Three healthy-looking trees were found in HLB ravaged orchards in Guangdong and Guangxi province, where all other trees planted at the same time or newly replanted were either dead or severely declining; these escape trees have been the focus of the follow-up efforts on this project. The two trees free of HLB symptoms from Guangdong had been propagated in the greenhouse at the Guangdong Institute of Fruit Tree Research facilities. Some were grafted with HLB affected branches for re-inoculation at the greenhouse, and others were planted in their research field to assess their reaction to natural inoculation with HLB. The other tree in Guangxi was transplanted to a protected location at the Guangxi Citrus Research Institute, and used to propagate more trees for inoculated by grafting infected material. Under observations for several months months, the propagated trees in the field surrounded by severe HLB disease and intense inoculum and vector pressure in Guangdong appeared not to show any HLB symptoms and no pathogen was detected by qPCR. Most of these propagated trees from the individual symptom-free tree found in Guangxi, inoculated in a protected greenhouse, were confirmed to be infected and some displayed HLB symptoms. Though they apparently were not resistant to inoculation, the question remains as to why the original source tree was not infected and symptom-free; the possibility of vector resistance in the host could be explored further. There are continuing observations on other grafting-inoculated trees propagated from the three trees at the protected greenhouse at both institutes, which could lead to a conclusion on the susceptibility, as well as propagation of enough materials for some transcriptome comparison to determine the molecular mechanism of survival. As a additional mission for this project, revisits to several commercial groves seen by us previously did demonstrate that psyllid control, together with other integrated management, is critical to minimize the HLB incidence rate and maintain the tree health and profitable production, from those successful and failed HLB management cases.
Three healthy-looking trees were found in HLB ravaged orchards in Guangdong and Guangxi province, where all other trees planted at the same time and newly replanted were either dead or severely declining; symptoms have been monitored and qPCR has been run multiple times, and thus far the original trees in the field remained free of HLB symptoms and CLas. After propagation and grafting inoculation, and observations over the past years, it was shown that the propagated trees from the one tree found in Guangxi could be re-infected by grafting in a protected greenhouse and expressed typical HLB symptoms. It is thought that this tree might possess some unknown mechanism for its survival in the field, perhaps ACP resistance or repellency. The two trees found in Guangdong appeared to remain healthy and uninfected in the field, which is interesting; however because of personnel changes in the institute, until now there have been no successful graft inoculations with infected budwood done in greenhouse. This greenhouse test is needed to determine its susceptibility. It is important to mention that propagated trees grown at the institute’s own field site, where HLB is widespread and barely managed, have remained asymptomatic. All the exchange of information with colleagues in China during this time period have been through emails, phone calls, and other meeting occasions when possible. No visits were made this year to the two institutes or the commercial groves, to directly observe the propagated and graft-inoculated trees, or to explore for new escape trees in abandoned or severely infected groves.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and data on disease progression have collected, through symptom expression and repeated PCR assays. We are now seeing striking differences in the rates of disease development as well as the severity of symptom expression; seedlings of several sources of germplasm have remained free of HLB symptoms and CLas detection. e=we have reinoculated these plants to continue to challenge them. Some accessions that were quickly infected, for example Daidai, have shown the ability to recover healthy growth flushes while retaining high titer values of CLas, as these plants were inoculated with CTV-free isolates in Carrizo citrange originally, they represent a new potential inoculum source for additional experiments in the future. New seedlings that have reached sufficient size have now also been inoculated with the original Carrizo HLB source. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges upon which we have done and will continue to do extensive genomic characterization, has been propagated and will soon be planted in a replicated field trial at the USDA-ARS farm on Picos Road, Ft. Pierce in collaboration with Dr. E. Stover. This population is of significant interest as the trifoliate orange and some of its hybrids have been shown to be very HLB-tolerant, and this experiment provides the opportunity to map genetic components responsible for the tolerance. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified and materials received for testing. Eleven crosses between 10 different susceptible and reputedly tolerant parents were made in spring 2010, and populations of at least 150 from each cross have been planted and now are growing in a DPI-certified propagation house, being prepared for replication and inoculation experiments. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
Two full genome sequences have been assembled and annotated, and made available to the citrus research community. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. Both genome sequence assemblies along with annotation are available at the Phytozome portal at JGI, as well as Tree Fruit GDR (citrusgenomedb.org). The Clementine haploid genome has been substantially improved in its assembly and consequent gene model predictions, annotations, and utility to the research community, consisting of 9 pseudomolecules, representing the 9 basic chromosomes of the haploid genome; it has been named Clementine v. 1.0, and is being used in comparative genomics studies to result in a high-profile manuscript describing the phylogeny of sweet orange. New citrus genome sequences have been generated by the Machado lab in Brazil (Ponkan mandarin, 4x coverage, using 454 technology) and the Gmitter lab and UF-ICBR (low-acid pummelo, 25x coverage by Illumina technology). These new genome sequences are being integrated and compared with sweet orange and the reference haploid Clementine v. 1.0. Regions within the sweet orange genome have been identified that represent mandarin/mandarin haplotypes, as have mandarin/pummelo haplotype regions. Work has proceeded on the other objectives of this project. The candidate genes for silencing were sent to our collaborator in Spain, and constructs were prepared to initiate silencing experiments to provide proof of the gene’s specific involvement in development of HLB disease symptoms. However, in assessing the constructs it was found that use of the original pHellsgate12 resulted in unstable inserts, though at least 3 candidate gene silencing sequences were cloned into it, and infiltrated into plants. However, due to the difficulty in obtaining the double integration events using this vector, we acquired new destination vectors. A total of 6 candidates have been cloned successfully now in a silencing vector and the pipeline to handle recalcitrant plasmids and to check for positive clones has been developed, so more candidates may be explored in the future if the silencing is successful in planta. Infiltration experiments are set up now, and we hope to know if the system works in the next few months. 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 are being prepared from ore than 150 individuals of a large mapping family, to integrate the sweet orange genome sequence with genetic and physical linkage maps, thus improving the quality of the orange genome sequence assembly and its annotation. Analysis of data from two microarray studies looking at differences in gene expression between sensitive and tolerant citrus types is proceeding.
Quarterly report October 2011: Two full genome sequences have been assembled and annotated, and made available to the citrus research community. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. Both genome sequence assemblies along with annotation are available at the Phytozome portal at JGI, as well as Tree Fruit GDR (citrusgenomedb.org). The Clementine haploid genome is under revision for substantial improvements in its assembly and consequent gene model predicitons, annotations, and utility to the research community. The current assembly consists of 9 pseudomolecules, representing the 9 basic chromosomes of the haploid genome, and was developed by integrating and anchoring BAC end sequences from the haploid BAC library with the high-density genetic linkage map constructed by the ICGC collaboration. There are 1398 scaffolds harbored on these 9 chromosomes, representing 301.4 million bases (MB), representing an estimated 98.9% of the full genome. The scaffold N/L50 numbers, 4/31.4 MB, are substantial improvements over the version 0.9. Comparisons of the haploid Clementine genome with that of sweet orange are underway, to understand better the phylogeny of sweet orange. A manuscript is being drafted by the ICGC and the sequencing center scientists involved in the project to incorporate both of these sequences into a single work, enhancing the value and utility of each. Work has proceeded on the other objectives of this project. A preliminary list of candidate genes for silencing was sent to our collaborator in Spain, and constructs were prepared to initiate silencing experiments to provide proof of the gene’s specific involvement in development of HLB disease symptoms. However, in assessing the constructs it was found that use of the original pHellsgate12 resulted in unstable inserts, though at least 3 candidate gene silencing sequences were cloned into it, and infiltrated into plants. However, due to the difficulty in obtaining the double integration events using this vector, we have recently acquired new destination vectors, pK7GWIWG2(II) and pK7GWIWG2D(II). Currently, cloning is being done in parallel using the three vector systems. Agrobacterium infiltration using the final constructs is to begin in January 2012. A comparative proteomic study was completed to understand the pathogenic process of HLB in affected sweet orange leaves. Using the isobaric tags for relative and absolute quantification (iTRAQ) technique, we identified 686 unique proteins in the mature leaves of both mock-inoculated and diseased sweet orange plants. Microarray analysis showed that stress-related genes were significantly upregulated at the transcriptional level. Moreover, the transcriptional patterns of some of these upregulated proteins were examined at different stages of HLB disease development, providing information that may be used for early, presymptomatic detection of CLas infections. We have mined the BAC end-sequences from Dvorak’s lab and identified SNPs, that are being screened and verified to build a GoldenGate assay platform for hi-throughput genotyping of a large mapping family, to integrate the sweet orange genome sequence with genetic and physical linkage maps, thus improving the quality of the orange genome sequence assembly and its annotation.
Quarterly report for March 2011: Two full genome sequences have been assembled and annotated. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. This genome was released through the Phytozome portal at JGI, as well as Tree Fruit GDR, at the International Plant and Animal Genome (PAG) Conference in January 2011. The current version, Citrus clementina 0.90, is based on ~6.4x coverage, and is a very preliminary product released to enable citrus research community access. BAC end sequences from the BAC library produced from the haploid have been included now, and a high-density genetic linkage map is being constructed by ICGC collaborators, to yield a chromosome-based assembly in the near future. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. This genome sequence is based on ~30x depth of sequence coverage and was assembled using Newbler software; it covers 319 Mb spread over 12,574 scaffolds. Half of the genome is accounted for by 236 scaffolds 251 kb or longer. The current gene set (orange1.1) integrates 3.8 million new ESTs (produced this year) with homology and ab initio-based gene predictions; 25,376 protein-coding loci have been predicted generating a total of 46,147 transcripts. The sweet orange genome also was presented at PAG in January 2011, and can be accessed through the portals indicated above. The 3.8 million sweet orange ESTs came from 17 different libraries that were produced and sequenced using the 454 platform. They represent various biotic/abiotic challenges including psyllid feeding on young seedlings, canker inoculation, and treatment with salicylic acid, among others. Substantial progress has also been made on the other objectives of this project. Studies comparing the time courses of gene expression in two sets of HLB-inoculated sweet orange and rough lemon plants, representing more susceptible and more tolerant types respectively, have been completed using Affymetrix and Agilent citrus chips (the latter developed by us at UF); some differentially expressed genes have been confirmed by RT-PCR. In addition, comparisons of carbohydrate metabolism and anatomical changes associated with gene expression differences in these same plants have been completed. For example, 4-fold induction of cell-wall-bound invertase activity was detected in symptomatic and asymptomatic leaves on diseased plants. Additionally,the expression profiles of starch breakdown genes indicated that the transcription of DPE2 and MEX1 was downregulated. Together with the reduction of maltose accumulation, it is suggested that the impairment of starch breakdown contributes to the starch accumulation in infected leaves. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from Brazil and Spain, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs. A preliminary list of candidate genes for silencing has been sent to our collaborator in Spain, and constructs are being prepared there to initiate silencing experiments to provide proof of their specific involvement in development of HLB disease symptoms.
Quarterly report June 2011: Two full genome sequences have been assembled and annotated, and made available to the citrus research community.. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. Both genome sequence assemblies along with annotation are available at the Phytozome portal at JGI, as well as Tree Fruit GDR (citrusgenomedb.org). BAC end sequences from the BAC library produced from the haploid have been included now, and a high-density genetic linkage map has been constructed by ICGC collaborators; the map consists of 9 linkage groups, corresponding to the basic chromosome number for citrus, and it contains 952 sequence-derived markers (SNPs from Clementine BES and EST-SSRs), covering 1112cM. This map is strongly anchored on a large diploid Clementine BAC library resource, as well, and it supports the alignment of the haploid Clementine whole genome sequence in the framework of the ICGC collaborative project. The map, BAC end sequences, and assembled sequence scaffolds will be integrated to yield a chromosome-based assembly. Work has proceeded on the other objectives of this project. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from Brazil and Spain, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs. A preliminary list of candidate genes for silencing was sent to our collaborator in Spain, and constructs were prepared to initiate silencing experiments to provide proof of the gene’s specific involvement in development of HLB disease symptoms. However, in assessing the constructs it was found that use of the original pHellsgate12 resulted in unstable inserts, though at least 3 candidate gene silencing sequences were cloned into it, and infiltrated into plants. We are seeking a better vector to use for these experiments. The PI Gmitter will travel to meet the collaborator in Argentina soon, to coordinate efforts and to resolve technical issues. The collaborator in Spain reannotated the previously UF-developed Agilent microarray, making analysis and interpretation of our time course experiments in sensitive and tolerant host citrus plants easier and more meaningful. We have initiated collaboration with Jan Dvorak’s group from UC-Davis, to utilize a BAC-based physical map of sweet orange and his BAC end-sequences in an effort to integrate the sweet orange genome sequence with genetic and physical linkage maps to improve the quality of the genome sequence assembly and its annotation.
Funds for this project have now been received. The antibody service provider has provided a quote for anti-NodT antibody production. Antibody production and screening will be initiated soon and is expected to take approximately 3 months.
Results from four sweet orange rootstock field trials exposed to HLB were summarized and submitted for publication. The studies identified rootstock differences in tolerance to HLB that were discussed in the scientific publication. I will present this information to growers in an appropriate upcoming forum to use in making management decisions. Fruit quality, yield, and tree size data were collected from eight early season rootstock field trials. Detailed fruit quality data were collected from a large grapefruit rootstock trial at multiple harvest times to assess rootstock influence on grapefruit quality early, midseason, and late in the season. Two replicated field trials with 35 new supersour selections were planted in Lake County and Orange County. Source trees of 150 new supersour hybrids were selected and first stage propagations made to increase trees for specialized disease, abiotic, and field testing. Cuttings were made of 100 advanced supersour selections in preparation for cooperative field trials. Another supersour rootstock trial with 800 trees was prepared for field planting. Cooperative work was continued with a commercial nursery to multiply advanced supersour selections for placement of trees into cooperative field trials with growers. Work continued to assess supersour tolerance of CTV and calcareous soils (high pH). Studies continue to assess citrus germplasm tolerance to Liberibacter – Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. Some trifoliate hybrid rootstocks, including US-802, US-812, US-897, and US-942 exhibit tolerance to HLB as seedling trees. Studies to compare the different components of tolerance in several rootstock selections were completed and a scientific publication is being prepared. Another study is underway to define the interaction of rootstock tolerance/susceptibility with scion tolerance/susceptibility. Collaborative work continues to study gene expression and metabolic changes associated with susceptible and tolerant plant responses to HLB, and to define genetic characteristics needed to prevent infection or avoid the damaging effects of the disease. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Selected anti-microbial and citrus plant resistance genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with increased resistance to HLB. Research is continuing to use HLB responsive citrus genes and promoters identified in the gene expression study published last year for inducing or engineering resistance in citrus. Thirty transgenic rootstocks with selected antimicrobial genes were propagated and entered into controlled greenhouse tests to assess tolerance to HLB. Data was collected from a field trial with selected transgenic rootstocks. Seed of the new rootstocks US-942, US-897, and US-802 was provided to the Florida Citrus Nursery Association for managed distribution to commercial nurseries.
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 transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene. The transgenic plants are currently under canker resistance test. We will propagate these plants for citrus greening test. We have shown that overexpressing the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker, suggesting that the salicylic acid (SA) signaling pathway plays an important role in citrus disease resistance. We recently established an Arabidopsis-Xanthomonas citri subsp. citri (Xcc) pathosystem with the support of a USDA special grant. Using the Arabidopsis-Xcc pathosystem, we found that mutants of the SA signaling pathway are more susceptible to Xcc. A manuscript about these results has been accepted by PLoS ONE. We are trying to generate citrus transgenic plants that accumulate high levels of SA. For objective 2, we are continuing the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Two quarts of seeds treated with gamma-ray irradiation at 50 Gy have been 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. Some shoots formed on these gamma irradiated seeds have been 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. We are also testing whether a direct genetic screen would work for identifying citrus greening-resistant varieties. We germinated gamma ray-irradiated Ray Ruby grapefruit seeds in soil and inoculated the seedlings with psyllids carrying greening bacteria. We are watching 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, but host plant resistance in Citrus and relatives to ACP has not been conclusively demonstrated to date. We found very low abundances of all life stages of ACP on two genotypes of Poncirus trifoliata L. in a field survey, so we tested whether 81 genotypes of P. trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) were resistant to ACP by determining whether these genotypes influence oviposition and lifespan of adults in no-choice tests. There was a higher abundance of eggs on the control (Citrus macrophylla Wester) than on all genotypes of P. trifoliata, except for the genotype ‘Towne ‘G’, and 15 of 34 genotypes of xCitroncirus sp. Lifespan of adults also was ~2-5 times longer on C. macrophylla than on P. trifoliata ‘Flying Dragon B’ and most of the trifoliate hybrids that also were resistant to oviposition. Our work is the first to conclusively identify resistance to ACP in citrus germplasm, but we must next identify the genotypic and phenotypic traits that promote resistance in order to create commercial varieties of citrus that reduce the population of ACP and lower the incidence of citrus greening disease. We additionally screened 8 cultivars of commercial citrus that had previously shown different levels of HLB infection in free-choice experiments in the field during May, July, and October 2011. Psyllid eggs were most abundant on short flush shoots and also varied among dates (most abundant in October and least abundant in May). Treatment with Admire insecticide did not influence abundance of eggs. The cultivar of citrus also was statistically non-significant, but the variation in egg abundance across cultivars likely is biologically significant. Abundance of eggs was noticeably high on two cultivars, Hamlin on Kinkoji and Ruby grapefruit on Kinkoji, and low on two cultivars, Temple on Cleo and Fallglo on Kinkoji. Abundance of psyllid nymphs also was influenced date (May > October > July) and by cultivar of citrus, but not insecticide. Abundance of nymphs was noticeably high on two cultivars, Hamlin on Kinkoji and Ruby grapefruit on Kinkoji, and low on three cultivars, Sunburst on Kinkoji, Fallglo on Kinkoji, and Tango on Kuharske. We are currently screening grapefruit trees that have been genetically transformed to express Lectin from the snowdrop pea. We are comparing rate of oviposition, nymphal development, and lifespan of adults on three varieties that express lectin to a grapefruit variety that does not. To date we have determined that expression of Lectin does not deter oviposition. Data on nymphs and adults will be available soon. The Fujian Academy of Agricultural Sciences has initiated no-choice experiments with Poncirus accessions and is adding different accessions of P. trifoliata, B. koenigii and other species outside the genus Citrus to their field studies. To date ARS and FAAS results are in general agreement about susceptibility of specific germplasm each group has studied.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or anti-psyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. We have about 50 new peptides that are now being screened. We are eliminating peptides that do not work and continuing to make and screen new ones. ‘ We have greatly improved our efficiency of screening. We are using small plants in order to screen faster. However, we have to balance psyllid damage with inoculation of HLB. We now are ‘pulse-inoculating’ plants by incubating them about 2-3 weeks with psyllids between intervals of no psyllids in the greenhouse. ‘ We have greatly improved the CTV vector to produce probably 100x more peptide. ‘ We have modified the vector to allow addition of a second anti-HLB gene. ‘ We have obtained permission and established a field test to determine whether the CTV vector and antimicrobial peptides can protect trees under field conditions. ‘ We continue to supply infected and healthy psyllids to the research community. ‘ We are testing numerous genes against greening or the psyllid for other labs.
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. A manuscript entitled ‘Overexpression of the Citrus CtNH1 Gene Confers Resistance to Canker Disease’ is in preparation. The CtNH1 plants have been propagated by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). No conclusive results can be reported at this time. Microarray experiments were conducted using the transgenic line CtNH1-1 and non-transgenic ‘Duncan’ grapefruit inoculated with Xac306. 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. A second manuscript describing these results is in preparation. 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. We will characterize these plants by Northern blot and propagate the lines with overexpressed CtNH1 for Las inoculation.
Huanglongbing (HLB) and Citrus Bacterial Canker present serious threats to citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters are used to generate numerous transformants of rootstock and scion genotypes. 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 continue to grow better than all controls at 8 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. 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 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. 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. 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. Unfortunately, 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, presumably due to high levels of phenolic compounds. Red citrus transgenics will be tested for HLB, ACP, and canker resistance. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 34 months and a repeated trial has been in the field for 22 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. Comparison of field-grown and greenhouse-grown valencia following graft-inoculation show much more rapid CLas development in greenhouse-grown trees. Several new collaborations are being explored to feed new HLB-suppressing transgenes and novel strategies into the citrus transformation pipeline.
A transgenic test site has numerous experiments in place 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 seventeen months. 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. 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 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. Additional plantings are welcome from the research community.
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