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.
Transgene Stacking for Long-Term Stable Resistance: Transgenic plants containing the NPR1 gene (best gene in our program for HLB resistance) stacked with the CEME transgene (best gene in our program for canker resistance) have been clonally propagated for further study (7 lines). Improving Consumer Acceptance: 1. The inducible cre-lox based marker free selection system: Molecular analysis of the putative marker free plants developed containing the cre gene driven by a Soybean heat shock gene promoter have demonstrated clean integration of the transgene in a majority of the regenerated plants. Leaky gene expression using this heat shock promoter system has however been observed in a few cases. This has not hampered our ability to regenerate marker free plants. This vector is being modified to incorporate the NPR1 gene from Arabidopsis, already proven to make plants resistant to HLB. 2. Transformation of Hamlin and W Murcott with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cells: We observed one of four scenarios when plants were analyzed using PCR 1) Most plants contains only the T-DNA of interest 2) Several plants contains both T-DNAs integrated into the genome 3) plants containing only the selectable marker T-DNA without any transgene of interest and 4) A few escapes that did not contain any transgene. Plants were obtained in a ratio of 6:2:1:1. Our results demonstrated the ability to produce marker free plants using this system, although we generated a number of escapes. Improvement of this protocol is currently underway to reduce the number of escapes and speed up the plant regeneration process. Induction of early flowering (Carrizo transformed with the FT gene): A majority of the plants flower prematurely in the tissue culture medium. These plants with apical flower development do not further develop in vitro. We are currently modifying the tissue culture medium to prevent in vitro flowering and successfully regenerate transgenic plants containing the FT gene for greenhouse evaluation. Transformation experiments are also underway with modified constructs containing weaker promoters driving the FT gene. Efforts to establish a new transgenic field site at the Southwest Research and Education Center: Working with Dr. Phil Stansly, we successfully renewed our transgenic field permit with APHIS to add this additional field site (the 4th site approved). Approximately 400 transgenic citrus plants were wrapped and tagged for planting during the next quarter. Field site preparation is underway.
“This project aims is to express molecules in plant that Disrupt the growth and ACP- transmission of CLas ” project narrative: Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detect AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Currently we are investigating the effect of expressing Lux-R in citrus plants on the level of many compounds, especially those presents in citrus. These compounds includes, but restricted to Indole-3-acetic acid (IAA), indole, g-amino butyric acid (GABA), salicylic acid (SA), Riboflavin and Lumichrome. These compounds can activate the Lux-R of many plant pathogens [1, 2, 3, and 4]. The level of these compounds is being measured in healthy and CLas-infected CLas expressing Lux-R as well as healthy and CLas-infected plants without Lux-R. We expect that the levels of some of those compounds will be reduced as possible binding to the Lux-R protein and/or bacterial cells. Identification of citrus compounds that mimic CLas N-Acyl homoserine lactone signal activities and affect CLas population density will highlight new strategies for the prevention of citrus greening disease. References 1) Spaepen S, Vanderleyden J, Remans R. 2007. Indole-3-acetic acid in microbial and microorganism-plant Signaling. FEMS Microbiol Rev 31: 425’448. 2) Lee JH, Lee J. Indole as an intercellular signal in microbial communities. FEMS Microbiol Rev: 34 (2010) 426’444. 3) Yuan ZC, Haudecoeur E, Faure D, Kerr KF, Nester EW. 2008. Comparative transcriptome analysis of Agrobacterium tumefaciens in response to plant signal salicylic acid, indole-3-acetic acid and g-amino butyric acid reveals signalling cross-talk and Agrobacterium’plant co-evolution. Cellular Microbiology 10(11): 2339’2354. 4) Rajamani S, Bauer WD, Robinson JB, Farrow JM, Pesci EC, Teplitski M, Gao M, Sayre RT, Phillips DA. 2008. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor. Molecular Plant-Microbe Interactions: 21 (9): 1184’1192.
“This project aims is to express molecules in plant that Disrupt the growth and ACP- transmission of CLas ” project narrative: Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detect AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Currently we are investigating the effect of many compounds, especially those found in citrus, on the activity of Lux-R of CLas. These compounds includes, but restricted to Indole-3-acetic acid (IAA), indole, g-amino butyric acid (GABA), salicylic acid (SA), Riboflavin and Lumichrome. Previous reports showed that those compounds can activate the Lux-R of many plant pathogens [1, 2, 3, and 4]. The E. coli strains carrying AHL reporter plasmids is being incubated with different concentrations of each compound and luminescence is measured after 0, 2, 4, 8, 24, and 48 h. The luminescence values obtained from each compared is also compared to that obtained from incubation of homoserine lactone standards with E. coli strain carrying AHL reporter plasmids. At the same time, we are measuring the levels of these compounds in different citrus cultivars (resistant and susceptible) to determine their role in citrus resistance to CLas. Using GC-MS, we are also studying the effect of CLas infection on the level of these compounds in citrus. In the next step, we will apply some of these compounds to healthy and CLas-infected citrus plants to confirm their roles in citrus-CLas interaction. References 1) Spaepen S, Vanderleyden J, Remans R. 2007. Indole-3-acetic acid in microbial and microorganism-plant Signaling. FEMS Microbiol Rev 31: 425’448. 2) Lee JH, Lee J. Indole as an intercellular signal in microbial communities. FEMS Microbiol Rev: 34 (2010) 426’444. 3) Yuan ZC, Haudecoeur E, Faure D, Kerr KF, Nester EW. 2008. Comparative transcriptome analysis of Agrobacterium tumefaciens in response to plant signal salicylic acid, indole-3-acetic acid and g-amino butyric acid reveals signalling cross-talk and Agrobacterium’plant co-evolution. Cellular Microbiology 10(11): 2339’2354. 4) Rajamani S, Bauer WD, Robinson JB, Farrow JM, Pesci EC, Teplitski M, Gao M, Sayre RT, Phillips DA. 2008. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor. Molecular Plant-Microbe Interactions: 21 (9): 1184’1192.
Project narrative: We aim to understand the specific interactions between Candidatus Liberibacter asciaticus (CLas) and the insect vector Asian citrus psyllids (ACP) to block the transmission. ‘The transmission process of CLas depends on the success of specific interactions between CLas and the insect vector ACP. The bacterium passes through the intestinal barrier to reach the hemolymph where they multiply then they must invade the salivary glandes in order to be inoculated in a new plant host while insect feeding. Passing these biological barriers needs specific interactions between CLas cells and the epithelial cells in the guts and the salivary glands cells.’ The molecular mechanism of citrus Huanglongbing (HLB) transmission by the insect vector, Asian citrus psyllid (ACP), is still largely unknown, and we have been investigating the protein-protein interactions (PPI) between ACP and the HLB associated bacterial agent, Candidatus Liberibacter asiaticus (CLas) by means of proteomics. The iron-transport protein, ferritin, is a highly stable, multi-subunit protein complexes, and it has been reported to play a key role in host-pathogen interactions of multiple vector-borne disease. In order to study the potential role of ACP ferritin in the CLas/ACP interaction, we first developed an extraction protocol to isolate and concentrate ferritin from ACP. Then the isolated protein complex was studied with two-dimensional Blue Native/SDS Polyacrylamide Gel Electrophoresis (2-D BN/SDS-PAGE) which we established earlier in our lab. After gel electrophoresis, a far-western blotting was performed to check the specific interaction between CLas and ferritin using CLas bacterium extracted from CLas-infected sweet orange plants. The signal of specific interactions observed on PVDF membrane was used to locate and isolate protein spots in the 2-D BN/SDS-PAGE gel copy (stained with silver staining) for protein identification by mass spectrometry. Our next step is to use far-western blotting to show the protein-protein interactions between CLas bacterium and the total proteins of ACP resolved by 2-D BN/SDS-PAGE, which could serve as a control and further validate our previous results. Our work should improve our understanding of the CLas/ACP interactions and contribute to the HLB management by providing potential targets of breaking the connections between the bacterial agent and the insect vector, thus slowing or even stopping the disease spread in the field.
Project narrative: We aim to understand the specific interactions between Candidatus Liberibacter asciaticus (CLas) and the insect vector Asian citrus psyllids (ACP) to block the transmission. Complexome In order to get a 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 is to find the proteins involved in the CLas-ACP interactions by studying the differences between the complexome of CLas-free and CLas-infected ACP. So far, we have established the Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) system in our lab to study the protein complexes (i.e. complexome) from CLas-free and CLas-infected ACP at their native status, and a further separation of the protein complexes in BN-PAGE by a second dimension of SDS-PAGE helped us obtain more detailed information on all the subunits or constituents of the protein complexes at their denatured status. By comparing the differences between the 2D BN/SDS-PAGE gel images from CLas-free and CLas-infected ACP, we have successfully located several protein spot candidates for protein identification by mass spectrometry. Our next step is to use far-western blotting to show the specific protein-protein interactions between the identified protein(s) and CLas, which will further validate our results from 2D BN/SDS-PAGE work. Our work should contribute to our aim of breaking the connections between the bacterial agent and the insect vector, thus slowing or even stopping the disease spread in the field.
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