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 1st year, 1st quarter progress report; our grant started December 1, 2018. In the last three months, we have processed all the paperwork needed to establish the grant and begin spending funds at ARS. We have identified a qualified and interested post-doctoral researcher, Dr. Tamara D. Collum, who we will be hiring with grant funding. However, all ARS hiring actions, even those using soft funds, are currently on hold at the Department of Agriculture level. Now that the department has a full-year budget, we hope this hold will be lifted shortly so we can bring Tami on board in the next couple weeks. Objective 1 (development of transgenic constructs) is close to completion and work has begun in the Stover lab on objective 2 (production of transgenic citrus lines). 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) inserts have been assembled and sent to Dr. Dawson’s lab for inclusion in CTV vectors and subsequent introduction into citrus.�