Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

SummaryGene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

While much study has gone into characterizing virulence factors that play a general role in disease, less work has been directed at identifying pathogen factors that act in a host-specific manner. Understanding these factors will help reveal the variety of mechanisms used by pathogens to suppress or avoid host defenses. We identified candidate Pseudomonas syringae host-specific virulence genes by searching for genes whose distribution among natural P. syringae isolates was statistically associated with hosts of isolation. We analyzed 91 strains isolated from 39 plant hosts by DNA microarray-based comparative genomic hybridization against an array containing 353 virulence-associated (VA) genes, including 53 type III secretion system effectors (T3SEs). We identified individual genes and gene profiles that were significantly associated with strains isolated from cauliflower, Chinese cabbage, soybean, rice, and tomato. We also identified specific horizontal gene acquisition events associated with host shifts by mapping the array data onto the core genome phylogeny of the species. This study provides the largest suite of candidate hostspecificity factors from any pathogen, suggests that there are multiple ways in which P. syringae isolates can adapt to the same host, and provides insight into the evolutionary mechanisms underlying host adaptation. P ATHOGENS cannot cause disease indiscriminately, but are generally capable of avoiding or suppressing defenses in only a relatively small set of hosts. Both pathogen factors and host factors govern these interactions, and which of these two is dominant likely depends on the specific interaction. Nevertheless, there is a growing body of data that clearly demonstrates that pathogens have evolved compatibility factors that facilitate the disease process in a host-specific manner. (Cornelis 2002;Abramovitch and Martin 2004;Alfano and Collmer 2004;Espinosa and Alfano 2004;Nomura et al. 2005). However, there are several key issues about pathogen host-specificity factors that are still unknown. For example, the general relationship between virulence factors and host-specificity factors is unclear, although it is likely that the latter are a subset of the former required only on select hosts. It is also not known if the factors that are responsible for pathogen compatibility on different host species are fundamentally different from the factors that determine compatibility among different cultivars of the same host species. We do not even know at what level host specificity acts most strongly. For example, hostspecificity factors may play important roles during initial colonization, during pathogen migration to the appropriate tissue or cell type, during the initiation of cellular interactions, during the maintenance of these interactions, or even at the point where the pathogen disperses to a new host (Levin 1996).To address these questions, we first must have a way of identifying host-specific virulence factors. A reasonable hypothesis is that strains that are able to in...

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