This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment of new scion and rootstock performance with a focus on HLB responses. Assessments of HLB field tolerance are continuously carried out in the vast collection of raw germplasm that we maintain, and new selections have been identified, and several previously found continue to hold up to HLB; additional evidence is accumulating supporting what may be differential sensitivity to HLB among sweet orange clones from the CREC program. We have essentially completed observations for the season on fifteen different individual rootstock trials planted throughout the citrus production regions of FL. Additional materials from the CRDF Rootstock Matrix list have been sent to the DPI PTP for STG and indexing, so certified materials can be made available to nurseries and TC companies for rapid increase of those trees for subsequent field trials and demonstrations. Individual Valencia trees on 70 rootstock hybrids (grown from grafted budsticks of HLB-infected Valencia) successfully made it through the greenhouse and ‘hot psyllid’ house phases of the HLB gauntlet and were planted at Picos Farm including 45 complex tetraploids, 15 robust Flying Dragon hybrids (ACPS potential), and 10 diploid sour orange-like hybrids. The first phase of an ‘interstock’ experiment was completed by budstick grafting interstock candidates (10 per selection) onto Swingle citrumelo rootstock liners. Candidates included HLB tolerant pummelos, polyploid pummelo/mandarin hybrids, a wide intergeneric hybrid, and sweet orange as a control. Six new rootstock trials were planted; five of these were with rootstocks introduced from outside of Florida and all showing some evidence of tolerance of HLB and one of our UF scion cultivars, Valquarius. The 6th trial was a major replanting of the grove at the PSREU in Citra, to introduce new mandarin cultivars, along with better tree size-controlling rootstocks for mechanical harvesting. Finally, a new cybrid sweet orange showing HLB tolerance and with exceptional juice quality (Rhode Red Valencia nucleus + Hamlin cytoplasm – Brix: 16; Ratio: 18.44; Juice %: 54.7; Lbs. solids/box: 8.7) was discovered.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment of new scion and rootstock performance with a focus on HLB responses. Assessments of HLB field tolerance are continuously carried out in the vast collection of raw germplasm that we maintain, and new selections have been identified, and several previously found continue to hold up to HLB; additional evidence is accumulating supporting what may be differential sensitivity to HLB among sweet orange clones from the CREC program. A new set of 25 HLB+ Valencia budstick-grafted hybrid rootstocks was rotated into the ‘hot psyllid’ house, following selection based on freedom from symptoms in the greenhouse test. Several new clones were entered into the DPI Parent Tree Program, including a cybrid sweet orange with exceptional juice quality and preliminary evidence of HLB tolerance, two early maturing Valencia somaclones, and twelve new promising rootstock hybrids with preliminary evidence of HLB tolerance. A total of 383 new transgenic citrus trees with potential HLB tolerance/resistance were planted at the USDA Picos Farm site under APHIS permit. Careful observations were made at a rootstock trial in Vero Beach from where several of the UF rootstocks already approved for release have been selected; a field day is being planned to highlight these and other promising rootstocks at this location. Hybrid rootstocks grown out from the previous season’s crosses have been prepared for field planting. Seedlings grown from seed collected from more than 3 dozen new candidate rootstocks were evaluated for trueness to type, and new seeds have just been collected from additional new candidates not evaluated previously.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks that are important for broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project significantly expanded the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in citrus defense control. We originally proposed a three-year research with three specific objectives: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. However only one-year funding was provided to support our research. With this support, our effort has been focusing on Aim 1. We have cloned 15 citrus defense genes affecting SA, ET, and/or JA pathways from citrus cDNA libraries and made overexpression constructs with the full-length cDNA clones of these genes in the binary vector pBINplusARS. While our focus is on gene cloning with the one-year support, we also initiated Arabidopsis transformation and testing disease resistance of the transgenic plants for these cloned genes (Aim 2). So far we obtained T1 seeds for 10 gene overexpression constructs of the cloned citrus genes and preliminary testing of transgenic plants overexpressing CsJAR1 or CsACD1 revealed promising results for future studies. In addition, citrus transformation with 10 citrus gene overexpression constructs has been initiated (Aim 3). In addition, we continue to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project was already terminated. Our recent analysis of transgenic citrus overexpressing CsNDR1 (five out of ten transgenic plants) showed that these plants had lower rates of HLB positive three months after inoculation psyllids carrying Liberibacter asiaticus, suggesting that CsNDR1 overexpression confers enhanced HLB resistance. This result is consistent with CsNDR1 overexpression in conferring enhanced disease resistance in Arabidopsis (Lu et al., 2013). We will follow up with these plants for a further test of HLB resistance in the next few months. Lu, H., Zhang, C., Albrecht, U., Shimizu, R., Wang, G., and Bowman, K.D. (2013). Overexpression of a citrus NDR1 orthodox increases disease resistance in Arabidopsis. Front Plant Sci 4, 157.
Huanglongbing (HLB) is the most serious threat to the U.S. citrus industry. Several transcriptome studies on citrus-HLB interactions have been published in recent years that provide a large pool of HLB-responsive candidate genes. These studies have identified many genes with respect to HLB infection but there is only a modest overlap in results which could be due to multiple factors such as lab-specific or genotype-specific effects. Interrogating all the transcriptome studies through meta-analysis can provide a better understanding that cannot be gained by analyzing single studies. A combined list of 7,412 differentially expressed gene probes was generated by using a Teradata in-house SQL script. Weighted gene co-expression network analysis was conducted and 21 modules with major hub genes were identified that show the greatest number of interconnections and contribute most to citrus-HLB interaction. We have shortlisted 2,000 candidate genes based on several criteria such as their expression patterns among different datasets, putative function and position as hub genes in the co-expression gene network. These candidate genes will be validated using high throughput target capture and massively parallel sequencing of targeted gene regions among susceptible and tolerant citrus genotypes. Toward this objective, we have designed a target capture system based on the Agilent SureSelect system. Different citrus accessions and relatives were collected and tested for their responses to HLB; they revealed a wide range of responses from susceptible to tolerant and resistant. DNA from these selected accessions will be isolated and used for the SureSelect library preparation. The bait of the library will be custom designed 120mer probes at every 60bp interval specific to the shortlisted candidates. The library will be sequenced using Illumina HiSeq 2000 to rapidly identify sequence variations in the candidate genes in selected genotypes. Phenotyping of these selected accessions is continuing in the field of Florida, using parameters such as the titer of CLas, and HLB symptom severity ratings. The titer of CLas in the HLB-infected plants was determined by TaqMan probe based realtime PCR, and the HLB severity on the plants was evaluated by experienced researchers. These phenotype data continue to be collected several times every year, and correlations among these data will be assessed before use in association analysis. From the preliminary investigation, we can see a great difference in HLB tolerance among these citrus species and relatives. Many HLB-tolerant citrus relatives were revealed from the preliminary investigation, including Citrus latipes, Poncirus trifoliata, and Severinia buxifolia. This study will lead to the identification of the genes most likely associated with HLB tolerance.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, both soil drench and foliar spraying of NAD+ have been performed. In the side-by-side experiment with the plant defense activator Actogard, soil drench provided good protection against citrus canker, whereas foliar spraying had limited effects. We are repeating the experiment and trying to find the best approach for NAD+ application. We have also been testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, newly generated transgenic plants are growing in greenhouse. Presence and expression of the transgenes have been tested. All transgenic plants are growing in the greenhouse and will be tested for canker resistance. Citrus homologs of the defense genes have been cloned and sequenced. The will be used for functionality test through complementation experiment.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, both soil drench and foliar spraying of NAD+ have been performed. In the side-by-side experiment with the plant defense activator Actogard, soil drench provided good protection against citrus canker, whereas foliar spraying had limited effects. We are repeating the experiment and trying to find the best approach for NAD+ application. We have also been testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, newly generated transgenic plants are growing in greenhouse. Presence and expression of the transgenes have been tested. All transgenic plants are growing in the greenhouse and will be tested for canker resistance. Citrus homologs of the defense genes have been cloned and sequenced. The will be used for functionality test through complementation experiment.
All of the research described in the previous report is still ongoing or is still being analyzed. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is concluded and is being analyzed. A study of CiFT3 transgenic tobacco plants treated with various growth regulators has been performed and all of the data have been collected except for the flowering dates of the nontransgenic control plants that have not yet flowered. The growth hormones produced striking and individually different phenotypes in each treatment. The data includes plant height and leaf number, size, and area. The endogenous ciFT3 promoter from sweet orange was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will hopefully activate the naturally present FT3 gene in citrus. The complete construct is completed and is being tested in tobacco for a rapid test before citrus experiments are started. This research was presented at the ASHS national meeting.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications; and 3) to create transgenic citrus cultivars with new constructs containing the resistant genes. A total of 25 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants were sequenced and analyzed. We mapped the RNA-Seq data to a reference genome, C. clementina using the bioinformatics program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24,275 genes of the originally predicted genes had been found to be expressed and a total of 10,539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to be expressed. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of ESTs varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 6 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component (PC) analysis study on the expressions of six samples. The results showed that the gene expressions were significantly different in resistant vs. susceptible citrus. A total of 686 differentially expressed (DE) genes between two groups using FDR threshold of 0.1 were identified. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We performed Gene Ontology (GO) enrichment analysis of DE genes. Genes associated with beta-amyrin synthase, cycloartenol synthase and Camelliol C synthase were significantly up-regulated in the HLB tolerant citrus trees while terpene synthase genes (CiClev10014707, Ciclev10017785) were down-regulated in the tolerant citrus trees. Some PR-protein genes were significantly up-regulated in the resistant citrus trees, including several TIR-NBS-LRR genes. Many cell wall degradation-related genes, such as cellulose synthase/transferase, cellulase and expansins were up-regulated in the susceptible citrus trees. Some glucan hydrolase genes were also up-regulated in the resistant citrus trees. These genes may play important roles in symptom development. The DE genes were also enriched in two classes of RLKs, LRR-RLKs and DUF26-RLKs. We have experimentally verified the expressions of 14 up-regulated genes and 20 down-regulated genes on three HLB-tolerant ‘Jackson’ and three HLB-susceptible ‘Marsh’ trees using real time PCR. 11 of 14 up-regulated genes and 18 of 20 down-regulated genes were validated. Further characterization is underway for these differentially expressed genes and their potential roles in HLB progression. Meanwhile we are making constructs of a few selected genes for citrus transformation.
We continued to computationally analyze genomes with the goal to suggest hypotheses about molecular mechanisms of Liberibacter pathogenicity. We added the genome of Liberibacter solanacearum (CLso-ZC1, 1192 proteins) to the analysis of Liberibacter americanus (Sao Paulo, PW_SP, 983 proteins), Liberibacter asiaticus (gxpsy, psy62, A4,1109 proteins), and Liberibacter crescens: (BT-1, 1378 protiens). Similarly to other Liberibacter, solanacearum genome contains 2 prophages, but they are not arranged in tandem. One of the prophages is highly similar, in agreement with the hypothesis that prophage proteins and their homologs integrated in the Liberibacter genome may be the cause of pathogenicity. Prophages of culturable (and possibly non-pathogenic or less pathogenic) L. crescens are less similar to those of other species. Comparative analysis of the genomes identified about 70 potential pathogenicity genes. 40% of them have homologs in the prophage region. Many contain predicted signal peptides, suggesting that they are exported. Some of these proteins are not of bacterial origin, but are more similar to Eukaryotic proteins, additionally reinforcing the evidence for their hypothesized pathogenicity. Some of the highlights are mentioned here. Hypothetical protein CLIBASIA 3975 is homologous to Protein tyrosine phosphatase (PTPc), which is a Eukaryotic protein not needed in Bacteria that functions in numerous eukaryotic signalling pathways. These phosphates are frequently exploited by pathogenic bacteria for virulence. In addition, CLIBASIA 3975 possesses a signal peptide and is expected to be a secreted protein. Hypothetical protein CLIBASIA 3630 is homologous to Von Willebrand factor type A (vWFA), a mainly extracellular eukaryotic domain that functions in immune defenses. vWFA in the pilus of a Streptococcal pathogen mediates adhesion to host cells, so it is likely that this protein is one of the pathogenicity factors. CLIBASIA 3975 also has a pilus domain TadG. Hypothetical protein CLIBASIA 1935 is similar to OmlA protein from Xanthomonas axonopodis pv. citri. Since X. citri is a citrus canker pathogen and expression of OmlA is enhanced when grown on citrus leaves, this protein may be a potential virulence factor. In addition it has a predicted signal peptide, suggesting that it secreted. More distant homologs of this protein are from the ‘BLIP’ (b-Lactamase Inhibitor) Fold. There is a loop in the protein structure that is known to insert in lactamase active site and inhibit it. Similarly located loop is present in CLIBASIA 1935, suggesting that this protein may be an inhibitor or some enzyme from Citrus.
We have found that cell penetrating peptides (CPPs) can be used to deliver molecular ‘cargo’ (e.g. protein or DNA) into a variety of plant tissues, including those of citrus. However, to date, stable genetic transformation with CPPs but without the use of Agrobacterium-based DNA sequences has not been achieved in citrus. The Foundation wanted us to concentrate on this specific objective due to the considerable regulatory issues surrounding the use of such bacterial sequences in transgenic plants. Fortunately, since we began this project, there have been a number of new genetic developments. Targeted DNA modification, or ‘gene-editing’, methods such as Zinc Fingers and TALEN technology have been developed. However, the most recent and exciting developments have been with the CRISPR/Cas system. This system not only allows for very precise, targeted gene editing, but gene over-expression or repression is also possible. Further, the CRISPR/Cas system is simpler to engineer than other available systems, and less likely to have nonspecific binding or to recombine out. Finally, this methodology will satisfy the main objective, which should allow engineering of citrus without the use of inserted non-native (e.g. bacterial) DNA sequences. We hope that the CRISPR/Cas system will allow transgenic plants to be free from regulatory issues (although, the USDA still has this under consideration). This quarter, we have concentrated on designing CRISPR/Cas vectors for use with CPPs and citrus tissue. This is still underway.
Based on good field performance and superior yield with severe HLB infection pressure on the east coast, the rootstocks US-1279, US-1281, US-1282, US-1283, and US-1284 were released by USDA for commercial use. Fruit yield of Hamlin trees on these rootstocks is 2-4 times the yield of trees on Swingle in the same trials, and the trees also have fruit that is larger in size and higher in sugar content. These promising new rootstock selections have been provided to Florida DPI for establishment of certified budwood sources, are being made available to the CRDF Product Development Project to establish large scale commercial field trials, and will also be used in multiple field trials with commercial growers with funding provided by the HLB-MAC project. A special permit has been obtained from Florida DPI to immediately begin establishing widespread commercial field trials using clean USDA sources of these and other new rootstocks. Based on this permit, cooperative arrangements are being made with commercial Florida nurseries for large scale vegetative propagation of these promising new rootstocks, as needed to meet commercial demand until adequate seed sources can be developed. A material transfer agreement was signed with Agromillora Catalana, SA, to immediately allow that company to begin rapid micropropagation of these most promising rootstock selections for use in Florida. Trees were prepared for planting of three new field trials with Supersour rootstocks later in 2014. About ten thousand new propagations of Supersour rootstocks were prepared for budding and planting in additional field trials in 2015. Work began to construct an additional greenhouse at USDA to propagate Supersour rootstocks for field trials. Cooperative work continued with a commercial nursery to multiply promising Supersour rootstocks to produce trees for medium-scale commercial trials. A Valencia field trial was planted with a commercial cooperator to evaluate performance of several promising Supersour rootstock selections alongside other good commercial rootstocks. Greenhouse studies continued to assess Supersour tolerance of CTV, calcareous soils, and salinity. Trees were planted into the field to establish seed sources for the most promising Supersour selections. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility will be presented at the HLB conference in February and is being prepared for publication. This work provides considerable insight into disease progression and the potential for improved management. Studies continued on defense-related genes and small RNAs associated with HLB infection, in collaboration with University of Maryland and University of California research groups. A study of localized defense gene expression in shoots and roots provided evidence of striking differences that are a major advance in understanding and yield strong insights into ways to overcome the disease. A new field trial was planted to evaluate grafting height effect on tree tolerance to HLB. Two replicated tests with US-942 rootstocks that overexpress the citrus defense gene CtNDR1, are showing significant reduction in Las infection for some of the transformed clones. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections showing increased resistance to HLB have been identified from groups transformed with other resistance genes, and are also being prepared for confirmation testing. One hundred new transgenic US-942 and Sour orange rootstocks were produced, targeting to increase tolerance to HLB by manipulation of the citrus resistance genes CtNHL3, CtNHO1, CtDIR1, CtAZL1, CtERF1, and CtFMO1.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of September 2, 2014, a total of 6,208 transgenic plants have passed through inoculation process. A total of 122,855 bacteriliferous psyllids have been used in no-choice inoculations.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over four years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. 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 are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. Exposure to canker inoculum showed remarkabe resistance in chimera compared to control. Canker infiltration showed greatly increased resistance in two chimera AMP and several thionin transgenics, at 107CFU/ml. RNA was isolated from transgenic plants containing chimera and thionin. RT-PCR showed gene expression in the transgenic plants. Further gene expression level was evaluated with RT-qPCR. Our results showed gene expression variation between different transgenic lines, from several fold to 35 fold. Transgenic lines containing D4E1 were evaluated with Xcc infiltration. All the transgenic lines with canker development at 105 CFU/ml while some transgenic lines show less canker development at 104 CFU/ml. Bacterial growth rate in transgenic lines containing D4E1, chimera and thionin was investigated by qPCR. Our results showed some transgenic lines containing chimera and thionin had low Xcc growth rate. More transformed Hamlin carrying chimera were generated and over 30 were confirmed positive by PCR. About 20 Hamlin transformed with thionin also were obtained. They will be tested by RT-PCR and replicated for HLB challenge. Putative transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 had increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
We have found that cell penetrating peptides (CPPs) can be used to deliver molecular ‘cargo’ (e.g. protein or DNA) into a variety of plant tissues, including those of citrus. However, to date, stable genetic transformation with CPPs but without the use of Agrobacterium-based DNA sequences has not been achieved in citrus. The Foundation wanted us to concentrate on this specific objective due to the considerable regulatory issues surrounding the use of such bacterial sequences in transgenic plants. Fortunately, since we began this project, there have been a number of new genetic developments. Targeted DNA modification, or ‘gene-editing’, methods such as Zinc Fingers and TALEN technology have been developed. However, the most recent and exciting developments have been with the CRISPR/Cas system. This system not only allows for very precise, targeted gene editing, but gene over-expression or repression is also possible. Further, the CRISPR/Cas system is simpler to engineer than other available systems, and less likely to have nonspecific binding or to recombine out. Finally, this methodology will satisfy the main objective, which should allow engineering of citrus without the use of inserted non-native (e.g. bacterial) DNA sequences. We hope that the CRISPR/Cas system will allow transgenic plants to be free from regulatory issues (although, the USDA still has this under consideration). This quarter, we have concentrated on designing CRISPR/Cas vectors for use with CPPs and citrus tissue. This is still underway.
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