Nucleocytoplasmic Distribution Is Required for Activation of Resistance by the Potato NB-LRR Receptor Rx1 and Is Balanced by Its Functional Domains

Abstract

The Rx1 protein, as many resistance proteins of the nucleotide binding-leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.

The Effector SPRYSEC-19 ofGlobodera rostochiensisSuppresses CC-NB-LRR-Mediated Disease Resistance in Plants

“…The full-length SW5F genes of tomato 'GCR161' were PCR-amplified as described before (Rehman et al, 2009). The regions of R-genes coding for the LRR domain were subcloned from existing plasmids: SW5A and -B (Spassova et al, 2001), SW5F (Rehman et al, 2009), Gpa2 (Rairdan and Moffett, 2006), and Rx1 (Slootweg et al, 2010). PCRamplification products were cloned into vector pRAP using specific restriction sites and confirmed by DNA sequencing.…”

“…tumefaciens strain MOG101 carrying vector pBINPLUS with 35S:4MYC: SPRYSEC-19 (described above), 35S LS :Rx1:GFP (Slootweg et al, 2010), 35S:GFP: PVX (Peart et al, 2002), or GPA2:GUS (Koropacka, 2010) were used for agroinfiltration of N. benthamiana leaves. Three days post infiltration, GFP expression of GFP-tagged PVX was visualized under UV light.…”

“…The 4MYC:GFP construct was used as a negative control for suppression. The following pairs of resistance genes and cognate elicitors were used to induce programmed cell death in leaves: Gpa2 / RBP1 (Sacco et al, 2009), Rx1 / cp106 (Slootweg et al, 2010), Cf4 / Avr4 (Thomas et al, 2000), Cf9 / Avr9 (Thomas et al, 2000), R3a / AvrR3a (Huang et al, 2005), Rpi-blb2 / AvrBlb2 , and BS4 (Schornack et al, 2005) / AvrBS4 (Ballvora et al, 2001). The following constructs of mutant CC-NB-LRR proteins were used to trigger an elicitor-independent programmed cell death: GG-GRR (Rairdan and Moffett, 2006), Mi-1.2(T557S) (Gabriëls et al, 2007), RGH10 , NRC1(D481V; Gabriëls et al, 2007), and INF1 (Kamoun et al, 2003).…”

Pepper Heat Shock Protein 70a Interacts with the Type III Effector AvrBsT and Triggers Plant Cell Death and Immunity

“…AvrBsT interacts with CaHSP70a in both the nucleus and the cytoplasm. The AvrBsTCaHSP70a complex promoted avrBsT-triggered cell death in N. benthamiana leaves only when localized to the cytoplasm by attachment of the NES (Slootweg et al, 2010;Choi et al, 2012). These results suggest that cytoplasmic localization of the CaHSP70a and AvrBsT complex is required for the positive regulation of cell death in plants.…”

“…To generate the BiFC constructs, cDNAs encoding AvrBsT and CaHSP70a without termination codons were PCR amplified and subcloned into the binary vectors pVYNE(R) (XbaI/XhoI) and pSCYCE (XbaI/XhoI) under the control of the cauliflower mosaic virus 35S promoter (Supplemental Table S1). Oligonucleotides containing NLS and NES sequences (Slootweg et al, 2010;Choi and Hwang, 2011) were inserted into the XhoI/KpnI site to create the NLS and NES fusion constructs (Supplemental Table S1). AvrBsT and CaHSP70a were coexpressed in N. benthamiana leaves by infiltrating A. tumefaciens strain GV3101 carrying each construct (OD 600 = 0.5).…”