1. Please state project objectives and what work was done this quarter to address them:Year-1 Generate CTV infectious clones that express different FT3s or downregulate negative regulators of flowering to inoculate into Citrus macrophylla. Prepare different citrus genotypes for inoculation with the generated CTV vectors.Our focus in the first quarter of this project was to generate the necessary CTV vectors to induce efficient early flowering in citrus1- Generate CTV expression vectors needed to induce efficient flowering in citrusFT3 cloning and sequence analysis:In previous work, we selected Citrus clementia FT3 gene to express from the CTV vector. In our current work, we selected to amplify the FT3 gene from Hamlin, an early flowering sweet orange variety. We worked with flower and mature leaf tissue and found that the RT-PCR amplification from mature leaves revealed a very weak band whereas amplification from flower sepals revealed a strong band. Thus, we used the RT-PCR product from flower sepals to clone FT3 into 3 different positions within the CTV vector. The different positions of insertion within the CTV vector will enable differential expression of the FT3 protein. We successfully generated vectors that express Hamlin FT3 gene as an insertion between CPm and CP, replacement of p13, and insertion between p23-3NTR. Sequence analysis revealed a consensus sequence among the different plasmids with minor variations in two plasmids. When compared with the Citrus sinensis FT3 genes theory sequence reported in the National Center for Biotechnology Information (NCBI), the cloned product was a hybrid between the two theory sequences. The variation in the FT3 gene cloned into the different CTV vector plasmids could produce variable efficiency in flower induction in citrus. We selected two divergent sequence as well as 6 consensus sequence vectors for amplification of virions in Nicotiana benthamiana before infecting into citrus.FT3 gene from Arabidopsis thaliana is robust in its ability to induce early flowering in different crop species including perennial hosts. Thus, A. thaliana FT3 gene was amplified by RT-PCR from the mature leaves of A. thaliana and cloned into the CTV vector in the position between CPm and CP. We have sequenced 6 CTV vector plasmids carrying A. thaliana FT3 ORF. Sequence analysis of the plasmids revealed 100% homology to A. thaliana FT3 ORF sequence (NCBI reference # AB027504.1).2- Generate CTV RNA interference (RNAi) vectors to depress targeted genesThere is a complex process to control flowering in plants, especially perennials. Environmental cues are perceived through receptors that instigate a complex cascade of reactions to prevent flowering and, at other times, promote flowering. They involve transcription factors, timing of gene expression level through microRNA, post translational processing, movement through the plant vascular system, and protein competition for the same ligand. As the silencing signal is mobile, to make citrus seedlings more conducive to flowering, we are directing citrus plant RNAi machinery to downregulate citrus genes that repress flowering. We generated CTV RNAi vectors to target:A. The Terminal Flower Locus (TFL) from C. sinensis. TFL is a suppressor of flowering and competes with FT3 for the FD transcription factor. TFLs from different citrus species and relatives have high homology. Thus, the same CTV RNAi vector could prime the plant RNAi machinery to downregulate TFLs of different citrus species and wild relatives.B. The APETALA 2 (AP-2) transcription factor is an activator of flower repressing genes and a repressor of flower activating genes. Thus, downregulation of AP-2 should make citrus seedlings more conducive to flowering.C. A temperature dependent interaction between FT3 and a membrane bound phosphatidylglycerol (PG) occurs that prevents its uploading into the phloem sieve tube, limiting its movement into the apical meristem where it induces flowering. We targeted the phosphatidylglycerol phosphate synthase 1 for downregulation by CTV RNAi vector to limit the effect of temperature on flower induction.D. Plasmodesmata are channels of communications between adjacent cells; however, they limit protein movement based on size. We are targeting two genes to increase size exclusion limit-1 and size exclusion limit-2 for downregulation by CTV vector either independently or together (3 vectors in total). ISEs from different citrus species and relatives have high homology. The aim is to improve the amount of FT3 loaded into the phloem which should help induce flowering more efficiently.3- Sequestration of microRNAMicroRNAs are noncoding RNAs transcribed by plants to control level and timing of mRNA of a set of genes. Plants also produce microRNA analogues which sequester microRNAs, preventing the mRNA of the targeted gene from slicing by the RNA-induced silencing complex (RISC). We are mimicking plant interventions against microRNA 156 and 157, which target inducers of flowering, by sequestering them through expression of analogues from the CTV vector. We have produced two CTV expression vectors which target sequestration of microRNA 156 alone or microRNA 156 and microRNA157 together.4- Propagation of transgenic rootstocksCTV vectors can accommodate foreign inserts for many years. However, in a few cases the foreign inserted sequences affect the replication or movement of CTV vector virions within the plant. If the Citrus or Arabidopsis FT3 open reading frame or protein affect CTV replication or movement, we are working on alternative approach to produce an efficient system that induces flowering in citrus by combining transgenic Carrizo rootstocks expressing FT3 (Soares et al., 2020) with CTV RNAi vectors to induce efficient flowering in citrus scions. For this purpose, we are rooting transgenic Carrizo rootstocks, which will be subsequently topped with different citrus scions, especially sweet orange scions.5- Propagation of different citrus genotypesWe planted seeds from different citrus genotypes, including Duncan grapefruit, Madam vinous sweet orange and Pineapple Sweet orange to be available for future bioassays.2. Please state what work is anticipated for next quarter:During the next quarter we aim to infect the different CTV vectors generated into C. macrophylla and test for stability of the vectors in citrus3. Please state budget status (underspend or overspend, and why):We are underspending because one person took a new job during the 1st quarter and we are planning to hire a replacement.