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.During the 2nd quarter of the third year of our grant, the Stover lab has completed production of the transgenics needed for this project. Many lines have been sent to the Rogers lab and the last few are growing well in the greenhouse and will be large enough to sent soon. The Rogers lab has continued small-scale no-choice psyllid inoculation experiments. ARS facilities are still at a maximum of 25% occupancy due to the COVID-19 pandemic; we are teleworking the remaining time. This continues to slow down progress on grant milestones. We are very much hoping to be allowed to move to the next phases of reopening soon, which will allow for much more rapid progress. In addition, the grant-funded post-doc, Dr. Tamara Collum has accepted a permanent position; her last day will be July 2, 2021. We wish her well and are glad she isn’t going far and will be available to answer questions while we hire another post-doc to continue most of the day-to-day work of this project.