Systemic infection of tobacco mosaic virus is limited by upward transport and phloem unloading without coat protein.

Coat protein (CP) is widely viewed as essential for systemic infection of tobacco mosaic virus (TMV), yet the mechanistic basis of this requirement remains incompletely resolved. In this study, we show that TMV can still achieve long-distance vascular transport in the absence of CP, but systemic infection becomes intermittent because the processes underlying "systemic movement" can fail at multiple, separable steps. By resolving movement into phloem loading, vascular translocation, and phloem exit, we show that CP deficiency does not completely block entry into or transport within the vasculature. Instead, CP loss imposes a strong, route-specific defect in bicollateral phloem, disproportionately reducing use of the adaxial/internal pathway associated with efficient upward spread, while downward movement through abaxial/external phloem remains comparatively permissive. In systemic leaves, CP-deficient TMV frequently produces vein/phloem-associated reporter patterns rather than broad lamina invasion, indicating that phloem exit/unloading is an additional, independent bottleneck. Enhancing RNA silencing suppression markedly enhances systemic viral accumulation and symptoms. However, it does not proportionally increase lamina invasion. This finding further supports the idea that exit and establishment are the limiting steps. Finally, among several helper viruses tested, only cucumber mosaic virus consistently alleviates this exit barrier, enabling CP-deficient TMV to escape the vasculature and establish lamina infection.