In order to examine how Candidatus Liberibacter asiaticus (Las) infection affects gene expression in citrus tissues, transcriptional response of leaf, stem and root tissues of Valencia sweet orange (Citrus sinensis) to Las infection was compared using Affymetrix microarray. The analysis revealed that Las reprograms several metabolic and cellular processes in citrus, and identified genes whose expression is regulated in a tissue-specific manner. Tissue specific regulation was observed for several genes and gene groups including those encoding cell wall pectins, transcriptional factors with MADS box and PHOR1 domains, G-proteins, legume-lectin family proteins, pectatelyases, SUT4, SUC6, BAP12, protein kinase THESEUS1 and vacuolar invertase, which were regulated only in leaves; major intrinsic protein family proteins, crinkly 4-like protein, SUT1, ERF5, CPRD2, CNGC1, CSLD4, and FERONIA regulated in stems; and NAT12, GLR4, DDM1, SCL14, APS kinase, resistance protein RGC2, CCR4-associated factor 1-related protein and Arabidopsis response regulator 1 regulated in roots. In another study, host response of Rangpur lime (Citrus . limonia Osbeck) which shows tolerance to the bacteria, to Las infection, was examined using suppression subtractive hybridization (SSH). Genes encoding senescence-associated protein, dehydroascorbate reductase, stress enhanced protein 1, protein kinases, miraculin-like protein 2, actin related protein 2/3 complex, checkpoint-like protein CHK1, ACC oxidase, type I proteinase inhibitor-like protein, xyloglucan endotransglycosylase, protease inhibitor, and PR10-related Cas s 1 pollen allergen were up-regulated, while genes encoding photosystem II subunit R, DNA binding protein, a putative zinc finger protein, SLL1 protein, CCR4-associated factor, metallothionein-like protein, cysteine proteinase, cinnamyl alcohol dehydrogenase-like protein were down-regulated. Many of these genes were unaffected in the susceptible C. sinensis, suggesting genotype dependent response to Las. Sequence analysis and comparison of the expression of a set of pathogenesis-related (PR) proteins is being done using quantitative real-time reverse transcription PCR. To examine the molecular response of citrus to infection by Las under field conditions, Affymetrix microarray analysis was repeated using samples collected from Valencia sweet orange in citrus grove. The analysis is currently undergoing to compare the microarray results from the greenhouse, and the citrus grove (two different times). This research has significantly advanced our understanding of how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Importantly, the manipulation of host response especially plant defense response by Las provides valuable information regarding how to manage HLB.
Using Liberibacter from the alimentary canals of psyllids as inoculum, we have tested over 560 media formulations for growth of liberibacter. Some formulations support prolonged survival of Liberibacter in culture. After 2-3 weeks in culture, masses of Liberibacter have been observed in biofilms on the psyllid alimentary canals used as a source of inoculum and streaming from the canals into the media. We will to continue testing different formulations of the media to determine if they will support planktonic growth of Liberibacter. Information obtained from the genome sequence of Liberibacter and by comparing that genome with the genome of the closely related Babaco bacterium from culture is being used to devise new media formulations. Other possible growth factors, such as diffusible signal factors produced by the psyllids, are also being evaluated.
1. Data mining for ACP continues for annotated transcriptomes (phase I) of the whole adult and gut ACP to identify candidate genes for functional analysis, with an emphasis targets for siRNA-mediated silencing. These data have been used to design RNAi solutions for bioassay. 2. Illumina sequence data for ACP ESTs (from PAVE) was analyzed based on R-stat and normalized expression levels. Pairwise comparisons were done for the Adult,Nymph and infected adult and infected nymph libraries, revealing a number of genes differentially expressed in the four pairwise comparisons, based on transcript copy number. Not all genes that are differentially expressed in the Adult and Nymph were exclusively expressed in either one. The varying range of differential expression suggests that there are well defined developmental cues. Expression of genes in all pairs and the fold change ranges from very obvious to very subtle differences. 3. Potato psylllid ESTs.The Six adult psyllid libraries (adults, dissected salivary glands and alimentary canal) were used to construct mRNA enriched from total RNA extracts (Trizol). The paired Illumina libraries were prepared for sequencing, subjected to DNA sequencing, and are currently being assembled by NCGR. DNA sequences will be organized in PAVE, our data management system that constructs a database and has tools for mining transcripts, and will allow for subsequent RNASeq quantification (Soderlund). 4. FISH probe localization was highly successful for Liberibacter solanacearum localization in dissected guts using the 16S rRNA, outer membrane protein, and abc transporter genes probes when fixation conditions were modified and the new fluorescent camera was employed. Testing with the 16S rRNA for Carsonella ruddii (primary endosymbiont, potato psyllid) as an internal control, are underway to demonstrate specificity. Optimization is underway using the 16S rRNA probe for salivary glands and the oral box. FISH results for whole psyllids indicated decolorization will be essential and that this may confound the technique’s utility for intact adult psyllids, however, parameters are being tested in an attempt to overcome this limitation. 5. Feeding studies are underway to optimize an artificial feeding assay for potato psyllid using a system developed for whitefly. Psyllids were found to remain alive on sucrose feeding solutions tested for 3-7 days.
1. mtCOI haplotyping. More samples are being received for mtCOI haplotyping from the R. Lee lab (USDA, CA) to explore baseline diversities in different locations, in an attempt to relate populations from the US and elsewhere in the Western Hemisphere to haplotypes from a primary region of endemism. All sequences in hand have been edited. 2. Time-course and transmission studies continue bioassay to assess transmission frequency in relation to Rt-PCR detection of Ca. Liberibacter solanacearum (AZ) and in Ca. L. asiaticus (FL) in individual psyllids reared on infected plant material. Results using dot blot hybridization were found to be less sensitive than RT-PCR but were generally comparable with a few exceptions; nonetheless we will continue the work using qPCR even though it is the more expensive method, results are robust. Five psyllids reared on infected tomato, and given a range of inoculation access feeds on tomato seedlings from 30 min, and 1,2,4,8,12,and 24 hr transmitted Ca. L. solanacearum 5-20%, 35, 25-30,70, 80, 90, and 95% of the time (20 plants per rep). Plants were assayed using RT-PCR and were scored for symptom development. Only the former data were used to calculate transmission frequencies because psyllid feeding causes aberrations in symptom phenotype that disallow Liberibacter-incitedsymptoms to be differentiated consistently. This above result was corroborated in two replicated studies and a third replicate is underway. Results suggest that once acquired psyllids transmit at relatively high frequency. The goal is to select individuals for light microscopy, and SEM-TEM ultrastructural observations to pin point localization following a range of different AAP and IAPs. We initiated two AAP time points, starting with 8 and 24 hours using a 24 hr IAP. IAP studies with ACP have proven too difficult owing to the apparent uneven distribution of bacteria in plants, which confounds reliable detection and transmission bioassay to further the localization effort. The goal to produce time course feeding populations for both ACP and potato psyllid is overly ambitious given the problems experienced obtaining ACPs with consistent Liberibacter titer, despite season or host. Even so, extrapolations from the more tractable potato psyllid study system, (an approach approved by the project Director) will clearly help clarify AAP and IAP time-points in relation to organs that are key to the transmission pathway. 3. As noted we are using the potato psyllid as a surrogate for the Asian citrus psyllid since they are closely related, both have digestive systems so similar that we can’t find any differences using the methods and instruments available to us (Cicero et al 2010), and, thirdly, the potato psyllid is easy to rear on tomato in our Arizona laboratory. Thanks to timely publications (Cicero et al 2010, Ammar et al 2011a, b), collaborative efforts, and FPRAC quarterly/annual reporting by the teams involved, the general motif for the transmission cycle of Ca. Liberibacter has been worked out in the potato psyllid. We are in publication mode now. Further, we are now developing the toolbox to answer more detailed questions, such as the infra-instar changes in bacterial titers, and the role of the pharate instar in the movement of bacteria from the midgut, where the anatomically operational beginning of the cycle occurs, to the stylets, where the cycle finishes. We are also moving to apply the methods, used for the above breakthrough, on the Asian citrus psyllid as a check to be sure no significant differences occur in that species.
In this fourth quarter, 208 plants of Valencia orange on Kuharske Carrizo rootstocks, inoculated with HLB infected buds on the 3rd week of April of 2011 using a standard inverted ‘T’ budding technique as describe by Ferguson (2007), were individually tested for HLB infection monthly, starting on July 11, 2011. Tree leaves per each inoculated tree were harvested to verify HLB infection by quantitative real-time PCR (qRT-PCR). Petioles and midribs of the citrus leaf were used (100 mg per sample) to extract total DNA using ‘Qiagen DNeasy Plant Mini Kit’. After DNA isolation the yield and purity of the DNA was estimated by measuring OD260 and OD260/280, respectively, with a NanoDrop Spectrophotometer ND-1000. The qRT-PCR for Candidatus.Liberibacter species was performed using 16S rDNA primer and probe sets as described by Li et al.(2006). The qRT-PCR consisted of 2 min incubation at 50 ‘C followed by 10 min incubation at 95 ‘C and 40 cycles at 95 ‘C for 15 s and 60 ‘C for 1.0 min. qRT-PCR data were analyzed using the Applied Biosystems software Version 1.4.0. The qRT-PCR results from July, August and September showed HLB infection on 0, 6, and 11 inoculated trees, respectively. Based on the low number of tree that acquired HLB infection 5 months after inoculation, starting on October, 2011 citrus trees will be re-budding with HLB infected buds. To obtain a base line of gene expression on leaves form HLB inoculated and HLB non-inoculated trees were sampled for RNA isolation on August 22, 2011. For control trees for each treatment, 10 mid-vein + petioles were pooled. For the HLB-infected trees, 18 were pooled (mid-vein + petiole only). A total of 448 frozen pooled samples were ground and immediately 1 g was weighed for RNA isolation and the rest was stored for back up as pre-ground RNA tissue. RNA was extracted from August 24 to August 30, 2011. The RNA extraction was done using the 3 day guanidine isothiocyanate (GITC) protocol and stored at -80 ‘C, gene expression will be determined by qRT-PCR. Literature cited: Ferguson J. 2007. Your Florida dooryard citrus guide. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Document HS 884. Li W., Hartung J.S. and Levy L. 2006. Quantitative real time PCR for detection and identification of Candidatus Liberobacter species associated with citrus huanglongbing. J. Microbiol. Methods 66: 104-115.
Since the recent release of the citrus genome sequences, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory. In some genes, however, we observed single nucleotide polymorphisms in our cloned genes, compared with the ones published in the citrus sequence database. We think that it might be due to the difference in the plants used in our lab from that used in the citrus genome sequencing. Nevertheless, we found that most published defense genes have full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes. The cloning of these genes is at various stages, some have been amplified from the cDNA library and cloned into pGEM vector while others have already been moved to the binary vector pBinARSplus. The genes newly cloned into the binary vector pBinARSplus include ctNHL1, ctMOD1, and ctJAR1. Since previously cloned ctEDS5 had many single nucleotide polymorphisms (SNP) from the published sequence, we recloned the ctEDS5, which now has fewer SNPs, into pBINARSplus. We have planted the corresponding Arabidopsis mutants plants and transformation will be conducted with the constructs, ctEDS5, ctNHL1, ctMOD1, and ctJAR1 soon. For ctNDR1 + ndr1-1 plants, we are continuously characterizing the defense phenotypes of the homozygous lines. So far we observed a range of defense phenotypes displayed by different individual plants, which is normal with independently transformed lines from one construct. Some plants with stronger disease resistance are also associated with minor cell death phenotypes. For ctPAD4 + pad4-1, ctEDS1 + eds1-2, and ctEDS5 + eds5-1 plants, we are trying to get more transgenic plants, obtain homozygous lines, and/or perform initial defense tests.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las is much shorter that we expected. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
The main objective of this project is to examine how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development and how it correlates with the distribution of the bacterium within infected trees. During this period of funding we established a psyllid colony for the use in our experiments. One portion of healthy psyllids is maintained on healthy Murraya paniculata plants in special insect cages and the other portion is exposed to HLB-infected citrus plants to generate HLB-infected psyllids that can be further used for inoculation of new citrus plants. Psyllid transmission experiments were initiated using already available HLB-infected plants. Additionally, we inoculated new sweet orange and grapefruit plants using the infected psyllids and graft-inoculation with HLB-containing buds to generate more plant material that can be used in our further experiments. The newly inoculated plants will be monitored for development of HLB infection and then used for transmission experiments. To examine acquisition of the bacterium from different types of flushes, healthy psyllids (as nymphs and adults) were placed on either a young growing flush or an older symptomatic flush of an infected tree. Psyllids were secured on those flushes by using small traps made up of mesh material. After 21 days traps were removed and psyllids were analyzed by PCR with HLB-specific primers. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. However, TEM observations of the material extracted from psyllids demonstrated that psyllids fed on different types of flushes contained different forms of Las: psyllids fed on the young flushes contained mostly rod shaped bacteria, while bacteria extracted from psyllids that fed on the old symptomatic flushes were mainly present in a round form. We are now conducting experiments that will determine the viability of the bacteria acquired during psyllid feeding on different tissue sources. Another interesting observation was made during these experiments. Psyllids fed on the old symptomatic tissue exhibited phenotypical abnormalities. Our next step is to assess whether these developmental abnormalities are due to psyllid feeding on the symptomatic diseased tissue or similar phenotype can be obtained from incubation of psyllids on old flushes of healthy plants. During this period we also began experiments in which we are examining what types of flushes are more susceptible to psyllid inoculation. In these experiments infected psyllids are placed on different types of flushes of healthy plants. Plants are grouped in two sets: first set in which only young flushes are exposed to psyllids and second set in which psyllids are placed on old flushes. Psyllids are kept for a period of 21 days on those plants. The experiments are in progress. Percentage of plants that become infected will be analyzed.
The initial indexed mature material that was maintained in vitro for a long time did not adapt properly after planting in soil. The plants have already 3 months and they have very long leaves and are growing stunted, most of them are not behaving like normal plants. We are going to continue monitoring them the next couple of months to see if they recover and come out of this stage. The new batches of indexed mature material introduced from May to July 2011 are being grafted on Swingle citrumelo, Macrophylla and C. Volkameriana that were growing originally inside the laboratory. This material will also be used to establish mother plants and to establish the first batch of plants that will give us the material for mature transformation experiments. The rootstocks that we started planting when the growth room was finished are not ready for grafting yet. Only a few plants coming from this material were available to graft and the whole group will be ready in October 2011. The Growth Room is still under “modification”. It took almost 5 months to be able to have completely access to the computer program, however it is still an issue to get access for users that come and go which is the case with the personnel we currently have. The Citrus Research and Education Center does not have enough IT help to assist in a timely fashion with the several needs required for this project. The IT people usually respond within weeks instead of hours to any problem we may have. The humidifier in the small room is still not working properly. The company responsible for the job was not able to coordinate the different subcontractors to finalize the job. The warranty in this case will not work and we will need to pay for them to finish this task. Another problem that we are currently facing is related with filtrations among the different areas where the floor meets the walls but also window sealing. The water is moving between growth rooms and between the growth room and the office. The caulking applied to close the gaps and to provide a seal between the concrete floor and the panel walls is not working. The subcontractor came and applied a new coat of caulking but it was not enough to stop the filtration. It is still happening as of today. Water leaking through the window has not been addressed yet. We also experience problems with tripped breakers and bad electrical connections of some lamps. We are still monitoring the situation.
The causal agent of greening, Candidatus Liberibacter asiaticus ( L asiaticus) is a fastidious phloem-inhabiting gram-negative bacterium. It is transmitted by Asian citrus psyllid (ACP) during feeding. Transmission mechanisms of Las have been intensely investigated recently, yet key information gaps still existed. We investigated whether Las is transmitted between infected and uninfected ACP adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male ACP to uninfected females at a low rate (< 4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of seven days or more was required to detect the bacterium in recipient females. In addition, Las was detected in the unlaid eggs and offspring of infected females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy but were absent in ovaries from uninfected ACP females. The size of the rod shaped structures varied from 0.39 to 0.67 'm in length and 0.19 to 0.39 'm in width. The spherical structures measured from 0.61 to 0.80 'm in diameter. Although, the overall percentage of sexual transmission of Las from males to females appears small, it could be a highly significant contributing factor to pathogen spread given that thousands of psyllids colonize individual trees. Collectively our results demonstrate that Las is transmitted from male to female ACP at a low rate during mating. These results suggest that the presumed causal agent of HLB may spread within populations of the vector even in absence of infected host plants. These findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of ACP, even in the absence of infected host trees. Detection of Las in female psyllids reared on Las-free citrus plants after mating with infected males strongly suggests that this plant pathogenic bacterium was transferred sexually from male to female insects during mating. This was also confirmed with lack of transmission from females to males, or between same sex pairs. While this phenomenon has been demonstrated experimentally for endosymbiotic bacteria of insects, this is the first report of sexual transmission of plant pathogenic bacteria among insect vectors. The distribution of Las appears to be ubiquitous throughout the hemolymph and organs of infected psyllids, although the bacterial titer is highest in the alimentary canal and salivary glands. We observed a low bacterial titer (Cq values 37 to 39) in 2.3% and 8.9% of individual male and female genital samples, respectively, suggesting a low bacterial titer in the reproductive organs of ACP. Cq values could also be high because of the small amount of tissue that is processed during DNA extraction even though there may be a high bacterial load relative to the size of the organ examined. If the overall titer of bacteria in the infected psyllid donor is low, the likelihood of bacteria occurring in the reproductive organs should also be correspondingly low. Detection of Las in the unlaid eggs and offspring of infected females is congruent with previously reported transovarial transmission of Las by ACP. Sexual transmission of Las provides additional, albeit indirect, evidence that the relationship between the pathogen and ACP could be mutualistic rather than pathogenic.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our library is estimated to contain 2.1 x 10 7th primary, unique antibody clones. Because our antigen was individual psyllids from Florida infected with high concentrations of ‘Ca. Liberibacter asiaticus’, the library contains antibodies for both the pathogen and the vector insect. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. These approaches are described in previous reports. We have now obtained a unique set of scFv antibodies including ‘B665, B705, B717, B734 and B743’ that bind to a portion of the major outer membrane protein, OmpA; B900, B905, B913, B932, B1037, B1071, B1072, B1075 and B1083 that bind to flagellar protein FlgL; B947, B968, B969, B982, B984, B1096, B1114, B and B1115, B1119 and B1128 that bind to flagellar protein FlhA; B435, B466, B467, B468, B475, B500, B547, B556, and B557 that bind to a pilus protein; B442, B443, B479, B494, B520, B1191, B1194, B1199, B1202 and B1212 that recognize a protein that polymerizes cell surface polysaccharides.We have also isolated a number of scFv that recognize the TolC protein of ‘Ca. Liberibacter asiaticus. We isolated these scFv by using genomic sequence data to clone and purify specific proteins to beused as capture antigens. Portions of these proteins predicted by software to be exposed on the cell the surface were cloned. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins have been purified. Emphasis is on recovering the scFvproteins in native, soluble form, which was difficult, but we have now been able to accomplish it. Thus we have developed and demonstrated a protocol that will allow us to isolate scFv antibodies that, in principle, will recognize any proteins from “Ca. Liberibacter asiaticus” or the insect vector, Diaphorina citri. Several of these proteins have also been successfully used in Das-ELISA assays and in dot blot assays of plant and insect extracts containing ‘Ca. Liberibacter asiaticus’ from Florida. Premature termination of scFv proteins has been encountered in the soluble expression system. This is because ‘stop’ codons accumulate in the phage selection system because the host E. coli is an amber suppressing strain. But these ‘stop’ codons become a problem for production of soluble scFv. Therefore we are checking all clones by sequence analysis to be sure premature stop codons are not present. This approach allows production of soluble scFv. Several of these antibodies detect antigen in plant but not in insect extracts. scFv antibodies may be useful as labels for ultrastructural studies of infected plants and insects, advanced detection assays, and possible even for HLB control through a ‘plantibody-based’ approach.
The main objective of this project is to manipulate calcium signals by over-expressing calcium signal modifier genes (CSM) from citrus to develop broad spectrum disease resistance. During the fiscal year 2010-2011 we produced 17 transgenic C-22 rootstocks, three Valencia and six Hamlin oranges over-expressing the CSM-1 gene. Previous grapefruit plants produced with this gene Were tested for disease resistance and shown to be resistant to citrus canker, Phytophthora nicotianae and Alternaria alternate and to the toxin tentoxin. Furthermore we engineer a new Agrobacterium binary vector with a citrus lectin gene to be used for genetic transformation during the fiscal year 2011-2012. As part of the transformation procedure, we are trying to accomplish genetic transformation without the use of antibiotic, and using only citrus genes. We were able to find a substance that increase the regeneration capacity of citrus stem segments used for genetic transformation and at the same time have the potential to be used for selecting transgenic plants and replace the antibiotic selection system. We are currently optimizing the system. We performed several crosses of Rio Red X Hirado Buntan pummelo and Rio Red X Wilking tangor. Hybrids will be recovered in November 2011.
In our initial schedule, the mature transformation facility (lab and greenhouse) at the CREC in Lake Alfred had to be implemented during the first year of the project. An existing laboratory was modified to fulfill the requirements of a tissue culture facility. The laboratory is now fully operative. Regarding the greenhouse, it became impossible to accommodate the budget to our plans for constructing the outstanding facility we requested. Alternatively, a growth room was designed profiting an existing structure at the CREC. The growth room construction was initiated in October 22nd 2010. The projected date of completion was February 11th 2011. Technically it was finalized by mid-April, however there were several technical/operational problems that came out during the following 4-5 months, especially regarding the refrigeration system (environmental conditions were not estable; some air handlers were not producing the air the manufacturer claims, in some cases the thermal expansion valve was changed because it was defective, air filters were not the ones we requested and they were changed, the humidifier in the small room is not located in the appropriate place for working properly), computer program (growth room technician still doesn’t have access to the program though this is currently being solved), water elimination after irrigation is defective, soil sterilizer (it needs a special accommodation to work ‘safely’). A generator should be purchased; without it, any prolonged electricity cut could jeopardize the whole project. The manager from Florida (Dr. Cecilia Zapata) completed her training in Spain during the first year, moved to Florida and has been working hard to set up the mature transformation facility at the CREC during the whole second year. Two part time OP technicians were hired to work on tissue culture and on plant preparation and a third OP technician was hired to work care at the growth room, under the supervision of the manager (and the PI at the initial stage). Another technician has been recently hired to help in the growth room and the lab because one of the OP technicians is leaving soon due to personal reasons. The Spanish lab has been monitoring the progress of the Florida facility. The PI and his manager at the IVIA greenhouses traveled to Florida last March 2011 to supervise the growth room construction and to set up healthy citrus germplasm bank establishment. The PI and his greenhouse manager will travel again to Florida next October 2011. An IVIA scientist with experience in mature citrus transformation will travel to Florida to help setting up the facility tentatively next November 2011. It is programmed that the IVIA scientist will spend three months in Florida (November 2011-February 2012). In Spain, mature tissues from the three sweet orange types (Hamlin, Pineapple and Valencia) plus Carrizo citrange were readily transformed. For our second objective, improving citrus tree management, we proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. We have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange over-expressing either CsFT or CsAP1 flowering-time genes already established in the greenhouse. We have characterized these transformants at the molecular level and continue characterizing them phenotypically in detail in the greenhouse. Moreover, for generating a dwarf-dwarfing rootstock, we have incorporated a construct aimed to induce RNA interference to downregulate the expression of a crucial gene in gibberellin biosynthesis, CcGA20ox1, in mature Carrizo citrange.
Objective 1: Comparative methodology was initiated to compare phloem plugging and collapse with different staining techniques. Phloem samples were collected and prepared from healthy and HLB affected trees. Preliminary results suggest that a rapid survey of plugging and starch accumulation is possible for general surveys of functional and non-functional phloem. Objective 2: Transformation experiments to produce plants that over-express the citrus ‘-1,3-glucanase gene: Experiments to validate the stability of the ‘-1,3-glucanase gene in transgenic greenhouse plants were initiated. PCR analysis on 9 of 10 plants showed the ‘-1,3-glucanase gene band, indicating stable transformation. All recovered transgenic clones (mostly Valencia, Duncan and Carrizo) were micropropagated, producing 4-5 replicates of each clone for further testing, including HLB challenge. Embryogenic callus transformation with the ‘-1,3-glucanase gene: More than 200 GFP+ somatic embryos were recovered from sweet orange OLL#20, and > 100 transgenic shoots were transferred to rooting medium. About 50 GFP+ embryos and shoots from Jin Cheng sweet orange and 20 GFP+ embryos and shoots from Valencia were also recovered ‘ which should result in stable transgenic plants in these important cultivars. Objective 3:To identify the putative genes involved in HLB disease development and psyllid transmission, quantitative reverse-transcriptional PCR (QRT-PCR) assays using total RNA isolated from infected plants and psyllids were conducted. Gene specific primers were used to check the expression of 506 genes in Ca. L. asiaticus. The genes showing a differential expression of two fold or more in either the plant or psyllid were categorized into Clusters of Orthologous Groups of proteins functional categories. Potential virulence related genes including hypothetical genes, which were overexpressed in planta, were selected. Differential expression of these selected genes were also evaluated in susceptible and tolerant varieties of greening infected citrus. Several ABC transporter genes and genes involved in protein export, along with many genes involved in porphyrin and chlorophyll metabolism, were upregulated in the plant. Most of the genes that were overexpressed in the psyllid included those involved in cell motility. The genes overexpressed in planta identified in this study will also be screened by transient assays on Nicotiana benthamiana plants. The results from this study will be useful in identifying the potential virulence genes involved in symptom expression of this pathogen in planta.
For the construct containing the ctEDS1 gene in the binary vector pBINplusARS, we selected the T0 seeds and obtained so far 15 independent T1 transgenic plants. The T2 plants will be planted to select for homozygous lines and also for an initial disease resistance test. For ctNDR1 transformation of the ndr1-1 mutant, we currently obtained 11 homozygous lines. We reported earlier that some of the lines showed enhanced disease resistance. Now we are at a stage to systematically analyze the defense phenotypes of the ctNDR1 overexpressing transgenic plants, using the homozygous lines that we have. For ctPAD4 + pad4-1 and ctEDS5 + eds5-1 plants, we have obtained over 10 and 3 independent T1 transformants, respectively. We have planted the T2 seeds and will test the segregating T2 plants for disease resistance and harvest seeds from multiple individual plants for selection of homozygous lines. For ctEDS5 + eds5-1, we are also selecting more T0 seeds in order obtain additional transformants. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. We have moved the ctNHL1 cDNA fragment from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. For ctSID2, we only obtained the cDNA clone in the pGEM T-easy vector. However, we have had some trouble in moving this fragment into pBINplusARS. We are currently trying a few different approaches to address this problem. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes. The cloning of some of these genes is underway.
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