Dataset: Infectivity assays and ultrastructural characterization of Thalassia testudinum agroinfected with Turtle grass virus X (TGVX) in laboratory aquaria, February-May 2024

Site
Study sites were located in Tampa Bay, Florida, USA. The viral genome was sourced from naturally infected Thalassia testudinum at Terra Ceia Aquatic Preserve (27.5838°N, 82.6161°W), a shallow coastal seagrass meadow. Experimental host plants were collected from Lassing Park in St. Petersburg (27.7538°N, 82.6281°W), another nearshore seagrass habitat.

Sample Collection and Preparation
Young Thalassia testudinum plants were collected from Lassing Park, St. Petersburg, Florida, USA (27.7538°N, 82.6281°W) on March 4th, 2024. Collected specimens were transported in ambient seawater to the Knight Oceanographic Research Center Aquarium (University of South Florida), transplanted into 6-cm diameter pots with beach sand, and maintained in 10-liter aquaria. Artificial seawater was prepared using Instant Ocean at a salinity of 30 PSU (±0.5). The aquaria system was maintained at 30°C (±0.5) with constant aeration and a 16:8 light:dark photoperiod.

Prior to agroinfection, plants were screened for potexviruses using multiplex RT-PCR with specific primers targeting the TGVX coat protein (MBL5: 5′-CACAGATGAAGAGCTGACC-3′ and MBL6: 5′-TTCGATGAAGTAAGTGGCGG-3′) and degenerate primers for the potexvirus replicase gene (Potex-5/Potex-2RC), as described by van der Vlugt and Berendsen (2002). Mitochondrial nad5 gene primers (nad5-F/nad5-R; Menzel et al. 2002) were included as internal control.

Viral Source and Clone Construction
TGVX genomic RNA was extracted from naturally infected T. testudinum leaves collected at Terra Ceia Aquatic Preserve, Tampa Bay, Florida (27.5838°N, 82.6161°W) on February 5th, 2024 (Van Bogaert et al. 2019). Viral RNA was isolated following the protocol of Sánchez-Navarro et al. (2013) and amplified using the SuperScript IV One-Step RT-PCR System (Invitrogen) with genome-specific primers (MBL1 and MBL2) designed based on the full TGVX genome (GenBank accession MH077559; Van Bogaert et al. 2019). The full-length cDNA (6.3 kb) was assembled into a pLX mini binary vector (Pasin et al. 2018) using a BsmBI-based directional cloning strategy. Resulting constructs were transformed into Escherichia coli DH5α, screened by colony PCR, and validated via whole-plasmid sequencing with Oxford Nanopore technology.

Agroinfection Assays
The pLX-TGVX plasmid and an empty vector (mock control) were transformed into Agrobacterium tumefaciens strain C58C1. Bacterial suspensions were prepared in induction buffer composed of 10 mM MES (pH 5.6), 10 mM MgCl₂, and 200 µM acetosyringone, and adjusted to an optical density of 0.6 at 600 nm. Plants were agroinfiltrated on the abaxial side of two leaves using a needleless syringe. At 17 days post-infiltration, systemic infection was confirmed by RT-PCR detection of the TGVX coat protein from upper, non-inoculated leaves.

Experimental treatments were categorized as mock-inoculated, pre-inoculation (baseline control), and post-inoculation (TGVX-infected).

Microscopy and Symptom Analysis
Phenotypic changes were documented using photography, capturing leaf deformation, curling, and wrinkling in both infected and control plants.

Transmission electron microscopy (TEM) was used to visualize virus-like particles and evaluate cytopathological effects. Leaf tissue was processed using the leaf-dip method and standard fixation, embedding, and ultrathin sectioning protocols (Brandes and Wetter 1959; Alvarado et al. 2019). Observed abnormalities included chloroplast swelling, loss of thylakoid grana, formation of replication organelles, and laminar aggregates, consistent with potexvirus infections.

TEM analysis was performed on both published and unpublished samples to support figure-based interpretation as well as broader ultrastructural screening.

Image Data and Inventory

The dataset includes three categories of image data:

   1. RT-PCR gel electrophoresis images: Bands confirming presence/absence of TGVX coat protein and potexvirus replicase.

   2. Phenotypic photographs: Images showing visible plant morphology differences between mock and infected conditions.

   3. TEM images: Visual evidence of cytopathology caused by TGVX infection, including filamentous virus-like particles, swollen or disorganized chloroplasts, reduced thylakoid grana, membrane-bound replication organelles, and laminar aggregates.

Each image file is cataloged in the file inventory table (file_inventory.csv), which includes:

   - Filename and folder name

   - Experimental treatment group (mock, pre-inoculation, post-inoculation)

   - Image category (RT-PCR, phenotype, TEM)

   - Short description of observed features

   - Scale bar (when applicable)

   - Publication status 

The TEM component of this dataset is complete and includes all ultrastructural images generated during the agroinfection experiments. RT-PCR and phenotype images correspond to figures published in the associated manuscript, while some TEM images remain unpublished but are provided here to support broader reuse and interpretation.

Terms and parameter definitions (used in files within TGVX_agroinfection_turtlegrass_files.zip):

Plant_ID, "Unique identifier assigned to each individual Thalassia testudinum plant used in the experiment", unitless, NA
Treatment, "Type of inoculation applied to the plant; pLX-TGVX = virus clone treatment, Mock = empty vector control", unitless, NA
Days_Post_Agroinfection, "Time elapsed since agroinfiltration was performed on the plant", days, N/A
TGVX_RT-PCR_Result, "Detection of TGVX RNA in plant tissues using coat protein-specific primers; Positive = viral RNA detected, Negative = not detected", unitless, NA
Infection_Symptoms, "Visual symptoms associated with viral infection; Yes = symptoms present, No = no symptoms", unitless, NA
VLP_Observed_TEM, "Presence of virus-like particles as observed by transmission electron microscopy; Yes = VLPs seen, No = not observed", unitless, NA
Chloroplast_Alterations, "Presence of cytopathological changes in chloroplasts due to viral infection; Yes = observed, No = not observed", unitless, NA
RO_Presence, "Detection of viral replication organelles in tissue sections via TEM; Yes = present, No = absent", unitless, NA
Grana_Reduction_Level, "Qualitative classification of thylakoid grana reduction in infected chloroplasts; None, Moderate, or Severe", unitless, NA

Organism identifiers (name, Life Science Identifier (LSID), ncbi_txid):
Thalassia testudinum, urn:lsid:marinespecies.org:taxname:374720, NCBI:txid55497
Agrobacterium tumefaciens, urn:lsid:marinespecies.org:taxname:1501465, NCBI:txid358
"Escherichia coli DH5[alpha]", , NCBI:txid668369
"Turtle grass virus X", , NCBI:txid2292642