Nicotiana benthamiana often displays more intense symptoms after infection by RNA viruses than do other Nicotiana species. Here, we examined the role of RNA-dependent RNA polymerases (RdRPs) in N. benthamiana antiviral defense. cDNAs representing only two genes encoding RdRPs were identified in N. benthamiana. One RdRP was similar in sequence to SDE1͞SGS2 required for maintenance of transgene silencing, whereas the second, named NbRdRP1m, was >90% identical in sequence to the salicylic acid (SA)-inducible RdRP from Nicotiana tabacum required for defense against viruses.

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.

SummaryPlant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf 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 source tissue and its exit from veins in sink tissue was studied using a modi®ed virus expressing green¯uorescent protein (GFP). Using a surgical procedure that isolated speci®c leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV ®rst accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes 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 tissue does not preclude virus entry. During transport, the virus initially accumulates in speci®c vascularassociated cells, indicating that virus accumulation in this tissue is highly regulated. These ®ndings have important implications for studies on the identi®cation of symplasmic domains and host macromolecule vascular transport.

The differential symptom determinants of the Holmes' masked (M) and U1 strains of tobacco mosaic virus previously were mapped to the 5'-coterminal open reading frame (ORF) encoding the 126-kDa protein and the N-terminal two-thirds of the 183-kDa protein. Both proteins influence viral RNA accumulation, but the function of, and impact on, symptom formation by large domains within the 126-kDa gene, which are not conserved with sequences in analogous ORFs from other related viruses, are unknown. In the current study, cDNA clones representing each strain (i.e., MIC-TMV and U1-TMV) were mutated in these nonconserved domains to further define the nucleotides responsible for mosaic symptom induction on Nicotiana tabacum. Progeny virus of a mutant containing only eight nucleotide substitutions from the MIC-TMV sequence to the U1-TMV sequence within the 126-kDa protein ORF of MIC-TMV induced U1-TMV-like symptoms. Single or multiple substitutions among these eight nucleotides further defined residues critical for symptom modulation. Complementary substitutions in the MIC-TMV and U1-TMV sequences did not always yield progeny virus that induced complementary visual symptoms. Progeny of some mutants contained second-site spontaneous mutations at specific positions shown to influence symptom phenotype. For a subset of the stable site-directed mutants, there was no correlation between severity of systemic symptoms and chlorotic lesion size or virus accumulation in these chlorotic lesions on inoculated leaves.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, UlmCP15-17, and SNCO15, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of nonnative capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas UlmCP15-17 and SNCO15 do not accumulate by phloemdependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and UlmCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP ofwild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.The movement of plant viruses through vascular tissue is essential to allow their maximum accumulation and symptom development in the host plant (1). In crops infected with viruses, the greatest yield reductions are due to vasculardependent movement (2). Although a considerable amount of information is known about the cell-to-cell movement of plant viruses (3-5), much less is known about vascular-dependent movement (5-8). The majority of plant viruses, including tobacco mosaic virus (TMV), move through the phloem (1). The coat protein (CP) of TMV is critical for phloemdependent accumulation of this virus. Mutant strains of TMV that produce CP, but are defective in virion assembly, spread as labile infectious entities from cell-to-cell, but do not accumulate in tissue accessed by phloem-dependent movement (9). More recently, it has been possible to delete or modify the CP open reading frame of cDNA clones of TMV, and infectious transcripts from these clones yielded virus that moved cell-tocell but did not accumulate in upper uninoculated leaves unless complemented by CP expressed in transgenic plants (10)(11)(12)(13) Dolja et al. (20,22) determined that the N-terminal 29 residues and the C-terminal 18 residues of the tobacco etch potyvirus CP were necessary for phloem-dependent accumulation. Also, it has been suggested that the capsid of red clover necrotic mosaic dianthovirus, and not some other CP-containi...

The masked and U1 strains of tobacco mosaic tobamovirus differ in symptom phenotype and in phloem-dependent accumulation in tobacco. The symptom phenotype is determined by eight amino acids in the 126- and 183-kDa proteins that differ between the two strains. In this study, slow phloem-dependent accumulation of the masked strain was shown to be determined by these same eight amino acids, but some symptomatically severe mutants altered at specific positions within the eight amino acids were inefficient in phloem-dependent accumulation. Therefore, the appearance of severe symptoms does not require rapid phloem-dependent accumulation. There was no consistent relationship between the accumulation of virus coat protein, movement protein, or 126- and 183-kDa protein in inoculated protoplasts and the efficiency of phloem-dependent accumulation in stem tissue. Therefore, the difference in phloem-dependent accumulation between the masked strain, its mutants, and the U1 strain in most instances resulted from the functional competence of the 126- and/or 183-kDa proteins or a host response to their change and not from their quantities or the quantities or functional competence of the movement proteins or coat proteins.

We have developed a double-sided labeling technique for detecting viral proteins or RNAs in plastic-embedded leaf tissue by immunocytochemistry or in situ hybridization, respectively, and light microscopy. The signal from the target was enhanced by double-sided labeling when compared with single-sided labeling because sections were submerged in labeling solutions with both sides accessible to antibodies or complementary RNAs. The additional label was visible during microscopic analysis. Background signal was decreased since the tissue was probed and washed under conditions where folds and creases in the tissue were minimized. This technique uses the same equipment and chemicals as for single-sided labeling, and thus adjustments for reagent expenditures are not necessary. The procedure should be applicable to animal and plant tissue.

Virus invasion of minor veins in inoculated leaves of a host is the likely prelude to systemic movement of the pathogen and to subsequent yield reduction and quality loss. In this study we have analyzed the cell number and arrangement in minor veins within mature leaves of various members of the Solanaceae and Fabaceae families. We then monitored the accumulation pattern of several tobamoviruses and potyviruses in these veins at the time of rapid, phloem-mediated movement of viruses. Vascular parenchyma cells were the predominant and sometimes only cells to become visibly infected among the cells surrounding the sieve elements in minor veins containing 9 to 12 cells. In no instance did we observe a companion cell infected without a vascular parenchyma cell also being infected in the same vein. This suggests that the viruses used in this study first enter the vascular parenchyma cells and then the companion cells during invasion. The lack of detectable infection of smooth-walled companion or transfer cells, respectively, from inoculated leaves of bean (Phaseolus vulgaris) and pea (Pisum sativum) during a period of known rapid, phloem-mediated movementsuggests that some viruses may be able to circumvent these cells in establishing phloem-mediated infection. The cause of the barrier to virus accumulation in the companion or transfer cells, the relationship of this barrier to previously identified barriers for virus or photoassimilate transport, and the relevance of these findings to photoassimilate transport models are discussed.