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.
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. We have modified the CTV vector to produce higher levels of gene products to be screened. 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 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 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.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Six months after grafting, HLB- budded tissue continues to grow in all treatments, especially after spring flush. At this time (May 8, 2015), leaf samples were taken from the uninfected tree and sent for HLB analysis. This type of analysis will be conducted every three months. Meanwhile, all trees plus controls are being monitored for HLB symptoms.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Six months after grafting, HLB- budded tissue continues to grow in all treatments, especially after spring flush. At this time (May 8, 2015), leaf samples were taken from the uninfected tree and sent for HLB analysis. This type of analysis will be conducted every three months. Meanwhile, all trees plus controls are being monitored for HLB symptoms. Results from the budding material came back all positive, meaning that all trees had been exposed to HLB as intended. On July 2015, all trees from both treatments were sampled for HLB. In one set of trees, leaf samples were taken from both sides of the girdle to test for the potential transfer of CLas material across severed phloem cells. In the other set of trees, in which the girdle was placed in the main trunk, was also tested for HLB. In the tree with the girdle on one side branch, 23 out of 25 trees turned out HLB+. In the other treatment, 24 out of 24 trees turned out HLB positive. From the data, we conclude that genetic HLB signal is sub-cellular in nature and can be transferred between non-phloem living cells. This new finding is important in that it shows that HLB causing effector is not necessarily the entire CLas bacterium.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Six months after grafting, HLB- budded tissue continues to grow in all treatments, especially after spring flush. At this time (May 8, 2015), leaf samples were taken from the uninfected tree and sent for HLB analysis. This type of analysis will be conducted every three months. Meanwhile, all trees plus controls are being monitored for HLB symptoms.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Graft material was allowed sufficient time to connect and grow onto the tree, or to perish and be replaced. Budded material began to show signs of growth after 2 months of grafting with a high success rate. Once buds began to grow, trees were trimmed and kept under observation until sufficient growth takes place for HLB testing.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. To conduct tree girdling in specific patterns to severe direct phloem connections, several techniques were assessed in a preliminary experiment in the greenhouse. To start with, a technique to create spiral girdling was developed and chosen. Trees have been purchased and a total of 25 trees were girdled with this particular spiral pattern on the main stem. A similar girdling pattern is now being applied to a secondary branch on a second group of 25 trees. The trees are kept in a greenhouse and watered regularly. Once all treatments and controls are finalized, they all will be challenged with HLB-affected tissue and assessed for HLB by PCR regularly.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct have been completed at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette has been introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Fifteen (15) independent transformant lines resistant to the kanamycin selection marker and expressing the green fluorescent protein have been regenerated successfully into plantlets. Of these 15 lines, 10 are strong expresses of the green fluorescent protein (GFP) transgenic marker, indicating successful transformation and expression of the transgenic marker genes. The other 5 show spotty expression of the GFP in cells of all tissues examined. This could indicate gene silencing might be affecting GFP expression in these plants. The effect that this might have on FLT-antiNodT fusion protein expression are not known, but will be tested later. The plantlets range from 5 – 15 cm in height, with the smaller plants being younger than the larger plants. All of the 15 lines appear to be growing and developing normally, which is a good sign that the FLT-antiNodT fusion protein is not having any unexpected deleterious effects on the plants. Permits to move the plants from Florida to Pennsylvania have been obtained, in order to study the FLT-antiNodT fusion protein expression levels and phloem-localization of the FLT-antiNodT fusion protein. Plans are being made to test the HLB resistance of these transgenic lines in collaboration with Dr. Tim Gottwald at the USDA Horticultural Research Lab at Ft. Pierce, FL.
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 been preparing citrus plants for root treatment with NAD+. We are testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, about 15 more transgenic lines have been generated. We are currently characterize the transgenic seedlings. For the 20 transgenic lines generated previously, we have confirmed 15 of them containing the transgene. Expression of the transgenes have also been tested. These plants are growing in the greenhouse and will be tested for canker resistance. We are cloning the citrus homologs and will confirm the sequences of the genes before transformation.
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