Retracted: The Cf-9 Disease Resistance Protein Is Present in an ∼420-Kilodalton Heteromultimeric Membrane-Associated Complex at One Molecule per Complex

Abstract

The tomato Cf-9 gene confers race-specific resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding avirulence gene Avr9 . In tobacco, Cf-9 confers a hypersensitive response to the Avr9 peptide. To investigate Cf-9 protein function in initiating defense signaling, we engineered a functional C-terminal fusion of the Cf-9 gene with the TAP (Tandem Affinity Purification) tag. In addition, we established a transient expression assay in Nicotiana benthamiana leaves for the production of functional Cf-9:myc and Cf-9:TAP. Transiently expressed Cf-9:myc and Cf-9:TAP proteins induced an Avr9-dependent hypersensitive response, consistent with previous results with stably transformed tobacco plants and derived cell suspension cultures expressing c-myc-tagged Cf-9. Gel filtration of microsomal fractions solubilized with octylglucoside revealed that the Cf-9 protein, either as c-myc or TAP fusions, migrated at a molecular mass of 350 to 475 kD. By using blue native gel electrophoresis, the molecular size was confirmed to be ‫ف‬ 420 kD. Our results suggest that only one Cf-9 protein molecule is present in the Cf-9 complex and that Cf-9 is part of a membrane complex consisting of an additional glycoprotein partner(s). The high structural similarity between Cf proteins and Clavata2 (CLV2) of Arabidopsis, together with the similarity of molecular mass between Cf-9 and CLV complexes (420 and 450 kD, respectively), led us to investigate whether Cf-9 is integrated into membraneassociated protein complexes like those formed by CLV1 and CLV2. Unlike CLV2, the Cf-9 protein did not form disulfidelinked heterodimers, no ligand (Avr9)-dependent shift in the molecular mass of the Cf-9 complex was detected, and no Rho-GTPase-related proteins were found associated with Cf-9 under the conditions tested. Thus, Cf-9-dependent defense signaling and CLV2-dependent regulation of meristem development seem to be accomplished via distinct mechanisms, despite the structural similarity of their key components Cf-9 and CLV2. INTRODUCTIONA major goal in plant pathology is to understand the molecular mechanism of disease resistance. In gene-for-gene interactions, a plant disease resistance gene ( R gene) confers resistance to invading pathogens that carry the corresponding avirulence ( Avr ) gene. Despite the cloning of numerous R genes in recent years, progress in understanding R protein function in Avr perception and signal transmission has been comparatively slow. It has been postulated that R gene products are receptors for pathogen-encoded Avr components (Staskawicz et al., 1995;Ellis et al., 2000). However, direct physical interaction in an R/Avr combination has been reported only for the Pto and Pi-ta R gene products using the yeast twohybrid system (Scofield et al., 1996;Tang et al., 1996) and an overlay assay (Jia et al., 2000). This and several other lines of evidence suggest that R proteins are part of protein complexes, with additional protein partners participating in Avr perception and subsequent signal initiation ...

Recognition Specificity and RAR1/SGT1 Dependence in Barley Mla Disease Resistance Genes to the Powdery Mildew Fungus

“…Recent studies suggest that members of the intracellular and membrane-anchored classes assemble in preformed heteromultimeric recognition complexes in the absence of pathogens (Leister and Katagiri, 2000;Holt et al, 2002;Mackey et al, 2002;Rivas et al, 2002aRivas et al, , 2002b. With one exception, there are no documented examples of direct interactions between LRR-containing R and AVR proteins (rice Pi-ta and AVR-Pita from Magnaporthe grisea ; Jia et al, 2000).…”

Wall-associated kinase 1 (WAK1) is crosslinked in endomembranes, and transport to the cell surface requires correct cell-wall synthesis

“…The fusion protein was detected in the Triton-soluble fraction at day 1, but accumulated into the Triton-insoluble fraction by day 3. However, the serum, and hence the signal, was weak and therefore a WAK1-TAP fusion construct (tandem-affinity-purification tag also driven by the 35S CaMV promoter) was transformed into protoplasts; the peroxidaseantiperoxidase (PAP)-soluble complex was used to follow the TAP-tag and fusion protein (Rivas et al, 2002). WAK1-GFP was also transformed at the same time and used to visually follow the location of WAK1 in the cell.…”

“…Aliquots of the samples (30 l) for the Triton-soluble fraction were separated by SDS-PAGE using 8% gel and transferred onto nitrocellulose membrane. The blots were blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline supplemented with 3% (v/v) Tween 20, probed with peroxidase-antiperoxidase (PAP)-soluble complex (Sigma) (Rivas et al, 2002) at 1:5000 dilution for 2 hours (western blot) or overnight (dot-blot), and the signal was detected by chemiluminescence.…”

Modification, Development, Application and Prospects of Tandem Affinity Purification Method

“…Through the TAP strategy, Liu et al demonstrated that Hsp90 associated with the plant resistance protein N , which meant that Hsp90 plays an important role in plant defense (Kanzaki et al, 2003;Takahashi et al, 2003). In another study, the Cf-9 protein function in initiating defense signaling was also investigated by TAP (Rivas et al, 2002). The TAP applications described above were all carried out in a transient expression system of Nicotiana benthamiana.…”