High-throughput in planta expression screening identifies an ADP-ribosylation factor (ARF1) involved in non-host resistance and R gene-mediated resistance.

To identify positive regulators of cell death in plants, we performed a high-throughput screening, employing potato virus X-based overexpression in planta of a cDNA library derived from paraquat-treated Nicotiana benthamiana leaves. The screening of 30,000 cDNA clones enabled the identification of an ADP-ribosylation factor 1 (ARF1) that induces cell death when overexpressed in N. benthamiana. Overexpression of the guanosine diphosphate (GDP)-locked mutant of ARF1 did not trigger cell death, suggesting that ARF1 guanosine triphosphatase (GTPase) activity is necessary for the observed cell death-inducing activity. The ARF1 transcript level increased strongly following treatment with Phytophthora infestans elicitor INF1, as well as inoculation with a non-host pathogen Pseudomonas cichorii in N. benthamiana. In addition, ARF1 was induced in the interaction between the N gene and tobacco mosaic virus (TMV) in Nicotiana tabacum. By contrast, inoculation with the virulent pathogen Pseudomonas syringae pv. tabaci did not affect ARF1 expression in N. benthamiana. Virus-induced gene silencing of ARF1 in N. benthamiana resulted in a stunted phenotype, and severely hampered non-host resistance towards P. cichorii. In addition, ARF1 silencing partially compromised resistance towards TMV in N. benthamiana containing the N resistance gene. By contrast, and in accordance with the ARF1 gene expression profile, silencing of ARF1 transcription did not alter the susceptibility of N. benthamiana towards the pathogen P. syringae pv. tabaci. These results strongly implicate ARF1 in the non-host resistance to bacteria and N gene-mediated resistance in N. benthamiana.