Multiple intramolecular trafficking signals in RESISTANCE TO POWDERY MILDEW 8.2 are engaged in activation of cell death and defense

Abstract

The extrahaustorial membrane (EHM) is a host-derived interfacial membrane encasing the haustorium of powdery mildew fungi. Arabidopsis thaliana RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically targeted to the EHM via two EHM-targeting signals. Here, we demonstrate that proper coordination between the trafficking forces engaged via the EHM-targeting signals and the nuclear localization signals (NLSs), as well as the nuclear export signals (NESs), in RPW8.2 is critical for the activation of cell death and defense. We show that in the absence of pathogens, RPW8.2 is partitioned between the cytoplasm and the nucleus, and turned over via both the 26S proteasome-and the vacuole-dependent pathways. Enhanced cytoplasmic localization of RPW8.2 by tagging it with a NES led to lethal cell death. By contrast, enhanced nuclear localization of RPW8.2 by adding an NLS to it resulted in resistance to powdery mildew. Whereas expression of the NES-containing C-terminal domain of RPW8.2 in the cytoplasm is sufficient to trigger cell death, no such cell death-inducing activity is found with RPW8.2 variants that contain the two EHM-targeting signals along with the NES-containing C-terminal domain. In addition, we present evidence for the involvement of a leaf senescence pathway in RPW8.2-mediated cell death and defense. Taken together, our data suggest that RPW8.2 is subject to adjustment by distinct and perhaps coordinated mechanisms for its localization and function via interaction with the multiple intramolecular trafficking signals, which should provide further insights into RPW8.2-activated, EHM-focused resistance against powdery mildew.

ANNEXIN 8 negatively regulates RPW8.1‐mediated cell death and disease resistance in Arabidopsis

“…Confocal imaging showed that although YFP signal was mainly detected along the plasma membrane of the epidermal cells, it was also found in intracellular structures (Figure 8A). To further determine the subcellular localization of AtANN8‐YFP, we transiently co‐expressed it with an RFP nuclear marker obtained in a previous investigation (Huang et al, 2014) in leaves of Nicotiana benthamiana . While RFP signal from the nuclear marker was exclusively found in the nucleus, YFP signal from AtANN8‐YFP was seen in the cytoplasm with strong signal visible around the nucleus as a typical ER ring (Figure S3).…”

“…H 2 O 2 examination in situ production was performed according to Xiao et al (2003). To examine the accumulation of RPW8.1‐YFP or subcellular localization of AtANN8‐YFP, leaves were examined first under a epi‐fluorescent microscope (Nikon 80i) and then performed images acquisition using a confocal laser scanning microscope (Nikon A1; Nikon Instruments Inc., Chengdu, China) as described in a previous report (Huang et al, 2014).…”

“…The RPW8.2 expression is induced by the powdery mildew infection through a salicylic acid (SA)dependent feedback circuit and the RPW8.2 is specifically targeted to extra-haustorial membrane that encases the fungal feeding structure named haustorium (Xiao et al, 2003;Wang et al, 2009). While adequate expression and accurate localization of RPW8.2 is critical for activation of defense responses (Wang et al, 2010;Huang et al, 2019), also RPW8.2 is under negative regulation presumably for minimizing the cost-ofresistance associates with expression of RPW8.1 and RPW8.2 in absence of the pathogens (Orgil et al, 2007). Indeed, genetic screens identified an inositol phosphorylceramide synthase as a negative regulator for RPW8.2-mediated disease resistance and cell death (Wang et al, 2008).…”

Genome-wide identification, characterisation and functional evaluation of WRKY genes in the sweet potato wild ancestor Ipomoea trifida (H.B.K.) G. Don. under abiotic stresses

“…WRKY53 (ItfWRK21, − 23, − 35, − 59, − 70 and − 71) is an early factor in drought response, and it regulates stomatal movement and early events of leaf senescence by reducing H 2 O 2 content and promoting starch metabolism in guard cells [46]. WRKY6 (ItfWRK3, − 11, − 65, − 62, − 52.1, − 52.2, − 52.3, − 37.1 and − 37.2) participates in the control of aging and pathogen defence [47]. WRKY33 (ItfWRKY 28.1, − 28.2, − 80, − 83.1 and − 83.2) specifically interacts with W-box and responds to salt, cold and heat stresses [14, 48].…”

The CaAP2/ERF064 Regulates Dual Functions in Pepper: Plant Cell Death and Resistance to Phytophthora capsici

“…Disruption of the nuclear localization signals (NLS) in CaAP2/ERF064 may be an effective, alternative way to approach this, while the RPW8.2 protein confers broad-spectrum resistance against powdery mildew, and the cytoplasmic localization of RPW8.2 could trigger cell death. In contrast, the RPW8.2 protein localization in the nucleus could result in plant resistance to powdery mildew [43,44].…”