Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins. IntroductionPseudomonas syringae infects a wide range of susceptible plants and causes mainly localized necrosis in infected tissues. A given strain of P. syringae may infect only a few cultivars of a host plant, exhibiting a high degree of specificity. Host specificity is the basis for classifying various P. syringae strains into more than 40 pathovars (Gardan et al., 1999). P. syringae pathovar (pv.) tomato strain DC3000 (Pst DC3000 hereafter) infects tomato and Arabidopsis and causes necrotic spots surrounded by diffuse chlorotic haloes (Cuppels, 1986;Whalen et al., 1991; Katagiri et al., 2002). The molecular basis of pathogenicity of Pst DC3000, like that of the majority of plant pathogenic bacteria, is not well understood. An essential weapon in the virulence arsenal of Pst DC3000 is the type III protein secretion system, which is conserved in many Gram-negative plant and mammalian pathogenic bacteria (He, 1998;Galan and Collmer, 1999;Cornelis and Van Gijsegem, 2000;Staskawicz et al., 2001). In plant pathogenic bacteria, the type III secretion system is encoded by hrp (for hypersensitive reaction and pathogenicity) and hrc (hrp gene conserved) genes (Van Gijsegem et al., 1993;Bonas, 1994;Alfano and Collmer, 1997;Lindgren, 1997;He, 1998;Mudgett and Staskawicz, 1998;Hutcheson, 1999). This secretion system is responsible for the assembly of the Hrp pilus (Roine et al., 1997;Jin and He, 2001) and is thought to deliver virulence effector proteins directly into the host cell. Once there, the effector proteins are believed to modulate the physiology of the host cell to favour pathogenesis. However, different plant cultivars may evolve specific resistance genes to recognize individual bacterial effectors and turn them into elicitors of host defence responses. In such situations, these virulence effector proteins have been named avirulence (Avr) proteins (Leach and White, 1996).The expression of hrc, hrp and effector genes is tightly SummaryPseudomonas syringae pv. tomato (Pst ) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the...

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a cochaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI. Some of the effectors are recognized by host surveillance systems and trigger a strong resistance when their cognate resistance genes are present (2, 4). Often, this so-called ''genefor-gene resistance'' or effector-triggered immunity (ETI; ref . 4) is activated by an indirect interaction between the resistance protein and the cognate effector protein (5). Three proteins, HSP90, RAR1, and SGT1, play an important role in ETI by regulating the stability of NB-LRR resistance proteins (6-11), but they are not known for a role in PTI regulation. It is thought that the plant resistance gene products somehow sense the virulence activity of these effectors, rather than the effectors themselves, which in turn activates resistance. Supporting this hypothesis, several host proteins have been shown to interact with both effector and resistance proteins and are required for ETI (12-16). However, a role of these proteins in effectormediated virulence function remains to be demonstrated.The P. syringae effector protein AvrB enhances virulence on soybean and Arabidopsis plants lacking cognate resistance genes but triggers ETI on soybean and Arabidopsis plants carrying the resistance genes (17). The virulence function of AvrB is expressed as increased bacterial growth in soybean plants and leaf chlorosis in Arabidopsis plants. The virulence and ETI activity of AvrB have the same structural requirements, suggesting that the virulence function and ETI are intimately connected (17, 18). Therefore, host proteins required for AvrB virulence function may provide a molecular link between effector virulence function and ETI.Here we show that AvrB inhibits PTI through RAR1, a HSP90 cochaperone required for disease resistance gene functions. When expressed in plants, AvrB suppresses plant defenses and enhances bacterial growth in a ...

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Surface modification is an essential tool in tissue engineering using synthetic biomaterial scaffolds. The authors report in this study a simple approach to modify the surface hydrophobicity, roughness and chemistry of electrospun polycaprolactone (PCL) fibers using a combination of oxygen plasma treatment, sodium hydroxide treatment and arginine–glycine–aspartic acid (RGD) immobilization. The modified surfaces were characterized using scanning electron microscopy, atomic force microscopy, water contact angle measurement and X-ray photoelectron spectroscopy (XPS). Plasma treatment decreased the water contact angle. Sodium hydroxide treatment further improved the hydrophilicity and increased the surface roughness. XPS analysis confirmed the presence of amide bonds on RGD-treated fibers. The enhancement of proliferation of ligament fibroblasts within 1 week of culturing on both the plasma- and sodium hydroxide–treated fibers was most likely due to improved wettability by the oxygen plasma treatment. The alignment and penetration of cells on PCL fibers suggested that these materials could be potential scaffold materials for the regeneration of fibrous tissues.

In this laboratory exercise, students have an opportunity to evaluate the potential endocrine disrupting abilities of environmental chemicals of their choice using human cell culture. Over the course of 9 weeks, students learn how to aseptically handle and manipulate cells, perform and analyze a cytotoxicity assay and an enzyme-linked immunosorbent assay. Following completion of the module, the majority of students reported large or very large gains not only in laboratory performance, but also in understanding of the scientific literature and research process, as well as scientific communication skills. The student survey results imply that this authentic laboratory experience improves students' scientific literacy and prepares them for future careers in science.