1. Please state project objectives and what work was done this quarter to address them:
The goal of this project is to protect citrus from Candidatus Liberibacter asiaticus by inducing the phloem to produce anchored, single-chain antibodies that will bind and immobilize the bacteria, allowing the plant to destroy them by natural defense mechanisms. Anchoring is accomplished by expressing the antibodies as extensions of native, phloem-specific sieve element occlusion (SEO) proteins.
On Oct. 16, 2023, 16 genetically transformed and 10 control Carrizo plants were shipped from Cornell to the Levy lab in Florida. The DNA constructs included the phloem-specific SEO promoter driving expression, in individual plasmids, of three different binding proteins (OmpA, CpaF, KpsF) as extensions of the native citrus SEO protein, for example, SEOp: OmpA-SEO. In controls the binding proteins were omitted. Plants were shipped with an APHIS epermit, according to USDA procedures. In Florida, the plants, which arrived in excellent condition, were retested for inserted sequences, repotted and placed in a secure greenhouse for additional growth.
Since Carrizo citrange plants, though readily transformed, can have low CLas titer (William Dawson, personal communication) it was decided to use the transformed Carrizo as rootstocks and untransformed Pineapple sweet orange (Citrus sinensis L. Osbeck) as scions. The grafted plants are now almost large enough to be tested for CLas mobility. The scions will be infected either by grafting infected tissue to them or by exposure to CLas-infected Diaphorina citri. CLas mobility will be tested by analyzing the Carrizo roots.
The Turgeon lab also synthesized SEO-anchored antibody constructs (the 3 different antibody genes above) using the strong phloem-specific rice tungro bacilliform virus (RTBV) promoter (Dutt et al., Tree Physiol. 32:83 2012) which should increase the number of binding proteins in the sieve elements. In addition, the Turgeon lab made constructs for RTBV driven dual-binding (bivalent) antibodies (OmpA-OmpA, CpaF-CpaF, KpsF-KpsF). Dual antibodies have been shown to have extremely strong binding potential (Bannas et al. Front. Immunol. 8:1603 2017). These constructs are now being used for Duncan grapefruit transformation.
2. Please state what work is anticipated for next quarter:
The Levy lab will soon begin testing the grafted trees by exposure to CLas and by grafting CLas-infected tissue to the scions. In the Turgeon lab, additional transformed trees are being produced, using Duncan grapefruit and Valencia sweet orange. The DNA constructs have been made, and transformation has begun, but the trees will not be ready for testing within the time frame of this grant. In order of priority the constructs are: 1) 35S promoter driving the three, anchored single-chain antibody constructs (these transgenics are being produced in the Levy lab); 2) RTBV promoter driving the three, anchored single-chain antibody constructs; 3) RTBV promoter driving the three, unanchored, dual-antibody constructs, and 4) 35S promoter driving the three unanchored, dual antibody constructs.
3. Please state budget status (underspend or overspend, and why):
The budget status is an anticipated with funds neither underspent nor overspent.
4. Potential commercialization products
No commercialization is expected during this grant period although we believe that if these constructs prove successful in arresting CLas, they will provide direction for the reconstruction of a CLas-free citrus industry.