Project Number: 6034-22320-007-001-A
Project Type: Cooperative Agreement
Start Date: Oct 1, 2020
End Date: Jul 31, 2024
Funds provided to cooperator are to support the following objectives: 1. Genomic sequencing and bioinformatic analysis of up to 10 Agrobacterium sp. isolates and using this data to create new PHACT vector plasmids designs. 2. Development of up to 84 different PHACT vectors for producing plant endosymbionts. 3. Development of the Vmax expression platform to identify cell lines expressing CLas effector binding nanobodies. 4. Evaluate VMax expression platform for use in producing pilot scale quantities of therapeutic at least 3 peptides.
We have identified wild Agrobacterium tumefaciens isolates from Florida & California that form durable galls on citrus and tomato. We will sequence the genomes of these strains & develop new Transfer-DNA binary vectors for delivering cell activation genes & defense genes within the T-DNA. Whole genome sequence & Ribonuceic acid RNAseq data will be obtained for all Agrobacterium strains. Through comparison to available sequence data and gene functions from literature, at least 20 vectors will be designed and tested containing various combinations of PGR biosynthetic & putative plasticity genes. Tomato and citrus specific vectors will be developed & evaluated for gall formation and GFP marker gene expression. Obj. 3. CLas and the related C. L. solanacearum (Lso) infecting potato and tomato, secrete effector proteins that modulate host immunity and are presumed to be essential for CLas to colonize citrus and cause disease1, 57-59, including those that inhibit immune-related protease activity (SDE1), suppress host cell death (HPE1)60, and suppress ROS production through peroxidase activity. PHACT delivery of nanobodies that bind to CLas effector proteins may be a promising new approach to HLB control. Nanobodies are small and would likely move readily with symplastic flow from the PHACT attachment site into the tree vascular tissue. We will use Vmax to develop an antigen-specific nanobodies selection platform, focusing nanobody discovery on six selected CLas effector proteins, which will also be expressed in Vmax. We will use a hybrid approach of isolating a cDNA library of reactive VHH nanobodies after challenging camelids or elasmobranchs with CLas effectors. To identify best effector binding nanobodies, we will perform additional refinement through cell-surface based selection on Vmax. The nanobody encoding sequences will be cloned in a surface-display compatible vector and expressed in Vmax. Cells displaying nanobodies that bind antigen with high affinity will be selected by binding the cells with biotinylated antigen and isolated using streptavidin magnetic beads and an ELISA based assay. To test whether the selected nanobodies block effector function in vivo, we will co-express the nanobody and effector protein in N. Obj. 4 Research will focus on using Vmax for production of antimicrobial peptides. Antimicrobial peptides will be over-expressed and purified by in-frame fusion with an intein55 and a suitable affinity purification tag. To maximize the production of recombinant protein in the soluble fraction, we will test different Vmax strains, purification tags, media and growth conditions. We will also attempt to secrete the fusion protein into the growth media to simplify purification by appending an appropriate peptide tag. Secretion will also assist in the seamless transition from small volume benchtop cultures to “at scale” production. Therapeutic proteins, peptides and metabolite expression in Vmax will be assessed, as appropriate, using methods standard for protein detection and quantification. Peptides extracted from PHACT-biofactories or Vmax cultures will be directly tested in a screening pipline developed at our location.