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.
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.
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