Location: Subtropical Plant Pathology Research
Project Number: 6034-22000-045-032-A
Project Type: Cooperative Agreement
Start Date: May 1, 2026
End Date: Nov 30, 2027
Objective:
Objective 1: Characterizing the NMV-M/CFL (NMV) citrus isolate, including its host range and transmission. and Objective 4: Developing an expression vector of NMV-M/CFL with antimicrobial peptides and/or siRNA.
Approach:
1). Revealing the distribution in planta and subcellular localization of the NMV-M/CFL with and without Las co-infection:
For visualization of the virus and Las co-infection or the virus alone at subcellular location, we will use fluorescence in situ hybridization with virus-specific and Las-specific probes, respectively, and using transmission electron microscopy as described by Hilf et al., 2013 [28]. Since the virus infection attenuates the damage of phloem and vascular system by suppressing HLB symptoms, we will also examine the healthy state of citrus vascular system that is co-infected with Las and the virus using above-mentioned methods. Although we have some preliminary data indicated that NMV-M/CFL was likely systemically distributed in citrus plants, we will use RT-qPCR to investigate its quantitative distribution in different tissues, including new flush, bark, roots, and fruits.
2). 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.