A discovery-based undergraduate laboratory exercise to investigate the effect of potential endocrine disruptors on sex hormones using human cell culture.

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.

Type III protein secretion in Pseudomonas syringae.

The type III secretion system is an essential virulence system used by many Gram-negative bacterial pathogens to deliver effector proteins into host cells. This review summarizes recent advancements in the understanding of the type III secretion system of Pseudomonas syringae, including regulation of the type III secretion genes, assembly of the Hrp pilus, secretion signals, the putative type III effectors identified to date, and their virulence action after translocation into plant cells.

RAR1, a central player in plant immunity, is targeted by Pseudomonas syringae effector AvrB.

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 co-chaperone 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.

Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome.

Pseudomonas 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 "hrp box") in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria-Bertani broth. Expression of all eight genes 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.