Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.
This is the first year, 3nd quarter progress report; our grant started December 1, 2018. In the last three months, the post-doctoral researcher, Tami Collum, has started optimizing nucleic acid extraction protocols for citrus. For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) Dr. Dawson’s lab has all necessary constructs and has moved many of them into citrus. CTV-infected plants will soon be ready for shipment to Maryland. Again, the majority of our efforts in the 3nd quarter were focused on objective 2 (production of transgenic citrus lines). The Stover lab has performed Agrobacterium-mediated transformation of seedling epicotyls from all three citrus genotypes indicated in the grant (Carrizo, Poncirus and Hamlin sweet orange) with the His-FLAG tagged RPL18 (ribosomal protein L18) under the 35S promoter and all three phloem promoters pSUC2, pSUL and p396ss. Carrizo transgenic plants with three promoters are already acclimatized in the greenhouse: p35S::HF-RPL18 (12 plants), pSUL::HF-RPL18 (21 plants), and p396ss::HF-RPL18 (30 plants), with many plants >25 cm and suitable for taking cuttings for replication. Seven plants transformed with each promoter were evaluated for presence (PCR) and expression (RT-qPCR) of the HF-RPL18 gene, and 100% of the plants are expressing the gene. The newly transformed Carrizo with the pSUC2 promoter has been transferred to greenhouse and will be evaluated soon. Putative transgenic plants of Poncirus harboring the 35S::HF-RPL18 (12 plants) and pSUL::HF-RPL18 (10 plants) were moved to the soil. Poncirus plants with constructions p396ss::HF-RPL18 and pSUC2::HF-RPL18 are still in rooting medium (16 and 49 plantlets, respectively). Hamlin transformation was intensified in this quarter and many shoots have being transferred to rooting media, and one plant to soil. Since Hamlin has a much lower transformation efficiency, some transformations were repeated and also cotyledons have being used as a new transformation target explant for this genotype. Carrizo plants expressing the HF-RPL18 gene will be replicated and transferred to Ft. Detrick in the next quarter.