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. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. 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. 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. Use of this promoter may reduce the number of independent transformants needed. A new ubiquitin promoter from citrus (Belknap) is being tested. 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 are being tested further for suppression of CLas in culture, before creating transgenes. ARS-Albany (Belknap) collaboration is providing genes from Carrizo citrange sequence generated using USDA and now CA-CRB funds, and other citrus genomic data, to permit transformation and resistance using citrus-only sequences; citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and are being tested in citrus scions. Sequence data are being mined for citrus AMPs and defensins to test in-vitro and ultimately in-planta. Anthocyanin production genes,give bright red shoots (UGray collaboration) and are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes have been developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. 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 30 months and a repeated trial has been in the field for 18 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. Several new collaborations are being explored to feed new HLB-suppressing transgenes and novel strategies into the citrus transformation pipeline.
Seed from new crosses to develop rootstocks and scions with tolerance to HLB and other improved traits were planted in the greenhouse. Fruit quality, yield, and tree size data were collected from 15 rootstock field trials. Source trees of 627 new supersour hybrids were established in the greenhouse to begin propagation. Propagations from 205 different supersour rootstock hybrids were budded to produce trees for disease testing and field trials. More than 3000 budded greenhouse trees for supersour field trials were grown to planting size. An agreement was developed to begin cooperative propagation of supersour rootstocks with a commercial nursery for widespread field trials with cooperators. One new rootstock trial with Rio Red, Hamlin, and Valencia scions was planted in St. Lucie County to evaluate tree performance with standard and new rootstocks under intensive HLB and horticultural management practices. Data was collected from three field trials at the Whitmore Farm in Lake County to study inheritance of fruit quality factors in sweet orange-type material from populations of hybrids between high quality pummelo and mandarin parents. It was apparent from one trial examining the effect of tree manipulations that most tree growth manipulations that might shorten time to fruiting will also greatly increase sensitivity to cold damage. Studies continue to assess citrus germplasm tolerance to Liberibacter – Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. All citrus germplasm and cultivars become infected with Liberibacter, but different germplasm responds to HLB infection at different rates and with different symptom severity. Some trifoliate hybrid rootstocks, including US-802, US-812, US-897, and US-942 exhibit tolerance to HLB as seedling trees. Some hybrid selections resembling mandarin, grapefruit, and sweet orange also appear to exhibit some tolerance to HLB. A publication was prepared on differences in HLB tolerance among different rootstocks in field trials. One greenhouse experiment to evaluate supersour rootstocks for tolerance to CTV was completed, and another greenhouse experiment was initiated to compare different rapid methods for evaluating CTV tolerance of supersour selections. Promising new scion cultivars were released, including seedless Pineapple and the seedless mandarin cultivar ‘Early Pride’. Cooperative trials continued and new trials established to provide more information on new scion performance and pollination effects. The new hybrid rootstock US-942 was released for commercial use because of outstanding performance in many trials. Seed of US-942, US-897, US-812, and US-802 was provided to the Florida Citrus Nursery Association for managed distribution to commercial nurseries. 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. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial, insect resistance, and other genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker. Research is continuing to use HLB responsive genes and promoters identified in the gene expression study published last year for inducing or engineering resistance in citrus. New studies were initiated to examine gene expression and metabolic changes associated with HLB disease development and apparent resistance to Liberibacter in particular selections. A publication was prepared to document gene expression changes associated with the HLB tolerance of US-897 rootstock. A greenhouse study is underway to compare the apparent HLB resistance of several different trifoliate hybrid rootstocks. These studies will provide additional insights about how to engineer HLB resistant cultivars.
In the second year of funding, the CCTF continued to maintain and improve the quality of service it offers, proving itself as a reliable partner and integral part of the wider research community engaged in fighting HLB and canker. CCTF has become truly known and recognized beyond the community of Citrus Research and Education Center (CREC) and that is reflected in the increasing percentage of orders coming in from main campus of University of Florida in Gainesville. Within the last year, CCTF received orders to produce transgenic plants by using following vectors: p33; p7; p10; pMOG800; pAS7; pAS13*; pNAC1; pMKK7; pMOD1; pSucNPR1; pWG19-5; pWG20-7; pWG21-1; pWG22-1; pWG24-13; and pWG25-13, and pWG27-3. This is the largest number of orders received during one year since the facility opened and it clearly describes high demand for transgenic Citrus plants. At the time this report is being written, the facility has already been informed of additional five orders (from UF researchers) and another two orders for which binary vectors for insertion of customer’s genes of interest were sent to Yale University. The initial goals of this project that were reached in the first year of funding were being met throughout the second year. Despite high flux of people, the number of employees was kept constant. That allowed the number of explants processed per week to stay at about 2500. This amount of processed material per week is sufficient for production of high numbers of shoots that are being screened for presence of transgene by using different methods. And this in turn creates situation where CCTF is capable of servicing multiple orders at the same time. Application of the new PCR-based screening method that we started using last year is proving to be extremely useful. By using PCR on small shoots and detecting those that are putatively transgenic before they get micro-grafted on the rootstock plants improved CCTF productivity. Considering the fact that many orders include the use of binary vectors with no reporter gene, introduction of PCR as screening tool has brought the efficiency of the facility to a new level that permits its present output. As a result, the production stayed on high level of above 400 plants per year. More importantly, the time needed for completion of order and delivery of transgenic plants has fallen to about 10 months for many vectors. Within the last quarter, additional plants were produced for the old orders: pHK (12) and pSuperNPR1 (2). However, most of the plants were produced for newer orders: pNAC1 (26), p33 (12), pMKK7 (16), pMOD1 (5), pAS7 (7), pAS13* (4), pSucNPR1 (3), pMOG800 (1), p7+p10 (15). Similar to last year, the plants that CCTF produced in the second year of funding belong to five cultivars: sweet oranges-Hamlin and Valencia, grapefruits-Duncan and Flame, and Mexican lime. All of the new orders received in this funding period had a goal of improving tolerance and/or resistance to Citrus pathogens. The production of plants for old orders listed here also continued: pCL1; p6; pN1*; pC5*; pNPR1; pSuperNPR1; pPiTA; pCIT108p; pCIT108p3; and pHK. This decisively confirms the relevance of this project for the overall effort to produce and challenge transgenic plants as soon as possible and present them to Citrus industry as prospective candidates for tolerance and/or resistance against huanglongbing (HLB), canker, and Citrus Tristeza Virus (CTV). All current orders are for faculty presently involved in research projects funded by CRDF to battle HLB, canker or CTV. Funding for the CCTF furthers the efforts of these research groups and brings some of their results into life by producing transgenic plants that carry genes with predicted protective roles against pathogen attack. Continued funding to CCTF will allow for this situation to continue by keeping production of transgenic material at high levels and uninterrupted.
Our group continued to make excellent progress in the second year of our project. Over the last twelve months we have accomplished the following: Testing TAL effector specificity: We have synthetically assembled six TAL effector genes from X. citri strains and have establishing a system to test their activity on our broad recognition or “super” promoter in transgenic Nicotiana benthamiana plants. We have also prepared constructs with promoters containing individual TAL effector binding sites to test their activity and specificity. Testing broadness of resistance: Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves with Bs3 promoter constructs. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. These studies concur with the preliminary results showing that the constructs limit X. citri growth and produce HR against a number of strains. Additionally, we have generated different versions of the constructs that are designed to detect TAL effectors in all known X. citri strains. We are currently testing these, as well as X. citri strains with single TAL effectors to isolate the role of specific TAL effector proteins in the disease and resistance process. We have isolated multiple new TAL effector genes from important X. citri strains, which we will sequence and analyze in the next project year. Production and analysis of transgenic grapefruit lines: Sixteen independent transgenic lines generated in the first project year have progressed through selection, shoot formation and rooting, and are now well established in soil. These lines were verified by PCR and have been used in several experiments to examine response to pin-prick assays with X. citri and controls. We have thus far tested nine of the lines, and all but one demonstrates some degree of canker resistance, with two showing strong resistance. We have also set up additional transformations on both cotyledons and epicotyls. In total, we have initiated transformations of 6,857 explants using seven promoter constructs, and the explants are moving through tissue culture and selection, with more than 200 additional plantlets in soil. Finally, we have consulted with industry personnel to identify the most industry-relevant commercial germplasm to transform, and as a consequence of this we have obtained seed of red grapefruit and mid-season sweet orange to test in transformation assays. Other outcomes: We filed a patent application in January 2011 on canker-resistant transgenic citrus, and we have drafted an initial manuscript of our results, to be completed following the outcome of pending experiments.
USDA Ft. Pierce (Neidz) Agrobacterium-mediated transformation of mature tissue explants: Transformation of mature internode explants from greenhouse trees has been demonstrated in four citrus types including Valencia sweet orange (1 plant), Ruby Red grapefruit (1 plant), US-942 (8 plants), and Etrog citron (8 plants). Current efforts are directed toward characterizing this system for routine transgenic plant production. Source of mature tissue: Four populations of adult phase trees were maintained in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). In vitro bud emergence and growth manuscript accepted for publication: A manuscript entitled, ‘Bud emergence and shoot growth from mature citrus nodal stem segments’ was accepted for publication by the journal Plant Cell, Tissue and Organ Culture. The paper documents the system developed for producing in vitro adult phase shoots from cultured nodes of greenhouse trees. Shoot regeneration from mature tissue explants: A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. New tissue culture method of Agrobacterium-mediated transformation of tissue explants: Preliminary results using alternative culture methods suggest improved transformation efficiencies. These approaches will be further explored. Mineral effect on shoots regeneration: Preliminary results suggest that mineral nutrition significantly affects in vitro culture response. The effects on transformation are currently being studied. University of Florida (Moore, Grosser, Gmitter) Efforts continue with greenhouse grown tissue (CREC) Rootstock effect on mature tissue transformation: the experiment conducted to determine if vigorous allotetraploid rootstocks could increase transformation efficiency was compromised by endogenous fungal contamination. We are now testing coconut fiber, sterile liquid nutrition, and low humidity in a clean environment for growing mature tissue explants in efforts to minimize problems with fungal contamination. Characterization of mature-tissue transgenic ‘Hamlin’ plants: recovered mature-tissue derived transgenic ‘Hamlin’ plants from previous experiments were propagated via micro-grafting for further characterization. Research continues on using cell penetrating peptides (CPPs) to deliver cargo (proteins, chemicals, plasmid) to existing citrus cells (Gainesville). Using the easily visualized GUS enzyme, we have found that we can efficiently get protein imported into a number of citrus tissues, using several different CPPs. Currently we are testing import of plasmid DNA, which should let us test clones and constructs before we do stable transformation. Based on a recent report on woody plants, we are also investigating whether we can produce cultures of rapidly proliferating cambial cells from citrus (Lee et al. 2010. Nature Biotechnology 28:1213).
We reported last quarter the cloning of the ctEDS1 gene in the binary vector pBINplusARS. We have already transformed the Arabidopsis eds1-2 mutant with this construct. The T0 seeds were harvested and will be selected for the transgenic plants in the next few weeks. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. The cDNA fragments of these genes are now in the pGEM T-easy vector and were confirmed with sequencing. The next step will be to clone these cDNA fragments to the binary vector pBINplusARS for plant transformation. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes for the next round of cloning and complementation analysis.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current control of ACP and HLB is inadequate, but identifying and incorporating traits from uncultivated Citrus spp. and Citrus relatives that confer resistance to ACP is a potential strategy to manage the disease. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae orange subfamily Aurantioideae, were assessed in the field in South Florida for resistance to natural populations of ACP. The majority of genotypes hosted all three life stages of ACP, however there were differences among genotypes in the mean ranks for eggs (F = 3.13, df = 86, P < 0.001), nymphs (F = 9.01, df = 86, P < 0.001), and adults (F = 4.21, df = 86, P < 0.001). Very low levels of ACP were found on two genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties. The field experiment was followed by no-choice tests in which female ACP had the opportunity to lay eggs for six days on five genotypes of Poncirus trifoliata, three genotypes from the Citrus genera that were not represented in the field, and a control (Citrus macrophylla) to determine whether any genotypes were resistant to ACP. Numbers of eggs on the five genotypes of P. trifoliata (means between 7-60) were lower than on the control (mean = 281.3; .2= 59.5, P < 0.001), which indicates that genotypes of P. trifoliata show some resistance to ACP. Numbers of eggs laid on the three genotypes of Citrus (means 129-200) were not significantly lower than on the control (.2= 4.37, P = 0.23). An additional 107 genotypes, including 81 genotypes of P. trifoliata and trifoliate hybrids, were planted mid-January and will be screened for resistance to oviposition by ACP in the coming months. Studies have been initiated to compare plant volatiles associated with plant genotypes that are readily colonized by the psyllid to those less colonized by the psyllid. Collaborators with the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated two experiments on resistance to ACP within the Rutaceae. Forty genotypes were evaluated in a free-choice experiment conducted in a screen house. Citrus tankan Hort. (cultivar Fuyouxuan Jiagan) was completely avoided by adults, and no eggs or nymphs were ever observed on this cultivar. No eggs or nymphs were observed on the following: C. reticulata Blanco (cultivars Bayueju, Xiang Ponkan, and Mashuiju); C. mitis (cultivars Chengshi Calamondin and Variegated Calamondin); C. sinensis (cultivars Navelia Navel orange and Skaggs Bonanza Navel Orange); C. grandis (cultivar Chandler Pummelo), and Fortunella hindsii var Chintou. The most heavily colonized genotypes included: C. reticulata Blanco (cultivar Fina Sodea Clementine); C. sinsensis (cultivar Fengcai anliucheng); and C. grandis Osbeck (cultivar HB Pummelo). A free-choice field experiment was established comparing 31 genotypes. Due to prolonged cool weather, no results have yet been obtained from this experiment. A delegation from FAAS will be visiting USDA-ARS during April or May to coordinate research efforts.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: In addition to the SSI4 obtained from Arabidopsis thaliana var. Columbia genomic DNA, we created two new constructs (5-4) AtSUC2/SSI4 and AtSUC2/ssi4 mutant derived from Nossen genomic DNA (lines obtained from Dr. Klessig). Out of 49 ssi4 transgenics, 18 showed a reduced stature phenotype. However, none of the ssi4 expressing transgenics displayed a severe dwarf phenotype seen when expressed using their native promoter. We visited Dr. Orbovic’s laboratory at the UF CREC at Lake Alfred and resolved a technical problem regarding the PCR-based screening of citrus transformants. Dr. Orbovic has subsequently identified transgenic citrus for our two clones: AtSUC2/snc1 and AtSUC2/ssi4 mutants. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to the single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. The PAD4/ssi4 transgenics were difficult to obtain. We screened three times as many To seeds to obtain 18 plants. From these, 50% developed the “smaller stature” phenotype. Conversely, almost all transgenic plants containing SSI4 (wt) developed normally. Of the 33 transgenics with the PAD4/SNC1 construct, the majority showed no detrimental effect on growth. However, from the 37 PAD4/snc1 mutant transgenics, 10 were smaller than normal. We obtained 27 transgenics with the PAD4-reporter and analyzed GUS expression. Originally, the PAD4 promoter was selected based on literature reports of its phloem-specific expression and wound-inducibility upon the insect feeding. Our results indicated four basic groups of PAD4/GUS reporter expression in transgenic Arabidopsis leaves as follows: 1) phloem-specific expression with no wound induction; 2) universal expression with strong wound induction; 3) restricted expression with no wound induction; and 4) strong wound expression only. Conclusions: Our GUS analyses of two phloem-specific promoters (AtSUC2 and AtPAD4) revealed that transgenic plants do not maintain strict phloem-specific expression. Moreover, we found no PAD4 plants with the expected phenotype of wound-specific expression limited to phloem cells. However, substitution of the native promoters for ssi4 and snc1 genes with AtSUC2 and AtPAD4 did result in a significant reduction in the severity of the growth retardation phenotypes. Evaluation of more transformants will give us a better picture of the feasibility of utilizing these two promoters to obtain the desired expression patterns in citrus.
Once we have been able to establish at IVIA the procedures and conditions to transform mature Hamlin, Pineapple and Valencia sweet oranges, we are in conditions to transfer the basic protocols to Florida. We believe they can be reproduced with little or no modification at the new laboratory that is being set up at the CREC. As we have also developed a genetic transformation system for mature Carrizo citrange, we are now incorporating a construct of interest into this genotype. The second objective of our project was to develop genetic engineering strategies to improve citrus tree management. In this sense, we proposed to reduce endogenous gibberellin levels in transgenic rootstocks to make them dwarf or semidwarf. Such rootstocks could provide reduced size to non-transgenic scion varieties grafted into them. With this aim, we have incorporated a hairpin construct into Carrizo citrange to silence an endogenous GA20-oxidase gene and them reducing bioactive gibberellin levels in growing shoots. After Agrobacterium-mediated transformation, the explants regenerated abundant callus and showed prolific shoots formation. Around 70% of the explants regenerated shoots in the light step. At this moment there are several transgenic (PCR-positive) shoots micrografted in vitro. They are still pending of grafting on vigorous rootstocks in the greenhouse and Southern blot verification in coming weeks/months. New experiments will be run with this construct and fresh starting plant material within the next couple of months. For improving citrus tree management, we also proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. This objective was initiated one year ago or so, and we have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. The PI and his greenhouse/growth room manager, Josep Peris, travelled to Florida last March 20-27th to visit the CREC and supervise the last steps of the growth room construction before been finalized (by the end of April, we guess). We suggested to revise minor details to make the facility more reliable and helpful for operators. We also short-trained the tissue culture technicians in horticultural practices. We checked substrate, seed stock and plant nutrition issues with the manager Dr. Zapata.
A comparative study of two susceptible hosts, Duncan grapefruit (DG, Citrus paradisi), and Rough lemon (RL, C. jambhiri) and two resistant species of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami has been conducted to evaluate the basis for resistance to Xanthomonas citri subsp. citri (Xcc). The type of resistance occurring in kumquats is a hypersensitive response (HR) that develops within 48-72 h. This is based on the phenotype of the lesion, histological changes at the cellular level of infected tissue and early expression of genes related to programmed cell death (PCD). In kumquats but not in DG, several genes linked to PCD (lipoxygenase, glutathione transferase, metacapcase, acid chitinase and peroxidases) are expressed at 4 h post-inoculation (pi) with Xcc at 108 cfu/ml. Later at 24 h, additional genes related to plant defense (e.g. betaglucanase) are highly expressed in kumquats but less so in DG and RL and their activity continues to increase up to 48 h pi. Additional sets of genes related to PCD and the host pathogen interaction will be investigated this coming year. A cybrid is an asymmetric hybrid that contains the nucleus of one parent in combination with the mitochondrion and/or chloroplast of the cytoplasm donor parent. Twenty cybrids of highly susceptible Red grapefruit (RG) and the more tolerant Valencia orange (VO, C. sinensis) as the cytoplasm donor, were screened for their susceptibility to Xcc. Tolerance inherited from VO appeared to be quantitative based on an intermediate lesion phenotype in selected cybrids. In contrast to the callus-like lesions typical for susceptible RG, lesions were more necrotic for VO and the cybrids. This lesion phenotype indicated cell death arrested the proliferation of Xcc. Populations of Xcc at 14 days post inoculation in cybrids (7.2 Log cfu), were similar to VO (7.6 Log cfu) and one log unit lower than RG (8.4 Log cfu). Expression of genes related to host pathogen interaction in VO and cybrids differed from RG. The contrasting pattern suggested a differential interaction of genes from the nucleus with the mitochondria and chloroplast genes from the cytoplasm donor. Mitochondria and chloroplasts have a central role in stress and PCD signaling. The response of cybrids to Xcc confirms inheritance of resistance from VO may be expressed at different levels depending on whether mitochondrial and/or chloroplast genomes are transferred in the cybridization process. Field trials with several Ruby red grapefruit cybrids planted in canker-affected locations on the east coast were showing less foliar disease incidence than the adjacent Red grapefruit trees, but were severely damaged by freezes. Additional material has been planted out in canker prone locations. Production of cybrids lines using a new callus line of Meiwa kumquat as the cytoplasmic donor is underway in the Grosser lab. The resultant cybrids will be evaluated for inheritance of HR resistance using the same approaches
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of 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 have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and 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 (in collaboration with D. Hall) is being conducted on a replicated set (8 plants of each) of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. It is anticipated that statistical analysis of CLas levels and symptoms will permit identification of material with significant resistance, for further testing. A series of promoters has been tested with the GUS gene to see how effective they are. As expected, 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. Use of this promoter may reduce the number of independent transformants needed. CLas sequence data were used to target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. Radiolabelled ATP is being used assess effect of identified peptides in E. coli expressing the CLas translocase. Collaboration with a USDA team in Albany, CA is: providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns; providing genes from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences; citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and are being tested in citrus scions. Anthocyanin production genes,give bright red shoots (Gray collaboration) and are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. 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 27 months and a repeated trial has been in the field for 15 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in the spring of 2011, and 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. An experimental attract/kill product, to disrupt citrus leaf miner (CLM) without disrupting ACP, was not effective last year. Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Several applications of Admire are being used to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance.
Previously we standardized quantitative real time PCR assays for 18 genes (AZI1, BLI, CHI, COI1, EDR1, EDS1, EDS5, JAR1, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1, RdRp, and SGT1) associated with SAR and plant defense. Using this technique and based on our previous results on defense gene expression during pathogen and PAMP inoculations we selected a few genes (AZI1, CHI, EDS1, NPR1, PBS1, R13032 and RdRp) that are differentially expressed during PTI/ETI/SAR and best characterize this response in citrus. We treated 24 ‘Carrizo’ citrange AtNPR1 transgenic lines with Candidatus Liberibacter asiaticus flagellin 22 peptide (L-flg22, as a proxy for the pathogen) and analyzed the expression of these 7 genes. The idea was to determine how the transgenic lines responded L-flg22, an inducer of PTI/SAR, and which transgenic lines showed an enhanced response compared to wild type plants. Several transgenic lines showed expression levels that were much higher for most of the genes compared to the wild type plants, further indicating that AtNPR1 seems to modify the defense response in citrus. We also studied the response grapefruit plants grafted on transgenic ‘Carrizo’ AtNPR1 plants infiltrated with L-flg22. The results are being analyzed.
During the past year we developed and standardized 8 more gene expression assays for the study of defense response in citrus using real time PCR. The 18 genes we have selected are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1, and SGT1), are targets of the regulatory SAR pathway (BLI, CHI, PR1 and RdRp) or are components of the jasmonic acid (JA) pathway (COI1 and JAR1) that works antagonistically to SAR. Several of these genes were previously undescribed for citrus, however our microarray studies indicated that these sequences were differentially regulated by chemical and pathogen treatment. Additionally, we continued to propagate more AtNPR1 ‘Carrizo’ citrange transgenic plants. To our previous lines (854, 857, 859 and 884) we have added 757, 775, 854, 890, 896 and also more of 857, the most promising line. We also studied the response of lines 854, 857, 859, 884 and wild type (WT) to Actigard (Syngenta Corporation), a commercial version of the SAR inducer salicylic acid (SA). We studied the effect of Actigard on gene expression levels either alone or followed by treatment with a Candidatus Liberibacter asiaticus Flagellin-like peptide (L-Fgl, as a proxy for the pathogen). In general, Actigard significantly induced some SAR genes (For example: CHI, EDS1, PBS1, RAR1 and RdRp) compared to water treated plants, these same genes were induced significantly higher in transgenic lines, specially 857 and these effects were observed as early as 6 hours after treatment . We are still analyzing the effect of the L-Fgl, although it seems to repress the expression of some SAR target genes, at least in the WT plants. We have not started the HLB inoculation experiments yet as the number of plants is limited and we wanted to characterize their response before the plants were permanently confined to a containment facility. However, we will initiate this part of the research as soon as we receive the last year of funding for this project.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. Results showed that the use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime, and a manuscript reporting this was accepted with revision for publication in PCTOC. Recovered transgenic Mexican lime trees 1.5 years after removal from tissue culture were girdled, which induced flowering about 4 months later; with complete flowering and fruit set in less than two years. Experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and Faryna Harvesting, yield and fruit quality data was collected from the St. Helena project. Approximately 10 acres of trees planted 3.0 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The best rootstocks identified to show positive affects on rapid tree growth with precocious bearing and good early fruit quality (higher lb. solids) were somatic hybrids Changsha mandarin+trifoliate orange 50-7, white grapefruit+trifoliate orange 50-7, and sour orange+Carrizo, and tetrazygs White#4 (Nova+HBPummelo x Succari sweet orange+Argentine trifoliate orange), Orange#13, Orange#14,Orange#18 and Orange#19 (Orange series all Nova+HBPummelo x Cleo+Argentine trifoliate orange). Trees on these rootstocks averaged more than a half-box of fruit per tree with juice brix values great than 11. These rootstocks will now be tested to determine if they can shorten juvenility in transgenic plants produced from juvenile explant. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. These will now be clonally propagated onto the rootstocks mentioned above for further study of early flowering and transgene expression. Horticultural manipulations on these plants will include the RES (Rapid Evaluation System) growth method plus girdling. Transformation with early-flowering genes: ‘Citrus has at least 3 FT genes. Cloning and characterization of all 3 (genomic clones) has been completed. We have put them into transformation vectors with a constitutive promoter and performed transformation experiments with Carrizo and Duncan. Although only a few transgenic plants were recovered, we know the constructs work because we had previously tested them in tobacco, where we recovered early flowering phenotypes, as well as some other phenotypic alterations. Our current theory is that expression with the 35S promoter is too strong in citrus (in some cases we got flowering directly on the initial explant!). We are currently testing a poplar FT with a weaker inducible promoter (a heat shock promoter shown to be inducible in Oregon); which will hopefully solve the promoter problem.
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