Over Expression of a β-1,3-Glucanase Gene in Transgenic Citrus in Efforts to Inhibit Phloem Plugging Caused by Citrus Greening Disease (Huanglongbing)

Citrus greening, also called Huanglongbing (HLB) or yellow dragon disease, is considered the most serious diseases of citrus. HLB, caused by Candidatus Liberibacter asiaticus, is a phloem-limited fastidious pathogen transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The main dehabilitating symptom of the disease is the blocking of the phloem by β-1,3-glucan callose, which leads to inhibition of photosynthate transport and starvation.  Transgenic approaches to achieve over-expression of the citrus β-1,3-glucanase gene using constitutive and phloem specific promoters were performed in efforts to counteract this effect. Citrus β-1,3-glucanase cDNA (1011 bp) (GenBank accession number AJ000081) was amplified from Valencia leaf and embryogenic callus using PCR with adding cMyc tag (to facilitate subsequent western analysis). The final PCR product (1059 bp, designated BG3) was purified, and following multiple sub-cloning ended in transformation vectors designated as p35SBG3 (constitutive promoter) and pSuc2BG3 (phloem specific promoter). Both plasmids contain GFP/NPTII fusion gene as a selectable marker and were transformed into Agrobacterium. Using a modified Agrobacterium-mediated transformation protocol, 40 transgenic sweet orange (Valencia and Vernia) shoots were regenerated containing p35SBG3 and 24 containing pSuc2BG3 to over-express the citrus β-1,3-glucanase gene. All have been micro-grafted to Carrizo citrange to expedite their greenhouse evaluation.  Twenty transgenic Carrizo citrange (rootstock) shoots were also recovered containing p35SBG3. More than 200 GFP positive somatic embryos were recovered from experimental sweet orange OLL#20 using an embryogenic callus transformation system with the β-1,3-glucanase gene. From these, more than 100 transgenic shoots were transferred to rooting medium and then micro-grafted. Also, about 50 GFP positive embryos and shoots from Jin Cheng and Valencia sweet oranges were recovered.  Regeneration of stable transgenic plants from these cultivars is underway.  PCR analysis on a subset of these transgenic plants revealed that 90% are showing the specific band for the BG3 gene. All recovered transgenic clones were micropropagated, producing 4–5 replicates of each clone for further testing, including the HLB challenge. A population of these transgenic plants containing BG3 will be moved to a ‘hot psyllid’ greenhouse for natural psyllid inoculation with HLB to determine if infected transgenic plants can maintain adequate functional phloem and normal development as compared to non-transgenic/non-infected control plants.  Molecular analyses for these plants including Southern and western blot analyses and RT-PCR are underway and results will be provided.