DEVELOPMENT OF ALL PLANT DNA CONTAINING HLB RESISTANT SCION AND ROOTSTOCKS FOR SUSTAINABLE CITRUS PRODUCTION

Citrus is a long lived deep-rooted perennial tree (Ford 1954). This tree consists of a grafted rootstock-scion combination and proper health of both is essential for productivity. In recent years, Citrus production has been challenged by many biotic stresses. This has resulted in significant decreases in production and as such juice and fresh fruit prices have been affected. The major biotic stress currently facing the industry is Huanglongbing (HLB), caused by the phloem limited bacterium Candidatus Liberibacter asiaticus (CLas) and vectored by the Asian Citrus Psyllid (ACP). HLB is a difficult disease to manage due to the non-specific nature of its symptoms, prolonged latency period in the field, irregular distribution of the pathogen in the plant, environmental effects on symptom expression, and the fastidious nature of the bacteria (Bové 2006; Manjunath et al. 2008). When a tree is infected, the options for the growers are limited to strong insect control, removal of infected trees to reduce inoculum, and use of certified disease-free nursery plants for replanting (Albrecht and Bowman 2008). Removal of HLB-positive trees is extremely costly, reduces production capacity and fruit yield, and makes citrus production unprofitable; most growers in Florida have abandoned this management tool. Many growers now are utilizing nutritional supplements to keep mature trees alive and to maintain some degree of productivity. However, for growers who are removing trees or who wish to implement such a strategy in the future, it is difficult to diagnose infection in a timely manner as CLas has a long latent period (often several years). The asymptomatic trees are a constant source of inoculum that continues to be spread by ACP. Under such circumstances, newly planted, pathogen-free trees may quickly become infected.It is estimated that the yearly direct economic loss from declining citrus production due to Huanglongbing (HLB) continues to be over $300 million dollars (Hodges and Spreen 2012). It is thought that without an effective strategy for control, this disease will be the single most important factor to cause serious and long-lasting damage to the citrus industry in the United States. It will also affect the livelihoods of hundreds of thousands of people employed in the citrus production and related sectors. The most sustainable, cost-effective approach for the management of HLB would be the utilization of HLB-resistant citrus scion and rootstock cultivars that will not be affected, or are substantially less affected, by the disease (Gottwald 2010). Research to engineer or incorporate HLB resistance into citrus cultivars has been recommended by the National Research Council as one of the top priority topics for addressing the HLB threat.The plant defends itself from biotic attack, primarily by two methods. The first is a physical level defense through the production of barriers such as trichomes that restrict pathogen infection. The second mode of defense is through a systemic plant defense mechanism (Guan et al. 2012). This defense mechanism is through the movement of long-distance signals to and from the rootstocks to the scion through the graft union. Long-distance signals potentially involve mRNAs, small RNAs and proteins (Kehr and Buhtz 2007). Some of the proteins involved in this long-distance signal transports are the defense-related phytohormones, particularly salicylic acid (SA) and jasmonic acid (JA). SA helps to activate plant defense mechanisms against biotrophic and hemibiotrophic pathogens, whereas JA is usually involved in defense pathways against necrotrophic pathogens and herbivorous insects (Bari and Jones 2009). There is increasing evidence that systemic defense mechanisms may play an important role in plant defense as a result of grafting (Guan et al. 2012). It is known that systemic defense responses can be triggered by localized tissue damage in plants (Schilmiller and Howe 2005). Direct evidence for this was observed in apples, where gene expression in the rootstock was positively correlated with disease resistance in the scion (Jensen et al. 2012). Rootstock-regulated gene expression profiling in apple trees also revealed genes whose expression levels were associated with fire blight resistance (Jensen et al. 2011)Genetic improvement of citrus, with genes that allow plants to defend themselves against pathogens utilizing systemic acquired resistance (SAR) has resulted in the production of transgenic canker resistant (Zhang et al. 2010) and HLB tolerant trees (Dutt et al. 2015). The Arabidopsis NPR1 (AtNPR1) functions as a signal modulator of systemic acquired resistance (SAR) (Cao et al. 1997; Cao et al. 1998a; Feys and Parker 2000; Yu et al. 2001; Yuan et al. 2007). NPR1 is a key regulator in the signal transduction pathway that leads to SAR since the NPR1 mutant in Arabidopsis fails to respond to various SAR-inducing agents and exhibits very low expression of several PR genes. The NPR1 gene may act as a regulator of the transcription factors that controls PR gene expression (Kinkema et al. 2000) and mediates the salicylic acid induced expression of PR genes and SAR (Clarke et al. 1998). Plants over-expressing NPR1 exhibit enhanced resistance to several pathogens (Cao et al. 1998b). Similarly, the Nicotiana tabacum derived salicylic acid?binding protein 2 gene (SABP2) displays high affinity for salicylic acid and plays a crucial role in the activation of systemic acquired resistance to plant pathogens (Forouhar et al. 2005).In addition to the SAR process, other genes have been studied in detail that play a role in biotropic pathogen tolerance. The VvNAC1 can provide enhanced tolerance to several pathogens when overexpressed in Arabidopsis (Le Henanff et al. 2013). The tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor also resulted in enhanced resistance against pathogen attack (Park et al. 2001). There are several others described in the literature that could potentially protect citrus against HLB.Agrobacterium is commonly used to insert useful genes into citrus to produce a transgenic plant that can potentially combat HLB and other abiotic and biotic issues. However there are other methods of Agrobacterium free genetic modification that has been utilized in citrus: protoplast based (Fleming et al. 2000) and biolistics based (Wu et al. 2016) for example. Additionally, the current transgenic technologies utilize promoter sequences of viral and bacterial origin, and marker genes including the commonly used GUS system and GFP (green fluorescent protein from jellyfish). This project will utilize both Agrobacterium and protoplast transformation technologies to develop a transgenic citrus that contains plant derived genetic elements (promoter, selectable marker, reporter gene and gene of interest) for HLB management.

Objective 1: Develop and test all plant DNA containing plant transformation constructs 1.1 Utilization of suitable DNA sequences for the development of plant transformation test vectors: Numerous citrus derived constitutive and phloem specific promoters have been identified, from the Phytozome citrus database and cloned from Citrus sinensis "Hamlin" and Citrus clementina 'Nules' genomic DNA. These promoters have been cloned upstream of a gus gene in the pCAMBIA derived binary vector pC2300-GUS. Similarly, a number of phloem specific promoter sequences from a PP2-type gene have been identified and isolated from Citrus sinensis 'Valencia'. Each sequence will be placed in various expression vectors that contain reporter markers such as the anthocyanin overexpressing VvMybA1 gene or the β-glucuronidase (GUS) genes, and the resulting vectors will be used in transformation and regeneration of GM citrus plants for expression analysis. Additionally, the anthocyanin overexpressing RUBY cDNA driven by a citrus derived embryo specific 6105 promoter will be produced.1.2 Development of a rapid gene evaluation system in citrus: The Agrobacterium transformation system is used as a routine plant transformation tool, even for difficult to transform citrus cultivars. This has enabled the production and testing of thousands of transgenic gene insertions, called "events" in a wide range of citrus cultivars. We are developing a robust juvenile tissue transformation technique for a precocious citrus cultivar called the 'Hong Kong' kumquat.This will enable rapid evaluation of transgenic constructs.1.3 Utilize the protoplast transformation system to deliver all plant DNA construct(s) into embryogenic callus: The protoplast transformation system has the potential to transform citrus with linear DNA sequences, but low efficiency of this technique is still a major concern. The current PEG mediated system is inefficient, and we will attempt to make improvements to the PEG mediated transformation system by modifying the various parameters necessary for successful PEG mediated transformation. In addition, other additional transformation systems will be evaluated. Nanoparticles can potentially aid to address the problem of poor transformation efficiency by protecting the DNA from the cellular DNAses and enhancing the DNA delivery into the nucleus. Different commercially available cationic lipid transfection agents will be used in this study.Various concentrations of plasmid DNA (1µg/µL) and cationic lipid transfection agents will be tested. The DNA-lipid complexes that form from the mixing of the two will be added in wells containing protoplasts, with approximately 150 µL of complex formulation in each well. After the transfection, cells will be maintained in dark at 28º C. Cell density will be calculated after 48 hours of treating the protoplasts with transfection reagent. Cells will be observed for cell health and rate of cell division between 3-10 days after transfection. Initial experiments to optimize protocol will be done using EGFP expressing plasmid DNA. An optimized protocol will be used to insert linear DNA pieces into the citrus genome (Obj. 3).Objective 2: Identify and utilize promising disease resistance inducing genetic sequences in citrus2.1 Develop single and stacked citrus transformation constructs that can further enhance the SAR process: In addition to the AtNPR1 and the NtSABP2 that have been well evaluated in our program, there are other genes involved in the SAR process and may aid to enhance the SAR process . Citrus is a long-lived perennial and it is indeed possible that in the long term, the Clas bacterium may be able to overcome the effects of a single transgene product. However, this can be attenuated by the stacking of two or more genes to act as a backup in case one fails. We will evaluate other SAR inducing genes either singly or stacked with either AtNPR1 or NtSABP2. Some of the genes that have been identified in our program as potential candidates include: DIR1 (a putative apoplastic lipid transfer protein), OBF5 (Encodes a basic leucine zipper (B-ZIP) containing protein that interacts with NPR1 to promote expression of salicylic acid induced genes), AZI1 (involved in the priming of salicylic acid induction and systemic immunity triggered by pathogen or azelaic acid) and SARD1, a protein that is required for activation of SA production as well as other defense responses. SAR induction will be monitored through the evaluation of PR1 and PR2 gene expression.2.2 Test other putative disease resistance inducing sequences from heterologous species: Several other genes such as the VvNAC1 or the NtTSI1 will be cloned in our plant transformation vector and inserted into commercially important rootstock and scion cultivars. PCR positive transgenic lines will be tested for HLB tolerance as outlined in Obj. 3.2.3 Identify putative disease resistance inducing sequences from the citrus genome and test their ability to protect citrus from HLB: We will utilize the publicly available citrus genome databases, as well as public citrus expressed sequence tags (ESTs) databases and the plant disease resistance gene database (PRGdb), to identify suitable homologs and transcription factors. The ORFsand regulatory elements and motifs, along with the genes will be determined by prediction programs. All potential genetic elements will be amplified from citrus cDNA, cloned, sequenced and incorporated into transformation vectors for insertion into citrus for testing. Constructs that have been produced for testing will subsequently be incorporated into the 'Hamlin', 'EV1' and 'EV2' sweet orange cultivars. Trees expressing high levels of the transprotein(s) (as determined by Western Blot) will be cloned, tested for HLB resistance in the greenhouse. Selected lines will be planted in the field for further evaluation.Objective 3: Produce and test all plant DNA containing GM citrus trees for HLB resistanceEmphasis will be given to transform scion and rootstock cultivars developed by the UF citrus breeding team, in addition to the most important conventional commercial varieties. DNA sequences will be inserted into citrus cells using our optimized protoplast transformation system. Linear DNA produced either through PCR amplification or through restriction digest of plasmid DNA will be utilized and protoplasts will be transformed with two sets of constructs. For visual selection of GM cells, a linear construct will contain a visual anthocyanin expressing reporter Ruby marker under the control of either the carrot Dc3 or the citrus 6105 promoter and terminator. The second will contain our gene construct(s) - either single or stacked under the control of either a strong constitutive or a phloem specific promoter and terminator sequences. Transgene(s) integration will be detected primarily by PCR. RT-qPCR will be carried out to select lines expressing high levels of the trans-protein and confirmed using western blot. Selected lines will be cloned for greenhouse / field resistance studies. Replicated greenhouse studies involving a 1 to 2 weeks no-choice exposure to CLas infected psyllids will be carried out. Subsequently the plants will be periodically checked for the presence of Clas by qPCR. SAR induction will be monitored through the evaluation of PR1 and PR2 expression. Once a potential HLB resistance candidate(s) is identified, permit for field planting will be applied for large scale field tests. Approximately 8-12 clones of each scion line will be grafted onto standard rootstock such as Kuharske citrange and planted in a randomized block design. The GM rootstocks will be budded with non-transgenic 'Valencia' sweet orange and planted. Leaf samples will be collected on a semi-annual basis and processed for qPCR.