@article{shymanovich_saville_mohammad_wei_rasmussen_lahre_rotenberg_whitfield_ristaino_2024, title={Disease Progress and Detection of a California Resistance-Breaking Strain of Tomato Spotted Wilt Virus in Tomato with LAMP and CRISPR-Cas12a Assays}, volume={4}, ISSN={2690-5442}, url={http://dx.doi.org/10.1094/PHYTOFR-05-23-0058-FI}, DOI={10.1094/PHYTOFR-05-23-0058-FI}, abstractNote={Use of tomato cultivars with the Sw-5 resistance gene cluster has led to the occurrence of resistance-breaking (RB) tomato spotted wilt virus (TSWV) strains globally, including California and, recently, North Carolina and Texas. We documented disease on tomato infected with either an RB strain from California (CA-RB) or a wild type (CA-WT) strain of TSWV on tomato with (cultivar Mountain Merit) or without (cultivar Mountain Fresh Plus) the Sw-5b resistance gene and detected virus incidence over time using microneedle RNA extractions and LAMP. We developed a LAMP/Cas12a assay for detection of the CA-C118Y mutation in a CA-RB strain and tested the assay with field samples. Disease in the susceptible cultivar was less severe with CA-RB than with the CA-WT strain. In contrast, the resistant cultivar had little disease when inoculated with the CA-WT strain but exhibited stunting of greater than 50% when inoculated with the CA-RB strain. In the susceptible tomatoes, the detection rates over time by LAMP reaction were higher in CA-WT than in CA-RB-inoculated plants. In resistant tomato, CA-RB remained detectable by TSWV LAMP over 14 days, whereas the WT strain was undetectable. A two-step LAMP/Cas12a assay differentiated the two strains in 1 h. Our methods were validated with samples from TSWV-infected North Carolina fields. A phylogeny of NSm gene sequences that included North Carolina field samples revealed two independent origins of the North Carolina RB isolates. The LAMP/Cas12 assay showed excellent detection of the CA-C118Y mutation. The TSWV LAMP/Cas12a assay is adaptable for in-field applications on either a smart phone platform or heat block. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .}, number={1}, journal={PhytoFrontiers™}, publisher={Scientific Societies}, author={Shymanovich, Tatsiana and Saville, Amanda C. and Mohammad, Noor and Wei, Qingshan and Rasmussen, David and Lahre, Kirsten A. and Rotenberg, Dorith and Whitfield, Anna E. and Ristaino, Jean Beagle}, year={2024}, month={Mar}, pages={50–60} }
 @article{oliver_rotenberg_agosto-shaw_mcinnes_lahre_mulot_adkins_whitfield_2024, title={Multigenic Hairpin Transgenes in Tomato Confer Resistance to Multiple Orthotospoviruses Including Sw-5 Resistance-Breaking Tomato Spotted Wilt Virus}, volume={114}, ISSN={0031-949X 1943-7684}, url={http://dx.doi.org/10.1094/PHYTO-07-23-0256-KC}, DOI={10.1094/PHYTO-07-23-0256-KC}, abstractNote={Tomato spotted wilt virus (TSWV) and related thrips-borne orthotospoviruses are a threat to food and ornamental crops. Orthotospoviruses have the capacity for rapid genetic change by genome segment reassortment and mutation. Genetic resistance is one of the most effective strategies for managing orthotospoviruses, but there are multiple examples of resistance gene breakdown. Our goal was to develop effective multigenic, broad-spectrum resistance to TSWV and other orthotospoviruses. The most conserved sequences for each open reading frame (ORF) of the TSWV genome were identified, and comparison with other orthotospoviruses revealed sequence conservation within virus clades; some overlapped with domains with well-documented biological functions. We made six hairpin constructs, each of which incorporated sequences matching portions of all five ORFs. Tomato plants expressing the hairpin transgene were challenged with TSWV by thrips and leaf-rub inoculation, and four constructs provided strong protection against TSWV in foliage and fruit. To determine if the hairpin constructs provided protection against other emerging orthotospoviruses, we challenged the plants with tomato chlorotic spot virus and resistance-breaking TSWV and found that the same constructs also provided resistance to these related viruses. Antiviral hairpin constructs are an effective way to protect plants from multiple orthotospoviruses and are an important strategy in the fight against resistance-breaking TSWV and emerging viruses. Targeting of all five viral ORFs is expected to increase the durability of resistance, and combining them with other resistance genes could further extend the utility of this disease control strategy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.}, number={5}, journal={Phytopathology®}, publisher={Scientific Societies}, author={Oliver, Jonathan E. and Rotenberg, Dorith and Agosto-Shaw, Karolyn and McInnes, Holly A. and Lahre, Kirsten A. and Mulot, Michaël and Adkins, Scott and Whitfield, Anna E.}, year={2024}, month={May}, pages={1137–1149} }
 @article{lahre_shekasteband_meadows_whitfield_rotenberg_2023, title={First Report of Resistance-Breaking Variants of Tomato Spotted Wilt Virus (TSWV) Infecting Tomatoes with the <i>Sw-5</i> Resistance Gene in North Carolina}, volume={107}, ISSN={0191-2917 1943-7692}, url={http://dx.doi.org/10.1094/PDIS-11-22-2637-PDN}, DOI={10.1094/PDIS-11-22-2637-PDN}, abstractNote={Widespread use of tomato cultivars with the Sw-5 resistance gene has led to the emergence of resistance-breaking (RB) strains of tomato spotted wilt virus across the globe. In June of 2022, tomato spotted wilt (TSW) symptoms were observed at two farms (A and B, within 15 miles of each other) in Rowan County, NC on several commercial TSW resistant tomato cultivars (all heterozygous for the Sw-5 gene). At farm A, ~10% of plants had symptomatic foliage with ~30% of fruit with symptoms, while at farm B, up to 50% of plants had symptomatic foliage with ~80% of fruit with symptoms. Visual symptoms included stunting, severe leaf curling and bronzing, necrotic lesions on leaves, petioles and stems, and concentric ring spots on fruit (Supplementary Fig. 1). TSWV ImmunoStrips (AgDia, Elkhart, IN) and reverse-transcription (RT)-PCR with NSm primers (di Rienzo et al 2018) confirmed the presence of TSWV in 12 symptomatic plants sampled across the two farms. Primers designed to detect Impatiens necrotic spot virus, groundnut ringspot virus, tomato chlorotic spot virus, tomato chlorosis virus, alfalfa mosaic virus, and tomato necrotic streak virus (ilarvirus, Badillo et al., 2016) failed to generate amplicons of the expected size from cDNA generated from these field samples. The amplicons from full-length NSm cDNA were sequenced from independent, single-leaflet isolates from the TSWV-positive plants (three from farm A, nine from farm B) with the expectation of finding an amino acid (aa) substitution associated with the Sw-5 RB phenotype identified previously in CA (C118Y, Batuman et al. 2017) or Spain (C118Y and T120N, Lopez et al. 2011). All three nucleotide sequences from farm A contained the NSm C118Y substitution reported in CA. All three sequences were 99% identical (including the C118Y mutation) to NCBI GenBank accession KU179600.1, a TSWV isolate collected from GA in 2014 with no cultivar information reported. The nine nucleotide sequences from farm B contained neither of the two previously reported aa substitutions associated with the RB phenotype. Instead, all contained a D122G substitution within a conserved region of the TSWV NSm protein reported to be involved in direct interaction with the Sw-5 protein (Zhu et al 2017). Likewise, Huang et al (2021) generated a D122A mutation in TSWV-NSm, resulting in failure to elicit a Sw-5 mediated hypersensitive response. Three NSm sequences retrieved from GenBank contained the D122G substitution (AY848921.1, HM015516.1, KU179582.1), however, this mutation was not implicated directly with RB phenotypes (Ciuffo et al., 2005; Lopez et al., 2011; Marshall, 2016). The RB phenotype was confirmed with the NC variants on 'Mountain Merit' (Sw-5) by two means of virus inoculation: mechanical, rub-inoculation with extracted sap from infected plants, and thrips transmission assays with lab colony-maintained,}, number={7}, journal={Plant Disease}, publisher={Scientific Societies}, author={Lahre, K. and Shekasteband, R. and Meadows, I. and Whitfield, A. E. and Rotenberg, D.}, year={2023}, month={Jul}, pages={2271} }