Objective 1, Mthionin Constructs:
Assessment of the Mthionin transgenic lines is ongoing. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic.
Data collection continues from the first round of field plantings (45 plants) of Mthionin transgenic Carrizo rootstock grafted with non-transgenic rough lemon. Initial results show transgenics maintaining higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. A large second planting of Mthionin transgenics went into the ground in April, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) and WT/WT controls (16 plants). The next significant data collection will be at one year in field, April 2020. Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) on transgenic rootstock are growing in the greenhouse
Additional Mthionin construct transformations have also been completed on 450 Valencia, 300 Ray Ruby, and 415 US-942 explants to provide additional transgenic material of these critical varieties.
Objective 2, Citrus Chimera Constructs:
Detached leaf assays, with CLas+ ACP feeding, have been conducted and repeated for lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, `73′, and `188′ using adjusted protocols to improve sensitivity and transmission rates (See section 4). Multiple lines from several constructs have been moved forward into greenhouse studies based on these results, as noted below.
Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many control plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All greenhouse experiments are now using an improved protocol to enhance inoculation. Through a combination of selecting smaller plants, more aggressively trimming larger plants and close observation, we have been able to extend the caged ACP infestation time from 7 days to 21 without severe mold or cage damage to the plants. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using the new protocol. At 3 months, control plants are now testing positive at twice the rate of the earlier inoculation and 6 month samples will be collected December 2019.
Plants are grafted and in the greenhouse for a field planting of ~200 `74′ and `188′ transgenics which is scheduled for spring 2020, with WT scions (Hamlin, Valencia, and Ray Ruby) on transgenic Carrizo root stocks. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT rootstocks are underway. These plants will be ready for planting fall 2020.
Seven new transformations, totaling over 3000 explants, have been completed to expand lines of Valencia, Ray Ruby, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs.
Objective 3, ScFv Constructs:
Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and, with all appropriate permitting now completed, will be moved to the field after hurricane season. An additional 129 rooted cuttings are propagated for follow up plantings.
The 3 month data from the 150 plants from the 2nd group of scFv lines (12 lines) that were initially no-choice ACP inoculated showed an insufficient infection rate. These plants have now been graft inoculated with HLB+ RL and are undergoing the first post-inoculation analysis. An additional 370 rooted cuttings were propagated for the third round of ACP-inoculations. From which, the first group of 54 plants large enough to use have been inoculated with the higher pressure 21 day protocol.
Objective 4, Screening Development and Validation:
A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.
An array of phloem specific citrus genes has been selected for investigation as potential reference genes to make comparisons focusing on phloem tissue only . Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer and potential therapeutic effects.
The best performing lines of Mthionin, chimeras `74′ and `188′ and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines) and Carrizo/Mthionin (2 lines) have been returned certified clean.
Objective 5, Transgene Characterization:
Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and His6/Flag tagged variants of scFv-InvA (22 lines) and scFv-TolC (18 lines) constructs have been generated and confirmed for transgene expression by RT-qPCR. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.