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. benthamiana 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.

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved ( i ) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn 5gus mutagenesis, ( ii ) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, ( iii ) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and ( iv ) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N ^ɛ -(indole-3-acetyl)- l -lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2 , hopPtoB2 , and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.

AvrPto and AvrPtoB are type III effector proteins expressed by Pseudomonas syringae pv. tomato strain DC3000, a pathogen of both tomato and Arabidopsis spp. Each effector physically interacts with the tomato Pto kinase and elicits a hypersensitive response when expressed in tomato leaves containing Pto. An avrPto deletion mutant of DC3000 previously was shown to retain avirulence activity on Pto-expressing tomato plants. We developed an avrPtoB deletion mutant of DC3000 and found that it also retains Pto-specific avirulence on tomato. These observations suggested that avrPto and avrPtoB both contribute to avirulence. To test this hypothesis, we developed an ΔavrPtoΔavrPtoB double mutant in DC3000. This double mutant was able to cause disease on a Pto-expressing tomato line. Thus, avrPto and avrPtoB are the only avirulence genes in DC3000 that elicit Pto-mediated defense responses in tomato. When inoculated onto susceptible tomato leaves and compared with wild-type DC3000, the mutants DC3000ΔavrPto and DC3000ΔavrPtoB each caused slightly less severe disease symptoms, although their growth rate was unaffected. However, DC3000ΔavrPtoΔavrPtoB caused even less severe disease symptoms than the single mutants and grew more slowly than them on susceptible leaves. Our results indicate that AvrPto and AvrPtoB have phenotypically redundant avirulence activity on Pto-expressing tomato and additive virulence activities on susceptible tomato plants.