Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the yo ung growing parts of the plant rapidly. Functional entry sites into the lea f vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in so urce tissue and its exit from veins in sink tissue was studied using a modi fied virus expressing green fluorescent protein (GFP). Using a surgical pro cedure that isolated specific leaf and stem tissues from complicating vascu lar tissues, we determined that TMV could enter minor, major or transport v eins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major v eins and petioles of the inoculated leaf and stems below the inoculated lea f. It also initially accumulated exclusively in internal or adaxial phloem- associated cells in stems above the inoculated leaf and petioles or major v eins of sink leaves. This work shows the functional equivalence of vein cla sses in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissu e does not preclude virus entry. During transport, the virus initially accu mulates in specific vascular-associated cells, indicating that virus accumu lation in this tissue is highly regulated. These findings have important im plications for studies on the identification of symplasmic domains and host macromolecule vascular transport.