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 this most recent quarter, the 2nd quarter of our 1st 6 month no cost extension (the original end date was 11/30/2021; we were granted one 6 month no cost extension and one 3 month one; the current end date is 08/31/2022), the Stover lab sent the last of the transgenic lines needed for the project to the Rogers lab. Now at least 4 high expressing lines for each of the 9 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 rooted cuttings available and ribosome pull-downs from the tissue collected. Work has progressed more quickly since the ARS 25% occupancy cap was lifted on Monday, March 28th. We have still not been able to identify a qualified and interested post-doc candidate and with less than 3 month left on the grant, now do not have time to hire someone. As many translatome RNA samples as possible will be sent for sequencing in July, leaving the month of August for data analysis.