Earlier in this project we evaluated 25 AtNPR1-transgenic ‘Carrizo’ citrange plants for their response to Candidatus Liberibacter asiaticus flagellin 22 peptide (L-flg22) as a proxy for the pathogen. Using this assay we identified several lines that had an enhanced defense response compared to wild type plants. The identified plants were propagated by cuttings a few months ago. During this period they have developed roots and new shoots and have been transplanted to bigger individual pots and fertilized. These plants will be further assayed for HLB resistance as whole plants and also as rootstocks of non-transgenic grapefruits. In total we have 20 different AtNPR1 transgenic lines propagated. Of those 20 lines 5 are currently in the containment facility being assayed for HLB resistance. There are 2 or 3 replicates of each line as individuals and as rootstocks, in addition to the respective non-transgenic controls for a total of 26 plants. This is the highest limit of plants we can maintain in the space available to us. All 26 plants have been grafted with HLB-infected inoculum kindly provided by Dr. Timonthy Spann (CREC-Lake Alfred). Prior to inoculation each twig used was assayed for HLB. We are employing two different real time PCR assays for the detection of HLB based on Li et al. 2007 (J Microbiol Methods 66:104) and Morgan et al. 2012 (Molecular and Cellular Probes 26:90). The latter was reported as a more sensitive method and is working that way based on our results. We used 3 ‘blind’ buds to inoculate each plant. If any of the buds did not take we grafted again to keep a constant number.
Dr. Pena and his greenhouse manager traveled again to Florida last October 2011 to continue evaluating the project. They started evaluating the establishment of the citrus germplasm. They checked the cultivars to guarantee that they were true to type since passing through in vitro conditions for cleaning the germplasm can cause somaclonal variation. Plants that were not true to type were discarded. They also found that the germplasm was very clean but plants were shorter than they should be for their age. They suggested changing the photoperiod in the growth room to be able to manipulate the plant growth. The suggestion was discussed with the facility coordinator and steps towards a reprogramming process to have a better control of the program have been initiated but at this date the programmer was not able to start the job. The germplasm was organized in lots that will be the material of origin of the mature in vitro experiments in the laboratory. Mother plants were also selected to be transplanted next year. The inventory was organized and I was trained in how to establish a ‘production’ schedule with the current germplasm and how to plan and manage the lots in the future. The greenhouse personnel was again trained in grafting. The personnel is taking too long to master the technique, and they are not consistent in the quality of the job they do which does not help to establish a calendar. Training, checking, and keeping the personnel on task in the growth room has been extremely difficult. Dr. Pena and his greenhouse manager also dedicated some time to check the infrastructure and growth room protocols. We found that the humidifiers are still not working. As a result of many months of waiting the solution was to bypass the filters to get enough water in the humidifiers and to place the program in manual daily. It seems like the system was not designed properly and a new set of filters will replace the current system next year. The facility coordinator is still looking for somebody to do this job. Growth room protocols were checked. The use of coconut fiber as proposed initially has been postponed until we master the current situation without major problems. Use of coconut fiber requires more trained personnel that is not available at this moment in the growth room. Once we master the current situation we will be able to move forward on this. We started doing citrus mature transformation experiments in the laboratory. We used A. tumefaciens with the pCambia 2301containing the GUS gene as a marker. The first experiment was done the first week of November with a small batch of Valencia 1-14-10 and we were able to obtain some positive plants. They are currently micro-grafted in vitro and will be ready to transfer to soil by the end of January 2012. We will evaluate efficiency of transformation after a few experiments are performed with the different cultivars.
Highly susceptible Red grapefruit (RG) produced abundant lesions at 15 dpi with cellular hypertrophy and hyperplasia typical of callused canker in compatible hosts. Similar lesion phenotype was observed in detached leaves and attached leaves. Valencia orange (VO) had less callus-like lesions, and more necrotic lesions. Numbers of lesions was greatest for RG (93) and least for VO (47). The different cybrids showed a variable number and phenotype of the lesions, 4 cybrids developed a higher number of lesions than VO (>50), 11 cybrids produced an intermediate number (25-50), and 5 cybrids formed a lower number of lesions than VO (<25). In contrast to the callus-like lesions for RG, less susceptible cybrid lesions were more necrotic as observed for VO. Thus, canker resistance appeared to be quantitatively inherited from VO based on an intermediate lesion phenotype in the selected cybrids. This was confirmed by Xcc population growth in Cy 3 (7.8 log cfu) and Cy 10 (8.1 log cfu), that was similar to VO (8.0 log cfu) and nearly one log unit lower than RG (8.7 log cfu) at 15 days post inoculation. Responses of genes related to host pathogen interaction in VO and Cy, differed from RG. The pathogenicity related proteins PR4, chitinase (CHI) and beta-glucanase (BG) were up regulated at 4 and 24 hpi in both cybrids. Higher expression of heat shock proteins (Hsp20) in the cybrids suggested a differential interaction of genes from the nucleus with mitochondria and chloroplast genes from the cytoplasm donor. Expression of genes related to programmed cell death and development of hypersensitive reaction to plant pathogens, like alternative oxidase (AOX), aconitase-iron regulated protein (IRP1) and ascorbate peroxidase (APX2) were up regulated at 4 hpi in the cybrids, as evidence for enhanced antioxidant activity. The response of cybrids to Xcc may be expressed at different levels depending on whether mitochondrial and/or chloroplast genomes are transferred in the cybridization process. Cybrids 3 and 10 produced a quantitative resistance reaction to Xcc resembling that in VO. Xcc population development in the cybrids was similar to VO and almost one log unit lower than in RG. More necrotic lesions in these cybrids and VO and lower Xcc populations in lesions suggests cell death occurred which reduced Xcc proliferation. Responses of genes related to host pathogen interaction, in VO and cybrids contrasted with those in RG. At the present time, several citrus cybrids are under evaluation in field trials in areas of endemic citrus canker.
The goal of this project is to genetically manipulate defense signaling mediated by salicylic acid (SA) to produce citrus cultivars with enhance resistance and/or tolerance to HLB and other emerging diseases that are challenging the citrus industry. Genetic engineering has been widely used to introduce disease resistance traits in crop plants, however, its application in citrus has been fallen behind due to the lack of adequate target gene information. With the recent release of citrus EST database and genome sequence, citrus researchers just begun to develop transgenic citrus with novel desirable traits. Since SA is known to play a central role in disease resistance against broad-spectrum pathogens in many plants, we chose to begin this project by focusing on genes positively regulating SA-mediated defense. We have three specific objectives in this project: Objective 1: Identify genes positively regulating SA-mediated defense in citrus Objective 2: Complement Arabidopsis SA mutants with corresponding citrus homologs Objective 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have made significant progress in the project in year 2010-2011 funding period as summarized below: 1. Bioinformatics analysis revealed that citrus and Arabidopsis share strong sequence conservation, most known Arabidopsis SA genes on our candidate gene list have homologous sequences available in the citrus sequence database. For some SA genes belonging to large gene families, we used phylogenetic analysis to identify the potential orthologs. 2. We have so far cloned ten citrus SA genes, among which six genes have been transferred to corresponding Arabidopsis mutants and are under analysis for defense responses. 3. Defense analysis indicates at least one citrus SA gene, CsNDR1, could complement disease susceptibility to Pseudomonas infection conferred by the Arabidopsis corresponding mutant, ndr1-1. CsNDR1 also rescued the HR defect of ndr1-1 in response to the avirulent strain P. syringae avrRpt2. The levels of disease resistance grossly correlated with the levels of transgene expression, suggesting dosage-dependent defense activation by CsNDR1 in Arabidopsis. A manuscript entailing function of the CsNDR1 gene in Arabidopsis is under preparation. 4. The citrus cultivars US-812, US-942, and US-802 were transformed with pBINplusARS constructs containing the citrus SA genes ctNDR1, ctEDS5, ctPAD4, and ctNPR1. Approximately 20,000 explants were transformed in 33 separate transformation groups. After micrografting regenerated shoots, transgenic plants are identified by PCR. Transformed plants are being regenerated and propagated to be used for replicated testing with HLB. It is planned to begin HLB testing transgenics with each of these SA pathway genes during the coming year. Citrus transformations will begin in the next three months with other constructs containing additional citrus defense genes ctACD1, ctJAR1, ctNHL1, and ctMOD1, and the corresponding transgenics will also be propagated and tested with HLB. Taken together, we have provided proof-of-principle data to demonstrate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes but also as a powerful tool to facilitate functional analysis of citrus genes. Several key SA regulators, when overexpressed in citrus, are expected to confer increased resistance to the greening disease and other emerging disease challenge the citrus industry.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. In addition to providing a degree of resistance to bacterial pathogens, overexpression of R proteins often results in in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: The transgenic citrus plants (Duncan grapefruit) containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well as transgenic control plants have been transported from the UF Citrus Research Facility (Lake Alfred) to our laboratory at the Microbiology and Cell Science Department. Out of the 53 transformants transported, 3 did not survive. The remaining 50 appear to be stabilized in their acclamation to our growth room environment. Currently, arabidopsis SNC1 (wt) and scn1 (constitutive mutant) transformants are being tested for resistance to Pseudomonas syringae (Psm 4326); however, since expression is largely limited to phloem cells, a more meaningful assay must include exposure to Liberibacter-infected psyllids. The design of assays and arrangement of the necessary collaborations are in progress. Our working hypothesis is that overexpression of the constitutively active mutants of the R protein genes Atsnc1 and Atssi4 will alert the endogenous innate immunity system of the plant and, thereby, provide resistance to Liberibacterium. In order to monitor the activation state in Arabidopsis lines transformed with the R protein constructs, we crossed (cross pollination) these lines with a homozygous line containing a reporter for the innate immunity response: the pathogen-inducible BGL2 (PR2) promoter driving the GUS reporter (kindly provided by Dr. Xinnian Dong, Duke University). Two homozygous AtSUC2/snc1 mutant and two homozygous AtSUC2/SNC1 wild type lines were crossed. Additionally, we crossed four other snc1, ssi4 mutant and wild type lines which had undetermined zygosity. In order to confirm that the PR2/GUS reporter line is functioning properly, activation tests are being conducted using salicylic acid, its analog INA, BHT, and pathogen P. syringae Psm4326 in induce reporter expression in the PR2/GUS reporter line. BTH (0.3 mM) and INA (0.5 mM) were the best inducers over the course of 72 hr. SA (0.5 mM ) induced GUS expression at 24 hr and plateaued for up to 72 h. Bacterial pathogen (Psm 4326) induction levels were the highest at 72 hr, but, overall, lower than those induced by other SAR agents. The use of these pathogen-inducible reporter lines will not only monitor the activation state of immune response of our R constructs, but they will also provide spatial information to confirm that phloem tissue is being activated. Two additional reporter lines to monitor the immune response are being developed to increase the sensitivity by using GUS plus (P2/GUS plus) and to monitor an additional pathogen-inducible promoter, PR5/GUS plus. Out of eight PR2/GUSplus transgenics (Arabidopsis, T1 generation), three showed constitutive expression (‘all blue’), while the remaining five showed either residual main vein expression, or no expression. From the five PR5/GUSplus transgenics, only one showed residual main vein expression, in line with published reports.
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.
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