METHODOLOGY: 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) have been transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; transformants expressing the tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter were also constructed as a control. We are in the process of exposing transgenic plants to CLas+ or CLas- ACP and leaves sampled 30, 60, 90, and 120 days later. Ribosome-associated mRNA is being 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. PROGRESS: During the three year and nine month grant period, our group has made at least four high-expressing lines for each of the nine promoter/genotype combinations and shipped them from the Stover lab to the Rogers lab. More than 150 rooted cuttings from these lines were exposed to CLas+ or CLas- ACP in no-choice psyllid inoculation experiments. Each rooted cutting was sampled four times, yielding more than 600 leaf samples for ribosome affinity purification and mRNA isolation. Approximately one-third of the samples have been processed and almost 60 have been sequenced. In a parallel approach, the tagged ribosomal proteins were cloned into a citrus tristeza viral expression vector. This vector and a control empty vector were moved into both Nicotiana benthamiana and Citrus macrophylla. Proof of concept experiments testing whether CTV can yield mRNA samples enriched for phloem genes appear promising. There were several issues that hindered our progress on project milestones. First, laboratory closures and occupancy caps due to the COVID-19 pandemic drastically limited personnel time in the lab and greenhouse. The Poncirus genotype is very slow-growing and the last Poncirus transgenic lines were not available until the end of year 3. The post-doc moved on to a permanent job after about 2 1/2 years on the project. In spite of extensive advertising, no suitable replacement post-doc candidate was identified. Additionally, mRNA yields from both of the citrus specific promoters were low and an ultracentrifugation step to concentrate ribosomes will be added prior to affinity purification. In summary, significant progress was made towards our goal of identifying phloem gene expression changes in response to CLas infection even though the final studies are not complete.