A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana Chia-Fong Wei 1,† , Brian H. Kvitko 2,† , Rena Shimizu 2 , Emerson Crabill 3 , James R. Alfano 4 , Nai-Chun Lin 2,5 , Gregory B. Martin 2,5 , Hsiou-Chen Huang 1 and Alan Collmer 2, * 1 Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40224, Taiwan, 2 Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA, 3 School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0118, USA, 4 Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, NE 68588-0660, USA, and 5 Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853-1801, USA Received 10 November 2006; revised 16 February 2007; accepted 23 February 2007. *For correspondence (fax +1 607 255 8835; email arc2@cornell.edu). † These authors contributed equally to this work and are considered co-first authors. Summary The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. bent- hamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti- effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000. Keywords: Hrp system, hypersensitive response and pathogenicity, plant defense, host specificity, Avr proteins. Introduction Pseudomonas syringae pv. tomato (Pto) DC3000 is a pathogen of tomato and Arabidopsis noted for its large and well-characterized repertoire of type III effectors: proteins injected into plant cells by the type III secretion system (T3SS). Strains in P. syringae are divided into 50 or so pathovars based largely on host specificity (Hirano and Upper, 2000). For example, Pto causes bacterial speck of tomato but is avirulent and elicits the defense-associ- ated hypersensitive response (HR) in tobacco, whereas P. syringae pv. tabaci (Pta) causes wildfire disease in tobacco but is avirulent in tomato. What controls such host specificity is not understood, although type III effectors are generally suspected (Alfano and Collmer, 2004). 32 ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd The Plant Journal (2007) 51, 32–46 doi: 10.1111/j.1365-313X.2007.03126.x