Figure 2.

Genomic organization of the ARF1 gene and analysis of ARF1 gene expression in Nicotiana spp. (a) Genomic Southern analysis suggests that NbARF1 is a member of a small gene family in Nicotiana benthamiana. Ten micrograms of total DNA were digested to completion with BamIHI, EcoRI and EcoRV, respectively, and hybridized with the ‘3′‐ORF‐UTR’ fragment (left) or full pSfinx:8‐51 insert (full‐length cDNA; right). (b–e) Northern analysis of ARF1 gene expression following biotic stresses in N. benthamiana and Nicotiana tabacum. Ten micrograms of total RNA were separated in a denaturing agarose gel, and rRNAs were stained by methylene blue on the membranes as loading controls. The probe used for Northern blotting was the ‘3′‐ORF‐UTR’ as used in (a). NbARF1 gene expression was induced in N. benthamiana following infiltration with a solution containing 100 nm of the Phytophthora infestans elicitor INF1 (b), inoculation with the non‐host pathogen Pseudomonas cichorii (OD₆₀₀ = 0.1) (c), but not following inoculation with the virulent pathogen Pseudomonas syringae pv. tabaci (OD₆₀₀ = 0.1) (d). The NtARF1 transcript level increased in N gene‐harbouring N. tabacum inoculated with tobacco mosaic virus following a temperature shift (e).