Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. ‘ A protocol for the accelerated production of transgenic plants has been published:Dutt M., Vasconcellos M., Grosser J.W. (2011) Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle). Plant Cell, Tissue and Organ Culture 107:79-89. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Seeds of precocious rootstocks (based on data from the St. Helena project) were harvested and planted for subsequent budding with transgenic precocious sweet oranges (Vernia and OLL series). Plans are underway to build a PVC-pipe scaffolding structure/rapid evaluation system (RES) in our transgenic greenhouse, similar to our successful RES in the field. This will allow horticultural manipulation of the precocious transgenic germplasm to demonstrate the reduced juvenility. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. Clonal propagation of these transgenic oranges onto the available liners of the precocious rootstocks mentioned above is underway. Transformation with early-flowering genes: ‘Duncan grapefruit plants transgenic for the poplar ft1 gene have been produced and are being tested for precocious and/or induced flowering using 2 different promoters. More experiments with the citrus ft constructs are also underway. Progeny plants of transgenic tobacco are being assayed for phenotype and transgene segregation.’ 122 transgenic Carrizo trees were generated following a co-transformation experiment using two vectors. The first containing 35S-cft1 and the second containing AtSUC2′ gus. The objective is to rapidly evaluate transgene expression in the fruit. PCR analysis revealed that 16 lines contained both cassettes. Plants have not flowered 12 months after transformation. Plants are currently being evaluated in an unheated greenhouse for cold stress in order to hopefully initiate early flowering in spring 2012.
In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae (orange subfamily Aurantioideae), were assessed in the field in Florida for resistance to natural populations of ACP. The majority of genotypes hosted all three life stages of ACP, however there were differences among genotypes in the mean ranks for eggs, nymphs, and adults. Very low levels of ACP were found on two genotypes of Poncirus trifoliata, ‘Simmon’s trifoliate’ and ‘little-leaf’. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties, so it may be useful in breeding programs as a potential source of genes that confer resistance to insects. The field experiment was followed by no-choice tests in which female ACP had the opportunity to lay eggs for six days on 46 genotypes of P. trifoliata, 35 genotypes of xCitroncirus sp. (hybrids of P. trifoliata and another parent species), 12 genotypes from the Citrus genera that were not represented in the field, and a control (Citrus macrophylla) to determine whether any genotypes were resistant to ACP. All genotypes of Poncirus trifoliata, except for one, and 14 of the genotypes of xCitroncirus sp. were resistant to oviposition by ACP, whereas the other genotypes were susceptible. Antibiosis-type resistance in genotypes of Poncirus trifoliata and xCitroncirus sp. against adult ACP was identified in no-choice tests: adult lifespan was greatly reduced on genotypes identified as resistant to oviposition. Currently we are testing whether development and weight of nymphal ACP are negatively influenced by the resistant genotypes. Studies also have been initiated to compare plant volatiles of the resistant genotypes to those of susceptible genotypes to ascertain whether presence or absence of certain volatiles confers resistance. We are also screening genetically-transformed plants (containing Lectin) for possible resistance to ACP. Collaborators at the Fujian Academy of Agricultural Sciences in China reported on two experiments investigating resistance to ACP within the Rutaceae. 53 genotypes of citrus were evaluated in a free-choice experiment conducted in a green house. Twenty of the genotypes had low abundance of adults, nymphs, and eggs, whereas the other 33 genotypes had moderate to high abundance of ACP. Of the 20 genotypes that had low abundance of ACP, several are in the Citrus genus and will be evaluated in no-choice tests to confirm whether they are resistant. A cluster analysis separated the 53 genotypes into three resistance classes. Among germplasm screened by both FAAS and USDA-ARS, there is general agreement for most genotypes with respect to ACP susceptibility but there are some differences. Two accessions of Poncirus trifoliata did not appear to be as resistant as those evaluated in USA, although the cultivars studied may not be same. A second free-choice experiment was initiated in the field with 71 genotypes of citrus, infestations of ACP have only been assessed once so far and psyllid pressure was so low that no conclusions can yet be made. We have recommended that FAAS concentrate during the upcoming winter months on no-choice studies with germplasm that has appeared to be resistant. Co-PI Duan is currently in China and will interface with FAAS on their research progress and future activities.
Over the past quarter, we have made progress in the following areas: 1. We have made progress with our Nicotiana benthamiana – GUS test system to examine effector specificity for induction. We have developed improved vectors and used these to demonstrate specific promoter activation by three distinct TAL effectors thus far. We have also tested a RACE method to map which UPT boxes in our test promoters are used. This method is still in development. 2. Novel TAL effectors from additional citrus canker strains have been isolated, and sequence analysis has been initiated. 3. Transformation of Duncan grapefruit has continued. At present, we have over 330 new candidate stable transgenic lines in soil, with six different promoter-gene constructs. We are systematically characterizing these on a molecular basis to confirm presence of the specific genetic elements transformed. Pathogen testing will begin shortly, and we will seek to identify the best performing transgenic lines. New Fall seeds will be available soon for additional transformations. 4. Transformed lines of sweet orange and Ruby Red grapefruit are at the rooting stage.
Over the past quarter, we have made progress in the following areas: 1. We have analyzed the contributions of individual PthA proteins by knocking out specific pthA genes from X. citri strain 306 from Brazil and testing them with GUS reporter gene fused to our super promoter Bs3 construct containing binding sites for 17 X. citri TAL effectors. Strains in which the genes pthA 1 and 2 were disrupted activated the reporter gene at levels nearly comparable to the wild-type strain. These results suggest that there is little significant contribution of these effectors to gene regulation. Strains disrupted for (i) pthA 2 and 3 showed a 25% reduction in GUS activity. (ii) pthA 1 and 3 showed a 50% reduction, pthA 1,2 and 3 showed a 60% reduction, and deletion of all four pthA genes showed a 98% reduction in GUS activity, similar to a type three secretion deficient strain, 306.hrpG. This results suggest that pthA4 is the principle effector in the activation of gene expression, with additional smaller contributions from the other three TALEs. These results confirm that combinations of UPT boxes allows triggering of engineered resistance promoters by more than one TALE which should reduce pressure on individual TALEs to evolve to evade detection. 2. A robust transformation system using either epicotyls or cotyledons has produced a large pipeline of transformed plant material. More than 800 Duncan grapefruit lines have been transformed, made shoots, and roots, and been transferred to soil, with six different promoter-gene constructs. 153 Ruby Red grapefruit transformants and 63 pineapple sweet orange transformants are now in soil. It is slow work, and further attrition occurs following molecular characterization to identify the desired lines. 3. Once plants are transferred to 4 inch pots and reach adequate size, the final line selection process can take place by pathogen testing. We have now begun analyzing new stably transformed Duncan plants containing the super promoter:resistance construct using the avrGf1 gene. Two candidate lines showed reduced pustule formation by pin prick inoculation, relative to a non-transformed control line, and a hypersensitive reaction to infiltration inoculation, compared to a water-soaked lesion in the susceptible control. These reactions suggest that the test construct is successfully conferring canker resistance in these stable lines. We are continuing to study these plant lines and additional stable lines that are reaching adequate size for pathogen testing. Lines identified from this analysis will be candidates for grafting and filed testing. These results support our hypothesis that a resistance construct based on a promoter containing multiple citrus TALE binding sites can confer transcriptional activation and disease resistance to canker strains.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, we have generated 20 transgenic lines of Duncan grapefruit, and the transgenic plants are currently under canker resistance test. After canker resistance test, we will identify transgenic lines overexpressing AtMKK7, then chose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we are repeating the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Another two quarts of seeds have been treated with gamma-ray irradiation. All seeds were irradiated at 50 Gy, as we previously found that this dose will not significantly decrease the germination rate of the seeds. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Shoots formed on these gamma irradiated seeds will be screened again on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. They all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xac in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. We have propagated the CtNH1 line by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into ‘Duncan’ grapefruit. Five transgenic lines have been obtained.
Funds for this project have not yet been received by Dr. McNellis. Penn State has assigned a fund number, but the Office of Sponsored Programs has not yet finalized a budget for the funds. Once funds are received, the development of the NodT antibody will be initiated immediately.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Discussions have been ongoing with Eliezer Louzada of Texas A&M to plant his transgenics wihc have altered Ca metabolism to target canker, HLB and other diseases. Jude Grosser will be planting ~250 additional trees on the test site next week. 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 will be 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.
During this period we standardized quantitative real time PCR assays for 3 more genes associated with pathogen defense in citrus. The new genes are part of the salicylic acid (SA) synthetic pathway (ICS1 and PAL1) and another pathogenesis-related gene (PR1b). We have now developed a battery of 21 defense-associated genes, in addition to the endogenous controls (18S and COX). This has allowed us to have a broad picture of the response in citrus to HLB and other inducers such as SA and flagellin 22 peptide (L-flg22) from Candidatus Liberibacter asiaticus. The assays evaluate genes associated with SA biosynthesis and signaling (AZI1, EDS5, ICS1, PAL1), PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity) (EDR1, EDS1, NDR1, PBS1, RAR1, SGT1), transcriptional regulation (NPR1, NPR3, R13032, R20540), the jasmonic acid pathway (COI1 and JAR1) and targets of the regulatory SAR pathway (BLI1, CHI1, PR1, PR1b, RdRp). The transgenic plants necessary for the HLB-infection assay have been acclimating in the containment facility and are being fertilized and treated with pesticide after a mite infection that has prevented us from furthering this part of the objectives.
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.
Hybrids were selected from populations of seedlings from crosses made the previous year. Selected SuperSour hybrids were identified by morphology, horticultural characteristics, and molecular markers for further study. Cuttings were made from 300 different SuperSour rootstock hybrids in preparation for disease testing and field trials. Cuttings made the previous time-period were grown-up in preparation for budding. A greenhouse system was developed to evaluate SuperSour hybrids for tolerance of high pH/calcareous soils. A project continued to compare graft inoculation and Asian citrus psyllid inoculation as methods for evaluating cultivar resistance or tolerance to HLB in the greenhouse. The results of this study will have important implications in focusing resources to be used for evaluating the HLB-resistance/tolerance of new conventional and transgenic cultivars. A study using the Affymetrix GeneChip citrus genome microarray, and that compared transcriptional response of susceptible citrus to the tolerant selection, US-897, was accepted for publication in the journal ‘Plant Science’. The study was focused on identification of genes associated with the HLB tolerance of US-897. The analysis identified several pathogenesis-related genes that were induced by Liberibacter infection of susceptible citrus, such as thaumatin, chitinase, phenylalanine ammonia-lyase, peroxidase, and a cytosolic copper/zinc superoxide dismutase. Microarray analysis identified 326 genes which were significantly up-regulated by at least 4-fold in the susceptible genotype, compared with only 17 genes in US-897. Notably up-regulated in susceptible citrus, but not US-897, were a Myb-like transcriptional regulator protein (generally associated with a salicylic acid-mediated hypersensitive response to bacteria) and phloem protein 2-B15 (PP2). Exclusively up-regulated in US-897 was a gene for a 2-oxoglutarate and Fe(II)-dependant oxygenase, an important enzyme involved in the biosynthesis of plant secondary metabolites associated with antimicrobial activity and plant defense. More than eight hundred genes were expressed at much higher levels in US-897 independent of infection with Liberibacter, some of them clearly also associated with disease resistance. Among these genes, a constitutive disease resistance protein (CDR1) were notable. One form of CDR1 was found to be expressed more than 200-fold higher in US-897 than in susceptible citrus. The involvement of these and other detected genes in tolerance to HLB and their possible use for biotechnology were discussed in the paper. Results of a study on early RNA and metabolic changes in HLB infected citrus, both tolerant US-897 and susceptible Cleopatra, were presented at the CHRP meeting in Denver, and provided significant insights into key metabolic pathways that may be targets for engineering HLB resistance. Metabolic changes were analyzed using gas chromatography coupled mass spectrometry. There were large differences between the metabolic constituents of non-infected and HLB-infected Cleopatra, with many compounds notably up-regulated in response to infection, including ornithine, proline, lysine, asparagine, and saccharic acid. Corresponding to the nearly non-existent symptom development of US-897 in response to HLB infection, few compounds were significantly different between non-infected and infected US-897. Also presented at the the CHRP meeting were the results of a study of volatile organic compounds produced by non-infected and infected Valencia trees. Samples were collected on polydimethylsiloxane coated magnetic glass twisters and analyzed by gas chromatography-mass spectrometry. Changes in volatile organic compound profiles were detected, but were not strong or consistent across time following infection.
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 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.
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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