A simplified disease resistance assay using YFP-expressingPotato Virus XinN. benthamianareveals a cell death-independent immune function of RBA1

Abstract

SummaryR (resistance) proteins, such as intracellular NLRs (nucleotide-binding leucine-rich repeat receptors), are integral components of the plant innate immune system (van Wersch et al., 2020). Host responses following R protein activation include the generation of reactive oxygen species, sustained increases in cytosolic Ca2+, transcriptional reprogramming and, typically, rapid host cell death at sites of pathogen infection, which together ultimately lead to pathogen growth restriction (Wang et al., 2023). To assess the activity of R proteins, agroinfiltration-mediated transient gene expression assays have been widely used inNicotianaspecies (e.g.,N. benthamiana). In these transient assays, host cell death is often chosen as an indicator of R protein activity from the host responses mentioned above, in part because of the ease of experimentation. However, the extent to which host cell death is a proxy for disease resistance signaling has long been debated, as host cell death and pathogen growth restriction can be uncoupled in several cases (Bendahmane et al., 1999; Coll et al., 2010; Heidrich et al., 2011, Maekawa et al., 2023). To assess the disease resistance activity of R proteins, bacterial growth assays have been employed in combination with transientRgene expression inN. benthamiana(Sun et al., 2021). Bacterial growth assays, however, require multiple experimental procedures, including agroinfiltration, pathogen infection and bacterial counts, which hinders high-throughput studies ofRgene-mediated disease resistance. Here, we report a simple plate reader-based assay to assessRgene-mediated disease resistance activity against PVX (Potato virus X) that expresses YFP (PVX-YFP). Unlike bacterial pathogens, PVX proliferation inN. benthamianais not restricted by the intrinsic activity of the EDS1 signaling pathway as previously shown by virus-inducedNbEDS1gene silencing (Peart et al., 2002) and as we consistently show in this study using aNbeds1gene knockout mutant. This feature would increase the sensitivity of the assay, allowing it to capture a weak-to-moderate disease resistance activity of R proteins, as the contribution of basal immunity to PVX via theNbEDS1 pathway is negligible. Using this assay, we show that a non-cell death-inducing mutant of the R protein of RBA1 (Response to HopBA1), which lacks 2′,3′-cAMP/cGMP synthetase activity but retains NADase activity, confers PVX resistance in an EDS1 signaling pathway-dependent manner.