<i>Orthotospovirus tomatomaculae</i> (Tomato spotted wilt virus [TSWV]) infects ovules and pollen to achieve vertical transmission in <i>Capsicum annuum</i>

ABSTRACT Orthotospovirus tomatomaculae (Tomato spotted wilt virus, TSWV) is an economically important plant pathogen reported to be transmissible by seeds. However, the pathways by which the virus enters the seed remain poorly understood. Here, we characterized the distribution and vertical transmission pathways of TSWV within reproductive organs of Capsicum annuum . Transmission electron microscopy (TEM) confirmed systemic distribution of TSWV throughout floral tissues, with virions detected in anthers, pollen grains, ovaries, ovules, and styles. Immunofluorescence histochemistry further revealed accumulation of the TSWV nucleocapsid (N) protein in the reproductive organs, with the strongest signals in anther septa, vascular bundles, and ovule integuments. Using reverse transcription-quantitative PCR (RT-qPCR) with an absolute standard curve, we detected the highest TSWV accumulation in anthers, ovaries, and styles, and lower levels in pollen grains. Cross-pollination assays demonstrated seed transmission via pollen (40%) and infected maternal tissue/ovules (30%), while self-pollination of infected plants resulted in 96% infected seeds. Testing of progeny seedlings showed infection rates of 20% (pollen-mediated), 30% (ovule-mediated), and 90% (self-pollination of infected parents). Together, these data support vertical transmission of TSWV through two reproductive routes, pollen and ovule, associated with providing a mechanistic framework for mitigating seed-borne spread in chili pepper. IMPORTANCE Tomato spotted wilt virus (TSWV) causes substantial yield losses worldwide, yet the mechanisms underlying its seed transmission remain insufficiently defined. We provide anatomical and molecular evidence that TSWV colonizes key reproductive tissues of Capsicum annuum , including anthers, pollen grains, ovaries, and ovules. Using controlled cross-pollination, we demonstrate that vertical transmission can occur through infected pollen (40% seed infection; 20% seedling infection) and through infected maternal tissues/ovules (30% seed infection; 30% seedling infection). Mechanistically, these data reveal two independent routes for vertical transmission: pollen-borne and ovule-borne. This finding directly supports two evidence-based management interventions: (i) the removal of infected male plants from seed production fields to block pollen-borne spread, and (ii) the selection of virus-free maternal lines to minimize ovule-mediated transmission. Incorporating both pollen- and ovule-associated transmission into epidemiological models and resistance breeding programs will therefore be critical for mitigating seed-borne TSWV losses in chili pepper.