we used our greenhouse-propagated CTV isolates, each containing a single virus strain, T36, T30 or T68. In addition, we used two FS field isolates collected by P. Sieburth in 2005 in Central Florida. These isolates were used to infect citrus plants that were later challenged with the SI vector. The performance of the vector was assessed by the assessment of the level of the GFP expression from the vector as well as the level of the SI CTV vector multiplication in the challenged plants. These parameters were compared to those in plants that had no primary infection and were inoculated only with the SI vector. As the result in these experiments, the SI vector was able to successfully superinfect trees pre-infected with the T36, T30 and FS isolates, with levels of the vector multiplication and expression similar to those the vector developed in citrus plants with no primary infection. This suggests that once on the field, the vector will successfully perform under the conditions similar to those created in this experiment. On the other hand, the SI vector, which contains the leader protease region from the T68 strain, failed to superinfect trees infected with T68. We will validate and address this observation in our future work. From the experiments in the Objective #2, we have learned that SI vector develops higher titers in sweet orange varieties, compared to grapefruit varieties, with the titers found in sweet orange being 2-3 times higher than those in grapefruit. This would suggest that levels of anti-HLB product produced in sweet orange will likely be higher than those in grapefruit. At the same time, we did not find major differences in the levels of the SI CTV vector multiplication between different cultivars of the same variety (e. g., Valencia versus Hamlin and Duncan versus Ruby Red) as well as between different rootstocks used (Swingle, Carrizo, and Citrus macrophylla were used as rootstocks). The examination of the temperature effect in the Objective #3 showed that optimal accumulation of the SI vector and thus optimal production of the gene of interest from the vector occurs at 25’C (77’F). Some decrease (~2-3 times) of the vector expression level was observed at 32’C (89.6’F) and a major decline (10 fold) was seen at 35’C (95’F) and above. In some cases, the vector could not be detected in the above ground tissues of the plants exposed to the latter temperature. So, the recommendation would be to conduct inoculations of field trees with the vector during the cooler seasons to provide the conditions for optimal initial virus vector multiplication and spread over the trees that are being treated. The experiments proposed under the Objective #4 are still ongoing. We expect to complete those in two-three months. In general, the presence of the HLB pathogen per se in trees prior to their challenge inoculation with the vector have not significantly affected the SI vector accumulation, especially in trees whose growth was not affected by the HLB pathogen. However, in trees that were severely affected by HLB, had strong disease symptoms, and stopped growing, the systemic movement of the CTV vector was impeded. The reason for this is that CTV moves with carbohydrates flow from mature source to newly developing sink tissues within a plant as the plant grows. Therefore, it could be expected that factors affecting plant growth affect CTV movement as well, which suggests that it likely would be a challenge to treat severely affected declining trees with the CTV vector. Additional results are being now evaluated.
The aim of this project is to identify important components of citrus for HLB pathogenesis. We will analyze the targets of four effector proteins produced by the HLB-associating bacterium Candidatus Liberibacter asiaticus (Las). Effectors are secreted proteins that perform essential virulence function in bacterial pathogens. Las possesses the general Sec secretion system, through which about 30 effectors can be secreted into the phloem. By characterizing the effector targets, this project will advance our understanding on basic biology of HLB pathogenesis. This knowledge will facilitate the development of control strategies. This project will focus on four Las effectors, which are highly expressed in infected citrus trees. We will determine their targets in citrus; these targets may play important roles in HLB development and symptom physiology. The main approach that we are using to identify the effector targets is yeast two hybrid (Y2H) screening. In the first year of this project, we cloned the four effector genes into an Y2H bait vector, which were then transformed them into the yeast strain AH109. We also constructed a citrus cDNA library using HLB-infected RNA samples. This library was subsequently normalized to exclude the highly abundant transcripts, which may bias the screen. At the end, we obtained a high-quality citrus cDNA library with more than 3 millions of primary clones. At the beginning of the second year of this project, we completed the Y2H screening using each of the four effectors as the bait. For each effector, more than 10 million yeast clones were screened in three independent experiments. Sequences of potential effector-interacting proteins were determined using Illumina sequencing. These sequences were compared to a non-selective cDNA pool generated from screens using a negative control protein as the bait. This practice effectively eliminated the non-specific interacting proteins. The rest of the sequences represent candidate proteins that specifically associate with a particular effector. These sequences have been aligned to the genome sequence of Citrus sinensis to obtain gene identities. After sophisticated statistical analyses, we came up with a list of potential targets for each effector. Our data suggest that one effector specifically targets E3 ubiquitin ligases, which are important enzymes that regulate protein stability. The second effector targets a class of transcription factors that regulate plant immunity and hormone signaling. The third effector targets peptidases, which also regulate protein stability; and the last effector mainly targets ion-binding proteins. These data suggest that the Las effectors potentially manipulate citrus immunity and metabolism, and thereby contributing to bacterial colonization and disease symptom development. Our on-going effort is to confirm the interactions between specific baits and their targets using other biochemical approaches. For this purpose, we have cloned the top five interaction candidates for each effector from citrus cDNA, and these candidates will be individually examined for their interactions with specific effector using in vitro and in vivo pull-down assays. We are also characterizing the localization of the effectors and the co-localization of effectors with their targets using confocal microscopy analysis.
Improving Consumer Acceptance: 1. In efforts to reduce juvenility in citrus, transgenic Carrizo citrange have been successfully produced expressing the clementine CFT3 gene. We have very small micrografted plants flowering in the greenhouse and these plants have been evaluated by PCR to confirm presence of the CFT3 gene. A few micrografted trees flowered immediately. 2. Following the successful demonstration of the inducible cre-lox gene system, the plant transformation vector has been modified to contain our NPR1 gene and Agrobacterium mediated citrus transformation is underway to incorporate this gene. 3. Transgenic plants containing our stacked transgenes are being clonally propagated for disease resistance evaluation and the first trees will be challenged for HLB resistance in spring 2015. 4. Plants in our Indoor RES structure have not flowered this year. It is possible greenhouse temperature may have played a role in the flowering process. We will attempt to keep the greenhouse unheated this fall in hopes of initiating flowering in spring 2015 Additional Resistance Gene candidates: 1. Transgenic plants containing antimicrobial gene LIMA-B were propagated for field challenge. 2. OLL-8 sweet orange and W. Murcott were transformed with the CtNH1 gene (NPR1-like), using our protoplast/GFP transformation system. Small colonies and embryos were regenerated from OLL-8 with GFP expression.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. In summary, a total of 15 independent transgenic lines now exist for the FLT-antiNodT fusion protein expression construct. These plants are now growing well and cuttings are being taken and rooted to produce multiple vegetatively-propagated plants for each line. This is essential to run multiple tests for the gene expression patterns and HLB resistance levels of each line.
This project aims to understand HLB pathogenesis by analyzing the citrus targets of four effector proteins of Candidatus Liberibacter asiaticus (CLas). Effectors are secreted proteins that perform essential virulence function in bacterial pathogens. They manipulate plant immunity and physiology for the benefit of colonization and disease development. CLas possesses the general Sec secretion system, which secretes about 20-30 effectors into the phloem. We hypothesize that Sec effectors of CLas target key components in citrus, and thereby contributing to HLB. Our bioinformatic and gene expression analyses revealed four Sec effectors of CLas that are highly expressed in infected citrus trees. In this project, we are identifying the direct targets of these effectors in citrus. These targets may play important roles in HLB development and symptom physiology. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109. We also constructed a citrus cDNA library using HLB-infected RNA samples. This library was further normalized to exclude the highly abundant transcripts, which may bias the screen. At the end, we obtained a high-quality citrus cDNA library with more than 3 millions of primary clones. In the second year of this project, we completed the Y2H screening using each of the four effectors as the bait respectively. For each effector, more than 10 million yeast clones were screened in three independent experiments. Sequences of potential bait-interacting proteins were determined using next generation Illumina sequencing. These sequences were compared to a non-selected cDNA pool generated from screens using control proteins as the bait. This practice effectively eliminated non-specific interacting proteins. The rest of the sequences represent candidate proteins that specifically associate with a particular effector bait. These sequences have been aligned to the genome sequence of Citrus sinensis to obtain gene identities. After sophisticated statistical analyses, we came up with a list of potential targets for each effector. Our data suggest that one effector specifically targets E3 ubiquitin ligases, which are important enzymes that regulate protein stability. The second effector targets a class of transcription factors that regulate plant immunity. The third effector targets peptidases, which also regulate protein stability. The last effector mainly targets ion-binding proteins. This is interesting because HLB symptoms resemble zinc deficiency, indicating that ion metabolism in citrus is altered by CLas infection. Taken together, we have found promising targets of the four effectors. Our data suggest that the effectors potentially manipulate citrus immunity and metabolism, and thereby contributing to HLB. Our on-going effort is to confirm the interactions between specific baits and their targets using in vitro and in vivo pull-down assays. We are also characterizing the localization of the effectors and the co-localization of effectors with their targets.
This project aims to understand HLB pathogenesis by analyzing the citrus targets of four effector proteins of Candidatus Liberibacter asiaticus (CLas). Effectors are secreted proteins that perform essential virulence function in bacterial pathogens. They manipulate plant immunity and physiology for the benefit of colonization and disease development. CLas possesses the general Sec secretion system, which secretes about 20-30 effectors into the phloem. We hypothesize that Sec effectors of CLas target key components in citrus, and thereby contributing to HLB. Our bioinformatic and gene expression analyses revealed four Sec effectors of CLas that are highly expressed in infected citrus trees. In this project, we are identifying the direct targets of these effectors in citrus. These targets may play important roles in HLB development and symptom physiology. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109. We also constructed a citrus cDNA library using HLB-infected RNA samples. This library was further normalized to exclude the highly abundant transcripts, which may bias the screen. At the end, we obtained a high-quality citrus cDNA library with more than 3 millions of primary clones. In the second year of this project, we completed the Y2H screening using each of the four effectors as the bait respectively. For each effector, more than 10 million yeast clones were screened in three independent experiments. Sequences of potential bait-interacting proteins were determined using next generation Illumina sequencing. These sequences were compared to a non-selected cDNA pool generated from screens using control proteins as the bait. This practice effectively eliminated non-specific interacting proteins. The rest of the sequences represent candidate proteins that specifically associate with a particular effector bait. These sequences have been aligned to the genome sequence of Citrus sinensis to obtain gene identities. After sophisticated statistical analyses, we came up with a list of potential targets for each effector. Our data suggest that one effector specifically targets E3 ubiquitin ligases, which are important enzymes that regulate protein stability. The second effector targets a class of transcription factors that regulate plant immunity. The third effector targets peptidases, which also regulate protein stability. The last effector mainly targets ion-binding proteins. This is interesting because HLB symptoms resemble zinc deficiency, indicating that ion metabolism in citrus is altered by CLas infection. Taken together, we have found promising targets of the four effectors. Our data suggest that the effectors potentially manipulate citrus immunity and metabolism, and thereby contributing to HLB. Our on-going effort is to confirm the interactions between specific baits and their targets using in vitro and in vivo pull-down assays. We are also characterizing the localization of the effectors and the co-localization of effectors with their targets.
We have been working to modulate the response of citrus to huanlongbing (HLB) infection, by targeting genes that are up-regulated, and contribute to phloem-plugging in HLB-affected citrus(Albrecht and Bowman, 2008; Folimonova and Achor, 2010; Kim et al., 2009). Our approach has been to use an engineered citrus tristeza virus (CTV) silencing vector (Folimonov et al., 2007; Gowda et al., 2005; Hajeri et al., 2014) to deliver truncated dsRNA sequences homologous to callose (CalS7) and phloem-protein 2 (PP2) implicated in phloem-plugging. These dsRNA molecules are expected to result in the down-regulation or reduced expression of CalS7 and PP2, such that phloem-plugging will be precluded or mitigated in citrus upon infection with HLB. The CTV vector has been engineered to express CalS7 and PP2 truncated sequences, and inoculated into citrus (Citrus macrophylla). Using conventional reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of the engineered virions has been verified in individual citrus plants. Plants positive for the engineered CTV vector were then graft-inoculated into other citrus varieties and tested similarly. The responses of these plants, and appropriate controls, to HLB infection are being tested by exposing young flush to an HLB-positive psyllid colony maintained in the laboratory for four weeks, and following the changes in mRNA expression levels of CalS7 and PP2 via quantitative RT-PCR (qRT-PCR) on a monthly basis. Two-Three months after testing positive for HLB, we have observed changes in CalS7 and PP2 mRNA levels in both HLB-positive treated and non-treated controls. Although the observed changes in PP2-silenced citrus are significantly lower than the non-treated controls, changes in CalS7-silenced plants remain highly variable at this time. We have observed variations in expression of CalS7 and PP2 even in healthy controls, and thus are unable to correlate these changes in expression with HLB-status. It is too early to draw conclusions based on the available data. However, the silenced plants exposed to HLB are beginning to show symptoms of infection, while the microscopy does not show phloem-plugging yet. It is possible that symptom expression is not directly the consequence of phloem plugging. Overall, our experiments are progressing well, and we have discussed our results so far at the just ended International Research Congress on Huanlongbing (IRCHLB IV) in Orlando, Florida. We intend to continue to follow the profile of CalS7 and PP2 expression in experimental plants via qRT-PCR, and to support our data with northern blot analysis to show silencing of CalS7 and PP2, and microscopy to reveal any phloem plugging.
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. As of October 15th, 2014, work is in progress regarding the greenhouse experiments described, above.
Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing” or cell-to-cell communication systems. Interestingly, the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We have confirmed the functionality of the CLas solo luxR by constructing a luxR gene promoter fused with a GFP reporter. This has resulted in a functional CLas luxR::GFP monitor strain E. coli similar to that reported by Kock et al (2005). This E.coli strain produces fluorescence if the luxR promoter binds to AHLs or to a eukaryotic signal. Several AHLs, including N-butanoyl homoserine lactone, N-hexanoyl homoserine lactone, N-3-oxo-hexanoyl homoserine lactone, N-3-oxo-octanoyl homoserine lactone and N-3-octanoyl homoserine lactone, as well as extractions from insect and from citrus plant, have been shown to activate CLas luxR. The plant derived extracts are likely to be structurally unrelated AHL mimics. As a response to infection by CLas, citrus may increase the production of its AHL mimic(s), which would bind to LuxR and possibly limit CLas bacterial growth by triggering cell aggregation and consequently limit bacterial movement in planta. The insect extract has a structurally related AHL which may be produced by the endosymbiontic bacteria and bind to CLas LuxR. As a result of this binding, Clas form biofilm on the surface of ACP gut. Currently we are investigating the effect of many compounds known to be signals for bacterial LuxR, especially those found in citrus phloem sap, on the activity of CLas LuxR. These compounds include, but not restricted to Indole-3-acetic acid (IAA), indole, .-amino butyric acid (GABA), salicylic acid (SA), Riboflavin and Lumichrome. We expressed CLas LuxR in citrus using the CTV-based vector system. LuxR expressing citrus showed equally distributed severe symptoms when infected with CLas. Rearing infected ACP on healthy LuxR plants resulted in a diminishing CLas population. Interfering with CLas cell-to-cell signaling may lead to new avenue of control strategies.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated. – The second cohort of advanced, tetrazygous citrus rootstock materials for en masse propagation have been identified and are being prepared to have cuttings taken from them. – In addition to the 1st & 2nd cohorts of tetrazygous rootstocks, promosing diploid rootstocks have also been identified and are being prepared to have cuttings taken from them. As of January 2015, plants are still growing in the green house and no significant changes to report.
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. As of January 15th, 2015, work is in progress regarding the greenhouse experiments described, above.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, as proof of concept to determine the root specific nature of the promoters (RB7, C1867 or SLREO), we have incorporated the promoter-gus sequences into N. benthamiana and Carrizo citrange and several plantlets have been regenerated. Testing of these plants to confirm the root specific activity of our promoters will be performed as they become available. In addition, we have initiated experiments to incorporate the plant transformation vectors containing the GNA, APA and ASAL genes driven by either the root specific RB7 promoter or the citrus derived C1867 promoter into Carrizo citrange. Stacked constructs, each containing the GNA, APA or ASAL genes with the CpTI gene driven by the SLREO promoter have been produced and are also being incorporated into Carrizo citrange.
In order to better understanding of the transmission mechanism of citrus Huanglongbing (HLB) by the insect vector, Asian citrus psyllid (ACP), we have been working on unraveling the protein-protein interactions (PPI) between ACP and the HLB associated bacterial agent, Candidatus Liberibacter asiaticus (CLas) by means of proteomics. Complexome is the whole set of the protein-protein interactions in a particular cell or organism. During the transmission process, CLas bacteria traverse inside the insect vector systemically and various PPIs and protein complexes (formed by protein constituents from both CLas and ACP) must be involved; therefore, our aim was to find the proteins involved in the CLas-ACP interactions. Several approaches have been used including protein-overlay assay (Far-Western) and Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) 1-Far-Western: we used phloem sap from infected citrus as source for CLas. The phloem sap was used to overlay ACP proteins that were separated by SDS-PAGE and trans-blotted onto PVDF membrane. The complexes were revealed using CLas-specific antibodies. We identified V-ATPase, ATPase-TER 94, beta-tubulin, and actin as binding proteins for CLas. 2- BN-PAGE We have established the BN-PAGE system in our lab to study the protein complexes (i.e. complexome) from CLas-free and CLas-infected ACP in their native status, and a further separation of the protein complexes in BN-PAGE by a second dimension of SDS-PAGE which helped us obtain more detailed information on all the subunits or constituents of the protein complexes at their denatured status. We have successfully located several protein spot candidates for protein identification by mass spectrometry. Among the identified proteins, ferritin subunits and transferritin formed complexes with CLas proteins. The identified proteins should be a good candidates for RNAi technology using CTV-based vector. Expression of dsRNA against those protein in citrus may reduce or block the transmission of CLas by its vector ACP.
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