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 30, 60, 90, and 120 days 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 1st quarter of our 1st 6 month no cost extension (the original end date was 11/30/2021; the current end date is 05/31/2022), the Stover lab has finally identified several high expressing lines of the p396 phloem-specific promoter in Poncirus. These will be shipped to the Rogers lab shortly, as soon as their BRS transgenic movement permit is renewed. APHIS has changed the on-line interface for applying for BRS movement permits and renewals are much delayed. At least 4 high expressing lines for each of the other 8 promoter/genotype combinations are in the containment greenhouse facility at Ft. Detrick and are being prepared for no-choice psyllid inoculation experiments.The Rogers lab has continued no-choice psyllid inoculation experiments on the many lines they have received from the Stover lab and continued ribosome pull-downs from the tissue collected. ARS facilities are still at a maximum of 25% occupancy due to the COVID-19 pandemic; we are teleworking the remaining time. This 25% occupancy cap will be lifted on Monday, March 28th, as long as the CDC community COVID metrics are in the ‘low’ category (which they are currently for Frederick County, MD). This will allow more rapid progress on grant milestones. Additionally, we are hopeful that the relaxed restrictions on in-person work will make our facility a more attractive place to work for potential post-doc candidates. Hiring a post-doc to help on the project will also allow more rapid progress on grant milestones.