In this proposal our objective is to find citrus versions for the two proteins that make up the functional components of a chimeric antimicrobial protein (CAP) previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have successfully identified a suitable replacement for the first component, the human neutrophil elastase (HNE). We obtained a close match with the tomato PR14a protein that is highly conserved in both Citrus sinensis (Cs) and Citrus clementina (Cc) genomes. We have completed the construction of two synthetic genes that encode this protein, one that just contains the coding region of CsP14a and the other is a chimeric version that contains the CsP14a coding region linked to the CecropinB (CecB) protein. We have also completed the construction constructing two CaMV35S expression cassettes to express both these synthetic genes so that the expressed proteins can be secreted. We have used CLASP to identify a citrus replacement component for CecB. Since CB has no enzymatic activity, we could not use a well-constrained motif like an active site. We chose instead the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. We have used an approach similar to that described earlier to identify a replacement for HNE to identify a replacement component for CB. However, instead of comparing the reactive atoms as was done for the HNE matching algorithm, in this case we match for the C-alpha atoms of specific residues based on the overall shape of CB. Thus, we adopted a slightly different computational flow in this case. First, we did a keyword search, ‘plants’ in http://www.pdb.org/ that yielded about 1000 proteins. Each pdb was expanded on basis of each chain. For example, PDB X.pdb with chains A and B resulted in PDB files XA.pdb and XB.pdb. This list was filtered based on a 80% similarity redundancy. Then proteins larger than 60 aa in length were ignored. APBS (Adaptive Poisson-Boltzmann Solver) software developed to examine electrostatic properties of proteins at a nanoscale) was run on each pdb (the ones that failed electrostatic analyses, a few, were excluded). We were able to extract 200 proteins which we then analyzed further using CB motif. The CB structure is characterized by two helices. We chose specific residues from these two helices such that the residues were polar, and had stereochemical matches. For example, Lys can be replaced by either Arg or His, and thus constitutes a good candidate. We chose, Lys10, Lys11, Lys16 and Lys29 as the input motif from CB, allowing Lys to be matched by Lys, Arg or His. Since the 52 aa H+ATPase (HAT) is large so we cannot have it cheaply synthesized as a protein. We are developing additional computational tools to better evaluate individual alpha helical domains so we can identify smaller proteins that can be more cheaply synthesized for testing. We have also begun making a CAP construct where CB is replaced by HAT. This will be expressed in plants to evaluate antimicrobial properties.
Our accomplishments are: 1) We have transformed juvenile explants of succari sweet orange and carrizo citrange using transformation enhancing genes (K and I genes) we have constructed. The use of the K gene leads to 7-15 fold increases in transformation efficiency while the use of the I gene had 4-9 fold increases in transformation efficiency when compared to a conventional Ti-plasmid vector containing no K or I gene. However, many adventitious shoots produced from the I gene containing Ti-plasmid vector are abnormal. 2) We conducted one transformation experiment using explants from adult trees but the results were not satisfactory due to some unexpected problems. We are preparing more tissues from adult trees grown in greenhouse for transformation using these constructs. 3) We are constructing the other transformation enhancing gene constructs and they should be done soon for testing.
Efforts continue to obtain data for further model development and validation.
Data have been gathered from three growers, and the data were prepared for analysis. A series of statistical models were fit to assess the association between yield and the application of enhanced ground and foliar fertilization in the presence of HLB. Some promising relationships were found. However, the data are sparse, and strong recommendations should not be made from these data alone.
Data have been obtained from three additional locations. Currently the focus is on getting the data into a common format. Then the data will be cleaned and further modeling will be conducted to explore the association between yield and enhanced ground and foliar nutrient programs in the presence of HLB.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. The girdling experiments have been finalized. All treatments were tested one last time and there was a high incidence of HLB transmission independent from the position of the budded material in relation to the partial girdle. Although there are still few trees that continued to be monitored, the results are clear that CLas (or an intrinsic signal) travels both in a longitudinal as well as a lateral way. The second set of grafting experiments, where graft bridges have been established between 2 independent plants, appear to be ready for the HLB-challenge. Two out of the 10 bridges appear unsuccessful, however. The remaining dead grafts were replaced with new material and are currently under observation. The initial 3 pairs of trees grafted under an initial project were tested once again. On this occasion, leaves from all branches were taken instead of one sample. The results were quite surprising in that in all trees, CLas had moved from the donor tree to the recipient tree through the xylem. This is the first data documenting xylem transmission of CLas. The anatomy of the citrus phloem has been completed and analysis of data is currently under way. A manuscript is being prepared for publication.
Understanding the proliferation and movement of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (citrus greening), within the citrus tree remains a major obstacle in the efforts to undermine the pathogenicity and destructive nature of the disease. CLas still remains an unculturable organism, in part due to the lack of information on the phloem contents and internal environment. Once established in the phloem sieve elements, CLas movement within a tree has been assumed to follow the photoassimilate stream. Our current belief of the CLas transport mechanism is dependent on a presumed bidirectional flow of phloem sap, with basipetal movement from the site of infection, proliferation in the roots followed by systemic distribution through the tree. For CLas movement to occur under the current theory, CLas must be able to move laterally around the stems, trunks, and young roots, and then travel bidirectionally within the phloem sap. However, determinations based on general phloem physiology, our limited understanding of CLas, and on current anatomical studies have exposed serious inconsistencies with the accepted beliefs of CLas phloem transport. For example, based on general phloem anatomy and our current microscopy observations, lateral movement of CLas around a stem appears improbable given the size of cytoplasmic plasmodesmatal connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from infected roots to healthy aerial tissues through the phloem conduits is difficult to reconcile without a reversal of the phloem flow, a condition not demonstrated for any evergreen species. The objective of this study is to define the biochemical properties and transport direction of citrus phloem elements that support CLas proliferation and allow movement within a citrus tree. The data will provide the basis for developing culturing conditions for the bacteria and for mitigating strategies based on movement of the phloem sap. The phloem sap composition part of the proposal is under way as planned. Samples are being conducted monthly from HLB and healthy trees gown in shade greenhouses. Samples are being stored at -80F until all samples are collected to be analyzed under the same conditions. Preliminary results indicate significant differences on both HLB+ and healthy at different times. Data on phloem sap movement in young tissues has been completed. Is likely that an additional experiment will be carried out using a different dye to monitor phloem sap. For the determination of phloem movement in stem tissue during different seasons, the designed electronic device went through a series of tests and improvements in data collection and transfer, as well as in energy use and storage. The devices have been installed in trees grown on the field and are collecting data every 20 minutes. Devices have been placed in “healthy” as well as HLB trees. Additional devices are being built to perform tests under control conditions in the greenhouse.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. The girdling experiments are well under way. Trees are frequently monitored for HLB symptoms and are routinely tested for HLB using PCR. HLB transmission has been confirmed in 40 of the 100 trees (20 trees per 4 treatments plus 20 controls). HLB transmission has been independent from the position of the budded material in reference to the girdled area. Many additional trees show classic HLB symptoms, but have resulted in PCR negative. These trees are being re-tested every 3 months. The second set of grafting experiments, where graft bridges have been established between 2 independent plants, appear to be ready for the HLB-challenge. Two out of the 10 bridges appear unsuccessful, however. These 2 pair of trees will be re-grafted while the eight remaining pairs will continue with experimentation. The anatomy of the citrus phloem has been completed and analysis of data is currently under way. A presentation on these matters was made at the Annual Meeting of the Florida State Horticultural Society.
Understanding the proliferation and movement of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (citrus greening), within the citrus tree remains a major obstacle in the efforts to undermine the pathogenicity and destructive nature of the disease. CLas still remains an unculturable organism, in part due to the lack of information on the phloem contents and internal environment. Once established in the phloem sieve elements, CLas movement within a tree has been assumed to follow the photoassimilate stream. Our current belief of the CLas transport mechanism is dependent on a presumed bidirectional flow of phloem sap, with basipetal movement from the site of infection, proliferation in the roots followed by systemic distribution through the tree. For CLas movement to occur under the current theory, CLas must be able to move laterally around the stems, trunks, and young roots, and then travel bidirectionally within the phloem sap. However, determinations based on general phloem physiology, our limited understanding of CLas, and on current anatomical studies have exposed serious inconsistencies with the accepted beliefs of CLas phloem transport. For example, based on general phloem anatomy and our current microscopy observations, lateral movement of CLas around a stem appears improbable given the size of cytoplasmic plasmodesmatal connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from infected roots to healthy aerial tissues through the phloem conduits is difficult to reconcile without a reversal of the phloem flow, a condition not demonstrated for any evergreen species. The objective of this study is to define the biochemical properties and transport direction of citrus phloem elements that support CLas proliferation and allow movement within a citrus tree. The data will provide the basis for developing culturing conditions for the bacteria and for mitigating strategies based on movement of the phloem sap. This project is well under way, with all preliminary work completed within this period. To determine phloem movement, two separate devices have been constructed following existing literature. To follow phloem sap movement in young tissue, a device consisting of 2 arm probes has been successfully tested. In one arm, a UV light source and fluorescent sensor are mounted on the tip, whereas the second arm probe contains just fluorescent sensor. An externally applied fluorescent phloem-mobile substance is excited upon passage through the UV and its fluorescence detected by both probes as it moves down with the phloem sap. Time is recorded and velocity can be calculated. For phloem movement in mature tissue, an electronic device was constructed. The device is based on heat transfer through the plant cortex. It contains 2 heat sensors that will determine time of heat transfer from a centrally located heat supply. The instrument is being tested in greenhouse trees. Once completed, other sill be constructed to commence with field experiments.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. As of this progress report the Valencia/Swingle trees have had HLB+ tissue grafted onto them. Approximately 90% of the grafted tissue has produced new flush and we are waiting for the remainder to produce new tissue. The girdling experiments have been completed and we are actively monitoring the wounds and the healing process to make sure that the redifferentiated tissue does not act as a pathway for CLas. We have hired a part-time employee to begin learning the microscopy techniques so they are well positioned to start the anatomical analysis once the trees are ready. Tissue samples have been collected and the SEM and light microscopy imaging are under way. Samples have been collected for PCR analysis. The second set of plants have been repotted and are now grafted together to form bridges between individual plants where either the phloem or the xylem is the isolated pathway. Tissue samples were collected for initial PCR analysis and samples will be collected in three month intervals.
The goal of this experiment is twofold, first to determine the effects of plant growth regulators on addressing vascular degeneration and fruit drop, and second to determine the effects of HLB and ACPS citriculture on drought tolerance. Preharvest fruit drop was monitored in a Hamlin (50% on Swingle, 50% on Carrizo) block located in Lake Alfred, FL from August to December 2013 with the final fruit drop count performed one week before harvest. At harvest the total remaining fruit on the tree were estimated to calculate a true percentage of the fruit dropped prior to harvest. The mean preharvest fruit drop for the block was approximately 42%. Data analysis is currently underway and suggest that preharvest drop was approximately 10% higher in trees with Carrizo rootstock compared to Swingle. We do not anticipate any significant differences between the plant growth regulator treatments with respect to preharvest fruit drop although additional statistical analysis is required due to the high variability in tree health within the trial. The harvested fruit was analyzed for size and quality. Fruit were smaller than average for all treatments and there were no significant differences between treatments. Phloem functionality data is currently being evaluated along with other measurements that will be available in a future report.
A number of genetic constructs have been received from various scientists, with and without supporting documentation (i.e. plasmid maps, sequence, publications). The genetic constructs for which we have supporting documentation have been put in the queue. The constructs without supporting documentation have been appropriately stored until information is received. As a new step in quality control, we are sequencing some plasmids, particularly those lacking the pVS1 replicon for stability in Agrobacterium. A genetic construct obtained from Dr. Mou has been transformed into mature scions of Hamlin, Valencia, Ray Ruby and mature rootstocks of Carrizo and Swingle. As advised, we have discontinued work with Pineapple sweet orange. Shoots will be pre-screened once they are bigger. Transgenic shoots of Valencia or Hamlin be micro-propagated and budded in different combinations with transgenic or wild-type Carrizo or Swingle immature rootstock. These trees will be submitted for disease screening to determine which have superior disease tolerance and whether transgenic rootstocks confer graft transmissable tolerance. Different cytokinins will be tested in scion and rootstock micro-propagation. Progress in being made towards increasing the productivity of the lab and the growth room. In the lab, mature scion and rootstock stem explants are being cut to 0.6 cm rather than the standard 1.0 cm for tissue culture. These smaller explants survive the Agrobacterium transformation protocol well and regenerate plantlets. This approach increases the explants available for each weekly transformation from ~600 to 1000. In the growth room, rootstocks are being budded with mature scion at an earlier age and the results look promising. Using this approach, we only need to transplant the rootstock on which the mature scion bud has opened. In an additional effort to increase our productivity, small experiments are being conducted to determine whether we can regenerate shoots from calli derived from mature leaf tissue after Agrobacterium treatment. Plantlets have been regenerated from the calli in one variety and are being elongated. These plants should still flower and fruit early because they have undergone the phase transition from immature to mature. We have also been able to root some varieties. NPTII immunostrip tests were conducted in sweet orange and grapefruit trees transformed with marker genes. Out of 47 transgenics tested, 34 tested positive for expression of the NPTII gene. Genomic DNA extraction and Southern blots to show transgene integration are underway.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Two different cytoplasmic viruses (CiLV-C and CiLV-C2) are now know to cause citrus leprosis. In addition a virus found in the nucleus, nuclear citrus leprosis virus (CiLV-N), also can cause citrus leprosis disease in North and South America. All types of the leprosis viruses are transmitted by Brevipalpus mites. We have continued mite transmission experiments at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD with endemic healthy Brevivalpus yothersii (syn. phoenicis) mites from Florida. Mite transmission experiments with the citrus leprosis affected citrus leaves from Mexico (CiLV-N) & Colombia (CiLV-C2) were done and two months after completion of the transmission experiments none of the citrus seedlings showed symptoms of leprosis. Leaf tissue from the experiments were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using CiLV type-specific primers and were negative. Using newly developed PCR primers we were able to prove the viruliferous status of Brevipalpus mites taken from infected lesions, killed in ethanol and shipped from Mexico (CiLV-N) and Colombia (CiLV-C2). We are continuing the use of these PCR primers to determine if Florida endemic healthy Brevivalpus Florida mites acquire the various citrus leprosis viruses. Additional collection of mites (preserved in alcohol) from Mexico have been sent to USDA cooperators to continue to compare their taxonomic status with those that do transmit in Colombia and elsewhere. In Colombia Guillermo Leon, continued work on the transmission and interactions of of CiLV-C2 and the vector, Brevipalpus phoenicis (Geijskes). The acquisition of the virus from citrus, then feeding on non-citrus host plants (some are hosts of the virus) and then feeding on citrus plants for virus transmission was evaluated. The appearance of the leprosis symptoms on Valencia orange plants was14-37 days after feeding, when the mites had previously fed on Swinglea glutinosa, Dieffenbachia sp. and Codiaeum variegatum for 2 to 20 days. When the mites were allowed to feed on Sida acuta, leprosis symptoms occurred in oranges between 18 and 46 days and when the mites fed on Hibiscus rosa-cinensis and Stachytarpheta cayennensis the symptoms appeared between 26-51 days after transmission. In conclusion it appeared that feeding on non-citrus hosts had no effect on virus transmission.
“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. Main findings since the start of project: 1-Using different bacterial biosensor, we partly identify these AHLs (number of Carbon). CLas biofilm formation on the surface of insect Gut confirms the presence of cell-to-cell communication in insect while the planktonic state of CLas in plant indicate the absence of this communication. 2- In plant, we found molecules that bind to LuxR but inactive its function (plant defense). We try now to characterize these molecule and study their effect on biofilm formation inside insect. We use purified molecule to feed infected insect through artificial diet system. 3-We produced citrus plants that express LuxR protein in the phloem sap in order to test I- If the acquired LuxR proteins in insect interfere with the biofilm formation in insect (cure the insect from CLas) II- if the expression of LuxR in plant induce biofilm formation (localize the infection in plant) We found that feeding infected ACP with CLas on the LUXR expressing plants reduce the bacterial populations in insect and reduced the infection rate significantly. This result strongly indicates that we can target this system to interfere with the insect transmission and the spread of Disease. We have identified molecules that are structurally non-related to AHL but reported in other system for their capacity to bind to bacterial LuxR such as GABA and Riboflavin like compound. We are testing these compound for their ability to bind to CLas-LuxR and induce the GFP fluorescence using our E. coli-LuxR biosensor. IN THIS REPORT: we studied the phloem sap composition of 14 different citrus varieties by GC-MS after derivitization with 2 different reagent to increase the limit of detection. we are analyzing these data right now to look for similarity in the composition in tolerant varieties and in susceptable varieties. this study will give us idea about what the citus phloem sap provide CLas with that may play as a signaling factor. We try to confirm our previous finding (GABA and Riboflavin like compound) and to link the presence of these compound with the susceptibility.
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