1a. Objectives (from AD-416):
To produce citrus scion genotypes which are resistant to huanglongbing and citrus canker through use of transgenic approaches.
1b. Approach (from AD-416):
Both constitutive and phloem-specific promoters are being used. In the near term considerable effort will continue on antimicrobial peptides (AMP), using genes of synthetic or plant origin which are effective in-vitro against Xanthomonas citri pv. citri and bacteria related to Liberibacter. To enhance AMP activity we will test peptides that include a component with high affinity for the Liberibacter membrane, attached to a potent AMP, and expected to be effective at lower concentrations. Promoter sequences, message stabilizers, and transcription enhancers will all be explored to enhance expression of key transgenes. Canker resistant transgenics should be possible using solely citrus genes, fast-tracking deregulation for cis-genics by using the native promoter. Transgenic plants will be tested for resistance in the laboratory, greenhouse, and field. We also will continue to reach out broadly within the research community to identify other transgenes that merit inclusion. Expression of peach wild-type "dormancy associated MADS box" in Hamlin should induce deciduous behavior, reducing the CLas load annually by dropping leaves, altering phloem physiology during dormancy, and synchronizing flushing for better Asian citrus psyllid (ACP) control. A unique ACP-supporting Poncirus may reveal genes for ACP resistance.
3. Progress Report:
This project is related to Objective 1: Create new genetic combinations of citrus, Objective 2: Screen germplasm for important traits and select superior individuals, Sub objective 1 D: Create new scions and rootstocks with potential resistance to huanglongbing (HLB) and Citrus Bacterial Canker (CBC) by genetic transformation. Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNA interference (RNAi)(to test possible reduction in vascular blockage even when Candidatus Liberibacter (CLas) is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 30 putative transgenic plants with citGRP1 were transferred to soil. They will soon be ready for RNA isolation and Real-time polymerase chain reaction (RT-PCR) to check gene expression. About 10 transgenic Hamlin shoots with citGRP2 are in the rooting medium for rooting. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting.In addition numerous putative transformants are present on the selective media. A chimeral construct that should enhance AMP effectiveness was completed and used to transform Hamlin. Some kanamycin-resistant shoots have already been obtained and rooted. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and 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 will be use to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Other targets identified in genomic analyses are also being pursued. 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 queued for testing with no-choice ACP and then free-flying ACP infection.