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, 2nd quarter progress report; our grant started December 1, 2018. In the last three months, we have brought on board our post-doctoral researcher, Dr. Tamara D. Collum. Objective 1 (development of transgenic constructs) has been completed. 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 the necessary constructs and has begun moving them into citrus. The majority of our efforts in the 2nd 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 putative transgenic plants with three promoters are already rooted and established in the greenhouse; transformation was initiated with the fourth construct. Putative transgenic plants of Poncirus are also in hand, with only one construct in soil and the other three in rooting and/or shoot induction and selection media. Hamlin transformation was initiated with three constructs; explants are still in shoot induction/selection media. shoots are already evident on the shoot induction/selection medium for p35S::HF-RPL18 (35 shoots). More in vitro germinated seedlings are growing for additional Hamlin transformations. Likely the Carrizo and Poncirus transformations already created will be sufficient for this project. Since Hamlin has a much lower transformation efficiency, some transformations will likely need to be repeated in the next quarter. Gene insertion and expression will be verified for many plants in the coming months. Plans are underway for transfer of the first plants to Ft. Detrick.