Location: Subtropical Plant Pathology Research
Project Number: 6034-22000-045-031-A
Project Type: Cooperative Agreement
Start Date: Aug 11, 2025
End Date: Nov 30, 2027
Objective:
Objective 4: Developing an expression vector of NMV-M/CFL with antimicrobial peptides and/or siRNA.
Approach:
1). The co-operator will clone the full-length stable antimicrobial peptide (SAMP) gene under the promoter of the coat protein, which is expected to produce the protein to a high level. GFP will be cloned in the same way as a control to monitor the level of expression and the systemic movement. They will evaluate the expression of the inserts in N. benthamiana plants both at RNA level using RT-qPCR and at protein level using Western blot analysis with antibodies against the SAMP peptide and GFP. Analysis to check whether the inserts are retained during replication as well as encapsulated in the viral capsid will also be performed. If successfully constructed, these expression constructs will be transferred to citrus plants to check their infectivity behavior again (replication rate, movement in plants and expression of insert sequences). After successful expression of the antimicrobial peptide or siRNA in citrus, we will evaluate their effects against Las or psyllids in a control greenhouse using graft-based inoculation.
2). In addition to expressing proteins such as antimicrobial peptides and GFP, NMV-M/CFL will also be an excellent vector for expressing small interfering RNAs (siRNA). We will utilize the NMV-M/CFL vector to express hairpin RNAs that target negative regulators of citrus immune responses. The hairpin RNAs are excellent triggers for siRNA generation. The cloning strategies are similar to that for expressing SAMP, but instead of using the full-length cDNA sequence, we will use 200-400 nt hairpin sequences of the target genes, such as VAD and DMR6. To avoid potential off-target side effects, we will choose the regions outside of the conserved domains. The recombinant viral vectors will also be tested first in N. benthamiana to examine the efficiency of siRNA formation, and then will be introduced into citrus plants for HLB resistance testing.