DOI: 10.1126/science.1085671 1230 (2003) 301, Science et al. Feng Shao, III Effector PBS1 by a Bacterial Type Arabidopsis Cleavage of www.sciencemag.org (this information is current as of July 3, 2007 ): The following resources related to this article are available online at http://www.sciencemag.org/cgi/content/full/301/5637/1230 version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/cgi/content/full/301/5637/1230/DC1 can be found at: Supporting Online Material http://www.sciencemag.org/cgi/content/full/301/5637/1230#otherarticles 11 of which can be accessed for free: cites 26 articles, This article 104 article(s) on the ISI Web of Science. cited by This article has been http://www.sciencemag.org/cgi/content/full/301/5637/1230#otherarticles 26 articles hosted by HighWire Press see: cited by This article has been http://www.sciencemag.org/cgi/collection/botany Botany subject collections: This article appears in the following http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS. c 2003 by the American Association for the Advancement of Science all rights reserved. The title SCIENCE is a Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Science (print ISSN 0036-8075 online ISSN 1095-9203) is published weekly, except the last week in December, by the on July 3, 2007 www.sciencemag.org Downloaded from

AvrPphB causes PBS1 cleavage in Arabidop- sis, independent of RAR1 and RPS5. The absence of this band in the pbs1-1 line is likely caused by nonspecific degradation re- sulting from activation of the HR. Given the susceptibility of rps5 mutants, these data in- dicate that cleavage of PBS1 is insufficient to induce disease resistance in the absence of RPS5, which suggests that RPS5 may func- tion downstream or independent of the cleav- age event. If PBS1 is a direct substrate of AvrPphB in planta, cleavage of PBS1 should be inde- pendent of host cell background. To test this, we coexpressed PBS1 and AvrPphB in tobac- co (Nicotiana tabacum), which belongs to a plant family (Solanaceae) different from that of Arabidopsis (Brassicaceae). PBS1 was cleaved in tobacco in the presence of wild- type AvrPphB, but not AvrPphB(C98S) (Fig. 1B). Previously, we identified a mutant PBS1 allele, pbs1-2, that blocks AvrPphB-induced resistance in Arabidopsis (14). Because the protein encoded by this allele, PBS1(G252R) (16), lacks kinase activity, we asked whether PBS1 kinase function was required for cleav- age. PBS1(G252R) was cleaved by AvrPphB in tobacco (Fig. 1B), which indicated that AvrPphB cleavage of PBS1 occurs without PBS1 kinase activity. This conclusion was confirmed by the cleavage of PBS1(K115N), which is mutated in the ATP-binding site and lacks kinase activity (Fig. 1B and fig. S1). We tested whether AvrPphB and PBS1 physically interact in planta using a coimmu- noprecipitation assay (15). We immunopre- cipitated total protein extracts of Nicotiana benthamiana, transiently transformed as above, with an HA-specific antibody, and we analyzed immune complexes using an AvrPphB-specific antibody. The protease in- active and wild-type forms of AvrPphB im- munoprecipitated with full-length PBS1 and the C-terminal cleavage product of PBS1, respectively (Fig. 2A). This shows that AvrPphB associates with a PBS1-containing complex in N. benthamiana. To address whether any other plant pro- teins are required for cleavage of PBS1 by AvrPphB, we simultaneously overexpressed epitope-tagged forms of AvrPphB and PBS1 in mammalian HEK 293T cells. AvrPphB, but not AvrPphB(C98S), cleaved PBS1 into a 28-kD C-terminal product and a 32-kD N- terminal product (fig. S2). The same PBS1 cleavage products were obtained when puri- fied recombinant AvrPphB protein was incu- bated with PBS1 synthesized in a wheat germ��� derived in vitro transcription and trans- lation system (fig. S3). To confirm that PBS1 Fig. 1. AvrPphB induces PBS1 clea- vage in planta, independent of RPS5, AtRAR1, and PBS1 kinase activity. (A) Dexamethasone-inducible constructs of PBS1-HA, AvrPphB, and a protease- inactive mutant form of AvrPphB (C98S) were transiently coexpressed in pbs1-1, rar1-20, and rps5-2 mutants of Arabidopsis accession Col-0 and in the rps5 null accession Ler (15, 30). Total protein extracts were subjected to HA- specific antibody and immunoblot analysis. (B) AvrPphB-dependent cleavage of PBS1 occurs in tobacco, irrespective of PBS1 kinase activity. PBS1-HA, PBS1(G252R)-HA, PBS1(K115N)- HA, AvrPphB, and C98S were transiently expressed in tobacco leaves as indicated, and total protein extracts were analyzed as in (A). Fig. 2. PBS1 is a substrate of AvrPphB. (A) PBS1 and AvrPphB coimmunoprecipitate in planta. The total protein extracts described in Fig. 1B were immunoprecipitated using an HA antibody affinity matrix (15). The immuno- complexes and the total extracts were subjected to im- munoblotting with an HA-specific monoclonal antibody (top) and an AvrPphB-specific polyclonal antibody (bot- tom). (B) In vitro cleavage of PBS1 by AvrPphB. Partially purified GST-PBS1(G252R)-His6 protein was incubated with purified AvrPphB-His6, or AvrPphB(C98S)-His6, or in blank buffer. Reactions were separated in an SDS���polyac- rylamide gel electrophoresis gel followed by Coomassie blue staining (left). Immunoblots from the same samples were made with GST antibodies (middle) and His anti- bodies (right). R E P O R T S www.sciencemag.org SCIENCE VOL 301 29 AUGUST 2003 1231 on July 3, 2007 www.sciencemag.org Downloaded from