The antibody developer, Creative Biolabs, Inc., has nearly completed screening for antibodies against the Candidatus Liberibacter asiaticus NodT protein. They have identified six high-affinity binding antibodies to the 30 amino acid peptide antigen used. The materials will be shipped to Dr. McNellis’ lab at Penn State University within the next few weeks. We anticipate beginning to screen the antibodies for their ability to detect the native NodT protein produced by Candidatus Liberibacter asiaticus starting in mid-August, working through the fall.
Quarterly report for April 2012. Two full genome sequences have been assembled and annotated, and made available to the citrus research community, the haploid Clementine sequenced by the ICGC partners using Sanger technology to produce the highest quality reference genome for all subsequent citrus genomics efforts; and the sweet orange, developed by UF, Roche/454, and JGI using the 454 platform. Both annotated assemblies are available at Phytozome and at citrusgenomedb.org. The Clementine assembly has been improved, assembled into the 9 basic chromosomes; it is designated Clementine v. 1.0, and is being used in comparative genomics studies describing the phylogeny of sweet orange and Clementine. New citrus genome sequences have been generated by the Machado lab in Brazil (Ponkan mandarin, 4x coverage, using 454 technology), the Gmitter lab and UF-ICBR (low-acid pummelo, 25x coverage by Illumina technology), and plans are in place for resequencing other genomes to contribute to the phylogeny study. The ICGC is to produce a high-profile manuscript to highlight the work done and the potential utility of the sequencing projects toward future research objectives. Work proceeds on the other objectives of this project. To demonstrate the potential of the silencing approach described in Enrique et al, 2001, as a rapid genetic tool to address HLB infection, the Gadea and Marano labs characterized the behavior and persistence of silencing signals. A total of 6 candidates were cloned, based on our microarray studies comparing sensitive and tolerant citrus, and >70 plants were inoculated with Agrobacterium during January-April 2012. No altered phenotypes have been observed, and based on the systematic problems we are looking at new strategies. We are identifying miRNAs induced in citrus-pathogen interactions, presumably regulating target genes involved in signaling pathways and metabolic events important for plant resistance. A real time protocol has been established to study miRNA and target expression in Citrus and thus validate computational or high-throughput experiments in a biological context. We have mined, screened, and verified SNPs derived from the BAC end-sequences from Dvorak’s lab and the GoldenGate assay platform for hi-throughput genotyping has been produced. DNA samples were prepared and >150 individuals of a large mapping family have been processed; data analysis is underway to anchor the orange genome to genetic and physical linkage maps, thus improving its quality and utility for HLB-targeted research projects. Analysis of data from microarray studies looking at differences in gene expression over time between sensitive (orange) and tolerant (rough lemon) citrus types has revealed substantial differences in host defense responses, and these are being associated with changes in metabolism and phenotypic responses. The Roose lab submitted DNA of 13 sweet orange x trifoliate orange progeny and two parents to evaluate the potential of genotyping by sequencing (GBS) to Floragenex, a company that performs this technique. The method used targets the non-methylated portion of the genome and is therefore likely to be gene-rich. The parents will be sequenced at 30x depth or greater and the progeny at 15-20x depth. If the results from this preliminary trial are acceptable, we will submit additional DNA samples for analysis.
Quarterly report for July 2012. The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, will soon be publicly available. New citrus sequences were generated by the Machado lab in Brazil (Ponkan mandarin, 454 and Illumina), the Gmitter lab and UF-ICBR (low-acid pummelo, Illumina), and Illumina datasets for Willowleaf/Avana mandarin, W. Murcott, Chandler pummelo, and Seville sour orange have been provided (Morgante, IGA-Italy; Talon, IVIA-Spain; and W. Roose-UCR). Comparative analysis has elucidated the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. The fine-scale characterization of citrus genotypes opens the possibility that ancient C. reticulata/C. maxima admixtures (such as sweet and sour orange) can be recreated by conventional breeding guided by a set of genome-wide markers, enabling incorporation of specific, limited genomic regions from other citrus or relatives to confer disease resistance, yet retaining the essence of marketable fruit phenotypes. A manuscript based on these results is under revision. Work proceeds on the other objectives of this project. We are identifying miRNAs induced in citrus-pathogen interactions, presumably regulating target genes involved in signaling pathways and metabolic events important for plant resistance. In order to set up protocols to validate microRNA expression in plant-pathogen interactions and identify target genes, we have performed a comprehensive analysis of the expression of 7 different citrus miRNAs in the context of 4 different Xanthomonas citri subsp. citri (XC) ‘ Citrus limon interactions. The results obtained so far reveal interesting expression patterns for some of the miRNAs. We have used the GoldenGate assay platform for hi-throughput genotyping of DNA from >150 individuals of a large mapping family; data analysis is continuing to anchor the orange genome to genetic and physical linkage maps, thus improving its quality and utility for HLB-targeted research projects. Analysis of data from microarray studies looking at differences in gene expression over time between sensitive (orange) and tolerant (rough lemon) citrus types has revealed substantial differences in host defense responses, and these are being associated with changes in metabolism and phenotypic responses. More genes were differentially expressed in HLB-affected rough lemon than sweet orange at early stages, but substantially fewer at late time points, possibly underlying differences in sensitivity to CLas. Pathway analysis revealed that stress responses also were distinctively modulated in rough lemon and sweet orange. Remarkably phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The pilot genotyping by sequencing (GBS) project has yielded promising results, and plans are being carried out to genotype a large segregating Citrus x Poncirus family. A large scale RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus, that weren’t seen previously in microarray studies, or to validate those already seen, has progressed; RNA samples have been sequenced using Illumina technology, and the massive data set is currently being analyzed.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
1. Monitoring genome-wide response to Candidatus Liberibacter asiaticus infections In response to pathogen attack, multiple defense mechanisms are triggered in the host plants, including basal defense and gene-for-gene resistance. So far, neither basal defense nor gene-for-gene defense mechanism has been reported in association with CLas infection. Because of the intracellular life style and lack of Type III effector genes of CLas bacteria, we hypothesize the basal defense resistance may play a more important role in these breeding lines. We expect that monitoring the gene expression differences among HLB-susceptible and HLB-resistant citrus trees using RNA-Seq will enable us to elucidate the HLB defense mechanism. We have identified the most suitable trees for the study and are now collecting and preparing samples. We have also identified and are co-ordinating with a company to perform the RNA-Seq. 2. Reconstructing and verifying the transcriptomic data After the next generation sequencing data is obtained, the first step will be to construct gene transcripts from these billions of short reads. This step includes a series of bioinformatics studies. Due to the enormous amount of reads, the bioinformatics study requires substantial computational infrastructures. The Palmetto cluster computer in Clemson University, which ranks 96th among the top 500 super computers reported in June 2011 (www.top500.org), can provide sufficient support for our bioinformatics studies. The constructed gene transcripts will be verified selectively using RT-PCR. 3. Identifying differentially expressed gene, gene modules, transcription factors under Candidatus Liberibacter asiaticus infections The transcriptome data provide a global monitor of expression changes related to Candidatus Liberibacter asiaticus infections, which open up opportunities for the elucidation of the gene, gene modules and gene networks responding to Ca. Liberibacter asiaticus infections. Here, we plan to conduct multiple computational analyses of our transcriptome data to identify resistance genes. First, the differentially expressed genes will be identified using statistical methods. Second, the gene co-expression networks and gene co-expression modules will be identified using the RMT-based method. Finally, the transcription factor(s) that directly controls each gene module will be determined. 4. Validating the identified genes under Candidatus Liberibacter asiaticus infections Transcription factors play an important role in defining gene co-expression modules. It is anticipated that transcription factors involved in Candidatus Liberibacter asiaticus infections will be identified by transcriptomic analysis. We will test whether an identified transcription factor indeed performs the physiological function as anticipated as a regulatory component of a co-expression module. 5. Creating transgenics of Carrizo, Hamlin, and Ray Ruby The most promising genes will be used in binary vector constructs to create transgenics of Carrizo, Hamlin, and Ray Ruby. It is anticipated that advances in citrus transformation techniques will verify similar efficacy for the Niedz mature tissue transformation protocol (Marutani-Hert et al., 2011) and the juvenile tissue approach we have been using (slight modifications of those published by Orbovic and Grosser, 2006). If this is correct, we will transition solely to mature tissue transformation techniques.
This report covers the first month of the project funding, and new data during this period is limited. Objective 1. Evaluate existing transformed lines: Experimental lines were generated during the first funding cycle, and one of our principal efforts is analysis and testing of candidate transformed lines of Duncan grapefruit to find stable transgenic lines that correctly express gene constructs. Objective 2. Expand stable transformations: This time of year seeds are poor, but we are planning how to best ramp up our efforts for transformation of Ruby Red grapefruit and sweet orange in September. Objective 3. Refine constructs: We have new genetic elements that we are incorporating into our expression constructs with the aim of improving resistance. Objective 4. Sequence more TAL effectors from additional canker accessions: We have identified several cankers strains that have particular phenotypes or geographies that may reflect differences in TAL effectors. We are working on isolating and sequencing these genes, with the particular goal of testing the effectiveness of our resistance strategy against these 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, besides transgenic citrus plants overexpressing the Arabidopsis MKK7 (AtMKK7) and NPR1 genes, we have also generated transgenic citrus plants expressing the Arabidopsis NAC1, MOD1, and EDS5 genes. These three genes have been shown to confer disease resistance in Arabidopsis. The transgenic plants are growing and will be propagated for canker and greening resistance test. In addition, we recently established an Arabidopsis-Xanthomonas citri subsp. citri (Xcc) pathosystem. Using this pathosystem, we have found that several genes of the SA signaling pathway function in nonhost resistance to Xcc. We are using the pathosystem to identify novel genes conferring nonhost resistance against citrus canker. For objective 2, we focused on the direct genetic screen for citrus varieties with increased resistance to citrus greening. More seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds were inoculated with psyllids carrying greening bacteria. The first batch of seedlings inoculated with psyllids carrying greening bacteria have been moved out, and we are monitoring the development of greening symptoms on the seedlings.
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, has spread to citrus growing regions nearly worldwide and adults transmit phloem-limited bacteria (Candidatus Liberibacter spp.) that are putatively responsible for citrus greening disease (huanglongbing). Host plant resistance ultimately may provide the most effective, economical, environmentally safe, and sustainable method of control. In earlier experiments we identified genotypes of Poncirus trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) that were resistant to ACP. We are now trying to identify the genotypic and phenotypic traits that promote resistance. We initially collected volatiles from one genotype of Poncirus trifoliata and two genotypes of xCitroncirus. We have since identified a susceptible genotype of P. trifoliata that is closely related to a resistant genotype of P. trifoliata and two pairs of closely related susceptible/resistant genotypes of xCitroncirus. These genotypes are ideal for identifying differences in volatiles among susceptible and resistant genotypes. Therefore, we collected volatiles using a SPME fiber and aerations from these genotypes and two susceptible control genotypes. The volatiles were analyzed using gas chromatography-mass spectrometry and we found clear differences in the volatile profiles. A chemist-collaborator is currently identifying all volatile compounds collected from the samples. We also plan to use these same genotypes to analyze the amino acids, sugars, flavenoids, carrotenoids, isoprenoids, and sterols in phloem. This will give us information about the underlying reasons why ACP avoid certain genotypes of citrus and help identify genes that can be used in citrus breeding programs to confer resistance to ACP. We have continued to screen grapefruit trees that have been genetically transformed to express Lectin from the snowdrop pea for susceptibility to ACP. We compared rate of oviposition, nymphal development, and lifespan of adult ACP on three varieties of grapefruit that express Lectin and one variety that does not. Expression of Lectin did not deter oviposition by ACP. However, our first two replications indicate that adult ACP likely have a shorter lifespan on the two varieties of grapefruit expressing the highest levels of Lectin. Additionally, our first two replications indicate that nymphs may develop more slowly or die at a higher rate on the variety expressing the highest level of Lectin. However, there has been wide variation in the results from the first two replications, so a third replication is currently in progress. We also used these trees to determine whether Lectin interferes with acquisition or transmission of citrus greening disease. Those results are currently being analyzed. ARS maintained contact with the Fujian Academy of Agricultural Sciences through emails and phone calls. ARS (Duan) is currently visiting FAAS in China and is on-site reviewing their research progress. FAAS concluded some no-choice experiments with Poncirus, xCitroncirus, Murraya, and Citrus accessions. In all cases where the same accessions have been studied, FAAS findings are in agreement with ARS’s concerning colonization by ACP eggs except for xCitroncirus (CRC 3957), which they report was more resistant. Among accessions studied by FAAS but not by ARS, ACP longevity was reduced on the following germplasm: xCitroncitrus (CRC 3881, CRC 3969, CRC 3957, CRC 1459), Citrus aurantium (CRC 3929), Citrus x Tangelo (CRC 3874) and P. trifoliata (CRC 838). These CRC accessions deserve evaluations under Florida conditions. In a free-choice settling experiment, adult ACP largely avoided Rhododendron simsii.
A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline. It appears prudent to replicate plants of each transgenic event and conduct challenges that last 10-14 months. Most of these plants in our program have been transformed with AMPs driven by several constitutive and vascular specific promoters. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. Several events grew better than all controls at 14 months after initiating the challenge, with 35% greater trunk-cross-sectional area increase than the overall experimental average and 64% greater growth than the mean of the controls, but do not show immunity to CLas development. These will soon be placed in the field for further evaluation. Forty four AMPs were screened in-vitro, a number of which were synthetics specifically designed to enhance efficacy against alpha-proteobacters. There appeared to be a ceiling of activity which could not be exceeded. The most active AMPs included Tachyplesin 1 from horseshoe crab, SMAP-29 from sheep, D4E1 and D2A21. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than the other phloem-specific promoters citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. D35S produces the highest level of expression but with great variability between events. Anthocyanin regulatory genes, give bright red shoots (UF Gray collaboration) and were tested as a visual marker for transformation, as a component of a citrus-only transgenic system. When antibiotics were left out of regeneration media, almost no red shoots were recovered. However, high anthocyanin apples are reported to have field resistance to bacterial fire-blight. Red citrus transgenics will be tested for HLB, ACP, and canker resistance. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and significantly suppressed CLas growth in culture. After verification these will be used to create transgenes. In our program, new constructs and resulting transgenics are in process, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), and a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA).
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for more than two years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. 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. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
This project has been an administration challenge from the beginning. It was impossible to get funds to our Brazilian colleague and difficult to get funds to our USDA partners. Dr. Machado had only requested funding for the first two years of the project. Since he could not get funding in Brazil, he asked me to support a “sandwich” student in my lab, which Dr. Turpin approved. This student is returning to Brazil this month after one year in my lab. She worked on controlling tissue contamination, which is our main problem with mature transformation, but with only partial success. Drs. Grosser and Gmitter have had the same difficulties at the CREC and do not feel that they should continue on this project without improved facilities. The part of the project that has been successful is the work on small penetrating peptides (CPPs) in the Moore lab. This technology allows an alternative to Agrobacterium-based transformation of citrus. The Agrobacterium method produces a low rate of stable transformants (~9%), takes several months, and commercialization is potentially difficult because of the negative views of bacterial genetic modifications. We propose an alternative method of transformation using cell penetrating peptides (CPPs). CPPs are positively charged short amino acid sequences able to simultaneously bind proteins and nucleic acids and deliver them across cellular membranes and cell walls. Many applications are available using CPPs including transient expression assays and gene silencing. We have developed a standard method for the transient expression of reporter genes (GUS and GFP) in citrus. Our data indicate that up to 50% of treated explants express GUS when CPPs are used alone. Several optimization steps have been tested. For instance, the efficiency is increased to 100% when CPPs are used in conjunction with a lipid reagent. Our main goal is to use this method to improve stable citrus transformation efficiency compared to the Agrobacterium method. We have produced 84/163 segments which survived kanamycin selection and produced shoots. We will use PCR and reporter gene analysis to confirm their stable integration. Further experiments, comprising RNAi and protein expression, will also be performed.
The first objective of this project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. A growth room was constructed instead of a greenhouse due to budget constraints. It took approximately 7 months to construct the growth room. It is currently operational after more than a year of troubleshooting. The water filtration system still needs some adjustments to be able to obtain a better water quality. The water quality is affecting the plant growth and the humidifiers. A generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and planted the rootstocks at the beginning of April 2011. Three (3) sweet orange varieties were indexed in vitro and micrografted. The cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA were adjusted to our specific environmental conditions and clone specificities. Mature Valencia, in our conditions, was very responsive to organogenic regeneration. We obtained positive plants, checked by PCR, and they are currently growing in the growth room. Hamlin was also transformed but was less responsive to organogenic regeneration, but we were able to obtain a plant currently growing in the growth room. Pineapple did not respond and we discarded the cultivar after the last batch in the production calendar was used. We are still waiting from results on this last experiment. We are currently introducing another clone of the same Hamlin cultivar 1-4-1 to improve its quality. A few of the initial mother plants were not as cleaned as previous determined and we introduced more plants using antimicrobial to guarantee cleanliness. It seems like yearly introductions may be necessary to maintain the quality of the material desired in the experiments. We are also cleaning the rootstocks Swingle Citrumelo and Carrizo to be transformed in future experiments.
Engineering Resistance Against Citrus Canker and Greening Using Candidate Genes 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 searched the citrus genome database (http://www.phytozome.org/citrus.php) with BLAST and identified nine genes similar to AtNPR1 or its Arabidopsis homologs. Among them, CtNPR1 (also named CtNH1) is the most closely-related to AtNPR1 based on phylogenetic analysis, supporting an orthologous relationship. The Figwort mosaic virus (FMV) promoter was used to overexpress CtNH1 in citrus. Previous studies in soybean showed that the FMV promoter is significantly stronger than the Cauliflower mosaic virus (CaMV) 35S promoter for gene expression (MPMI 21: 1027). Three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts have been identified. When inoculated with X. citri subsp. citri (Xcc), 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 Xcc 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. Some lines inoculated six months ago with Candidatus Liberibacter asiaticus (Las) in Dr. Yongping Duan’s lab showed interesting results: one negative control had displayed greening symptoms, while two CtNH1-1 plants had not. Inoculation of more CtNH1 plants are now in progress. A microarray experiment was conducted using CtNH1 and non-transgenic Duncan grapefruit inoculated with Xcc. A needleless syringe was used to infiltrate the leaves with the bacterial culture (OD600 to 0.3). Three time points were used for this study. For each time point, three replications were used. Data analysis indicates that at p value <0.01, a total of 451, 725, and 2144 genes were differentially expressed at 6, 48, and 120 hours post inoculation (HPI), respectively. Using the visualization tool Mapman 3.5.1, the differentially regulated genes (Log FC ' 1 and Log FC ' -1) were mapped to give an overview of the pathways affected. Interestingly, at 120 HPI, a large number of genes involved in protein degradation and post-translational modification were differentially regulated. Furthermore, numerous genes involved in signaling also showed differential expression at this time. The results indicate that a large number of genes involved in the regulation of transcription were up-regulated in the transgenic plants at 120 HPI, and also at 48 HPI, although to a lesser extent. The photosynthetic pathway was affected to a larger extent at 48 HPI, which is signified by a large number of genes involved in photosynthesis being up-regulated in the transgenic plant when compared to the non-transgenic citrus. 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. To date, ten transgenic lines have been obtained. They are ready for Las inoculation.
The citrus variety Carrizo, the single most important rootstock in the U.S., was generated by a ‘Washington’ navel orange x Poncirus trifoliata cross, Carrizo shows tolerance to diseases such as Citrus tristeza virus, foot rot and huanglongbing (HLB, citrus greening). We had previously generated a Carrizo genomic sequence database containing 3.5x coverage of the genome (USDA Public Citrus Genome Database, http://citrus.pw.usda.gov/). The primary objective of this proposal was to acquire additional sequence for this database, which is universally available for unrestricted use by research programs requiring citrus genomic DNA sequences. Funding was sought to increase the database depth from the previous 3.5x coverage to approximately 10x (454 GS FLX). Because of an increase in average read length, the sequence acquired significantly exceeded that indicated in the proposal (15x final coverage). This increased read length significantly improved segregation of the two genomes during assembly. Assembly of the sequence data was performed by the Roche 454 gsAssembler version 2.6 software program. Multiple program runs were done based on default or heterologous genome settings which influenced the final scaffold forming numbers; 39,966 versus 31,239, respectively. There were 262,573 total contigs formed and represented in the assembly. The software estimates in both cases predicted 9X coverage and a genome size of 673.9Mb indicating partial segregation of the Poncirus trifoliata and Citrus sinensis genomes. Assembled scaffolds are currently available on the USDA Public Citrus Genome Database web site (http://citrus.pw.usda.gov/). Candidate genes were predicted using the scaffold sequences as a source for the software program GENSCAN, which resulted in 69,580 candidates along with predicted nucleic acid and peptide coding sequences from intron/exon junctions. The Arabidopsis.mat matrix file was used for this processing. The candidate coding sequences were compared to the latest UNIPROT database using BLASTX to determine candidate identities; 31,496 (45.3%) sequences had matches with evalues better than 10E-30. Expressed Sequence Tags (ESTs) were collected from the citrusgenomedb.org consortium site; these were also publicly available from the NCBI resource. A total of 361,458 ESTs were collected and compared against the Carrizo citrange genome scaffolds. The EST sequences were derived from Citrus clemintina, C. sinensis, and Poncirus trifoliata sources. The GENESCAN candidate sequences with their scaffold assignment and coding sequenceorder on the scaffold were compared against the other citrus species scaffolds using BLASTN. The best matches were aligned and compared. Because of assembly difficulties associated with independent assembly of the Poncirus trifoliata and Citrus sinensis genomes, an additional approximately 20x coverage of the Carrizo genome will be acquired employing Illumina Sequencing technology prior to attempted alignment to citrus physical/genetic maps. The data acquired under this proposal is currently being analyzed to identify potential Poncirus-derived HLB-tolerance genes. A number of potential Carrizo-specific ‘R’ genes (NBS-ARC-Leucine-Rich Repeat Proteins) have already been identified for PCR amplification and transformation into citrus.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
