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A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt virus (TBSV) and Cauliflower mosaic virus (CaMV) strain W260, indicating that resistance to these viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusviruses, including Cucumber necrosis virus, Cymbidium ringspot virus, Lettuce necrotic stunt virus, and Carnation Italian ringspot virus. [ABSTRACT FROM AUTHOR]

In plants, RNA silencing is an important mechanism for gene regulation and defense that is targeted by proteins of viral pathogens effecting silencing suppression. In this chapter we describe a new assay to probe silencing suppressor activity using Agrobacterium infiltration of Nicotiana benthamiana and confocal microscopy. The key element in this assay involves the use of a reporter construct that is transiently expressed at a much lower level than free GFP, and this increases the sensitivity of detection of weak silencing suppressors such as the P6 protein of Cauliflower mosaic virus. Although initially developed for virus silencing suppressors, this technique could also prove valuable to characterize the potential for weak silencing suppressors in the effector repertoires of fungi, bacteria, nematodes, and oomycetes.