An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific silencing and is graft transmissible to commercial citrus varieties

Authors: Krueger, Robert; CHEN, ANGEL - University Of California, Riverside; ZHOU, JACLYN - University Of California, Riverside; LIU, SI - University Of California, Riverside; XU, HUAYING - University Of California, Riverside; NG, JAMES - University Of California, Riverside

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/7/2024
Publication Date: 11/21/2024
Citation: Krueger, R., Chen, A.Y., Zhou, J.S., Liu, S., Xu, H.K., Ng, J.C. 2024. An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific silencing and is graft transmissible to commercial citrus varieties. Phytopathology. 114(11):2453-2462. https://doi.org/10.1094/PHYTO-05-24-0167-R.
DOI: https://doi.org/10.1094/PHYTO-05-24-0167-R

Interpretive Summary: Viruses infecting plants can be engineered as "vectors' capable of production and/or delivery of therapeutics such as vaccines, antimicrobials, and anticancer compounds, to targeted bioimaging and theranostics. A vector based upon a Florida isolate of Citrus tristeza virus (CTV) was developed. However, due to phytosanitary regulations, this vector could not be used in California and a vector based upon a genetically closely related isolate of CTV was developed that was capable of systemically infecting and expressing green fluorescent protein (GFP) in Nicotiana benthamiana and Citrus macrophylla seedlings. The current report documents further development of the California-derived vector that was able to induce gene silencing in the host plant. These versions of the vector also appeared to accumulate to higher levels in the host than previous versions. This report also documents the ability of these vectors to infect commercial citrus varieties grafted onto CTV-resistant and -susceptible rootstocks.

Technical Abstract: A protein-expressing citrus tristeza virus (CTV)-based vector, pT36CA-V1.3, constructed from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of T36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.