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 3rd quarter of the second year of our grant, the Stover lab continues producing transgenic plants and the Rogers lab continues to propagate the transgenic lines. However, currently ARS employees have been ordered to maximize telework due to the COVID-19 pandemic. This has continued to slow down progress on the grant milestones. Still, this quarter 72 new transgenic plants were evaluated for transgene expression levels in the Stover lab. A number of high expressor were identified. There are now 20 Hamlin transgenic lines, 2 Poncirus lines, and 7 Carrizo lines ready for shipment to the Rogers lab once both labs are ready to reopen. The Rogers lab now has a CLas infected colony of Asian citrus psyllid and is ready to start exposing transgenic lines. We are very much hoping to be allowed to move to the next phase of reopening soon, which will allow for citrus shipments and CLas infections of transgenic lines.