The Workshop for the Design and Control of Dexterous Hands was held at the MIT Artificial Intelligence Laboratory on November 5-6, 1981. Outside experts were brought together to discuss four topics: kinematics of hands, actuation and materials, touch sensing and control. This report summarizes the discussions of the participants and attempts to identify a consensus on applications, mechanical design, and control.

<i>Rhynchosporium commune</i> is one of the most destructive fungal pathogens of barley worldwide. It causes scald, responsible for reduced grain quality and yield losses of up to 40%. This project aimed to identify genetic resistance in barley using two different approaches: an effector approach through the identification of important pathogen virulence factors and their barley targets, and a genomics association approach. Numerous secreted effectors have been identified in many phytopathogens including <i>R. commune</i>. <i>Rrs1</i> resistance, recognising the <i>R. commune </i>avirulence protein - AvrRrs1 (NIP1) has been deployed in the field to prevent infection but has soon proven ineffective. <i>R. commune </i>has managed to overcome this resistance by alteration or deletion of the <i>NIP1</i> gene as it is not essential for pathogenicity. However, our field trial data suggests that <i>Rrs1</i> remains an important component of resistance to <i>R. commune</i> in the field. Resistance genes recognising more essential <i>Avr</i> genes are likely to be more durable and as a consequence, the discovery of novel <i>R. commune Avr</i> genes is fundamental for the implementation of an integrated pest management approach to prevent this disease. Recent sequencing of the<i> R. commune</i> genome allowed identification of putative effectors. Expression of 26 potential effectors with low sequence variability in 9 sequenced <i>R. commune</i> strains have been analysed during barley infection. The best genes were selected for gene disruption and individual expression in barley cultivars and landraces using the Barley Stripe Mosaic Virus (BSMV) – based expression system to see if they are recognised by the plant. The work also focused on candidate effectors with putative functions. A putative protease inhibitor was chosen for functional characterisation but its function and importance for pathogenicity could not be confirmed. In addition, high amount of the candidate protein appeared to be toxic for barley and <i>Nicotinana benthamiana</i>. Two SA (salicylic acid)-related putative effectors were also chosen for further characterisation and revealed a direct link between the SA pathway of barley and <i>R. commune</i>. The results of this project suggest that <i>R. commune</i> might be able to manipulate the SA pathway of the host confirming the existence of a biotrophic phase of the fungus. The genomics association approach to identify resistance genes against <i>R. commune</i> in barley used a Genome Wide Association Scan (GWAS) using a combination of three years of disease nursery field trial data for a collection of over 500 elite spring barley cultivars. This analysis identified a number of quantitative trait loci (QTL) in barley genome regions previously shown to contain major resistance genes such as <i>Rrs1</i> on chromosome 3H, <i>Rrs2</i> on chromosome 7H, <i>Rrs3</i> on chromosome 4H, <i>Rrs4</i> on chromosome 3H, <i>Rrs13</i> on chromosome 6H, <i>Rrs14</i> on chromosome 1H and<i> Rrs16</i> on chromosome 4H, as well as novel QTL. The work was focused on <i>Rrs1</i> resistance.<i> R. commune</i> strains producing a type of NIP1 effector, recognised by barley lines containing <i>Rrs1</i>, were used to confirm the resistance in predicted <i>Rrs1</i> barley cultivars. The <i>Rrs1</i> interval has been narrowed down to 3 Mbp, and high resolution mapping led to the identification of 3 SNP markers which perfectly discriminated <i>Rrs1Rh4</i> lines from susceptible lines. These diagnostic markers will provide a useful breeding tool for improving the design of new varieties allowing the incorporation of the<i> Rrs1</i> resistance. This research takes us a step closer towards cloning the first barley major resistance (R) gene against <i>R. commune</i>, which is likely to be present only in<i> Rrs1</i> lines and have a kinase domain very similar to the one in a putative wall associated kinase found within the <i>Rrs1 </i>interval in the genome assembly of susceptible cultivar Morex. It will also help us to better understand <i>R. commune</i>-barley pathosystem and to identify further R genes.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

>Magister Scientiae - MSc / Xylophilus ampelinus, the causal agent of bacterial blight and canker of grapevines, has long been a threat to the table grape industry in the Western Cape, leading to severe economic losses due to the reduced productivity and shortened lifespan of infected grapevines. Very little is known about the genetic makeup of the organism, especially with regard to the factors that contribute to its pathogenicity. Generally, bacterial pathogens directly inject the effector proteins into host cells via Type III secretion system (T3SS). In the attempts to identify and characterize the T3 secreted effectors, different reporter plasmid systems have been used to study the secretion and translocation mechanisms the effectors employ during pathogenicity. The aim of the study was to generate a T3 reporter plasmid system for X. ampelinus that will allow the identification and classification of potential pathogenicity factors as members of the Type III secretion class of effectors. First, the avrBs1 family genes avrBs1 and avrA were identified and characterized. The two avirulence genes induced HR on Nicotiana tabacum leaves. Due to the relatedness of the X. ampelinus avr sequences to those of xanthomonads, and the fact that Xanthomonas avrBs1 has been successfully used in a number T3 effector studies, it was decided to construct an X. ampelinus T3 effector reporter vector based on the avrBs1 gene. The minimal segment of the X. ampelinus AvrBs1 protein C-terminus, sufficient for recognition inside host cells and also responsible for HR-induction was identified and characterized using Agrobacterium-mediated transient expression. The AvrBs157-413 HR-inducing domain was cloned in-frame with the 3x FLAG epitope, into a broad-host range vector. To test the reporter vector, the full length avrBs1 sequences of X. ampelinus and Xanthomonas campestris pv. campestris were cloned ahead of the 3x FLAG epitope and the constructs were transferred into XaΔavrBs1 knockout mutant to test for protein secretion. Furthermore, the reporter construct was tested for Type III protein translocation on Bs1 resistant pepper cultivar STAR 6657. Optimization of protein secretion and translocation assays is however required for the improved results. This might include the application of an alternative protein tag to identify candidate X. ampelinus T3SS effectors.

<p>The modification of proteins by the covalent attachment of ubiquitin is a natural process that crucially regulates a wide range of eukaryotic signaling outcomes. This process has been understood as the linking of the C-terminus of ubiquitin to the lysine residue of a target protein via an isopeptide linkage, catalyzed by the coordinated effort by E1, E2, and E3 enzymes. Importantly, ubiquitination has only been observed to be a eukaryotic phenomenon. In recent years though, intracellular bacteria, including human pathogens, have been observed to possess ubiquitin-interacting proteins in their genomes. These proteins serve to subdue and manipulate their hosts’ ubiquitin signaling for their own benefit. While some of these proteins act within the eukaryotic context, more recent findings reveal the existence of prokaryotic enzymes that catalyze ubiquitination using mechanisms never before seen in nature. These remarkable processes utilize different cofactors and target different amino acid residues of both ubiquitin as well as substrate protein. The findings reported in this Thesis involve structural and biochemical studies on two new ubiquitinating proteins, the only two proteins known to catalyze ubiquitination outside of the canonical pathway. Both proteins are present in the genome of the intracellular human pathogen <i>Legionella pneumophila</i>: the SidE family, which catalyzes ubiquitination via a mechanism combining ADP-ribosylation and phosphodiesterase activities, and MavC, which utilizes a mechanism reminiscent of transglutaminases. Key insights provided in this document include the discovery that SidE enzymes can modify multiple ubiquitin moieties within a ubiquitin chain, and that modified ubiquitin chains are resistant to hydrolytic cleavage from many deubiquitinating enyzmes. Also, the development of a robust, continuous assay for SidE-catalyzed ubiquitination using a synthetic substrate is described. The catalytic action of MavC, which differs from both canonical E1/E2/E3 ubiquitination and SidE ubiquitination is also here elucidated. The crystal structure of MavC in complex with its ubiquitinated product is presented and provides an atomic view into the basis of substrate recognition. These findings bring to light a new dimension of host-pathogen interactions, where pathogenic ubiquitinating enzymes have appeared to arise from convergent evolution. The regulation of these pathogenic enzymes by other effectors is also discussed, as well as biochemical studies of these regulators. Further, these findings describe possible new drug discovery strategies, as well as possible techniques for discovering similar enzymes in organisms besides <i>Legionella</i>.</p>

The genus Phytophthora contains a large number of species that are known plant pathogens of a variety of important crops. Phytophthora sojae, a hemibiotroph, causes approximately 1-2 billion dollars (US) of lost soybean world-wide each year. P. infestans, the causative agent of the Irish potato famine, is responsible for over 5 billion dollars (US) worth of lost potato each year. These destructive plant pathogens facilitate pathogenesis through the use of small secreted proteins known as effector proteins. A large subset of effector proteins is able to translocate into host cells and target plant defense pathways. P. sojae Avr1b is able to suppress cell death triggered by BAX and hydrogen peroxide. The W-domain of Avr1b is responsible for this functionality, and is recognized by the Rps1b gene product to induce effector triggered immunity. These oomycete effector proteins translocate into host cells via a highly conserved N-terminal motif known as RXLR-dEER without the use of any pathogen encoded machinery. In fungi an RXLR-like motif exists, [R,K,H] X [L,F,Y,M,~I] X, that is able to facilitate translocation without pathogen encoded machinery. Both functional RXLR and RXLR-like motifs are able to bind phosphatidylinositol-3-phosphate (PtdIns- 3-P) to mediate entry into host cells. The use of novel inhibitory mechanisms has shown effector entry can be blocked either by sequestering PtdIns-3-P on the outer leaflet of plant and animal cells or by competitive inhibition of the binding pocket of the RXLR or RXLR-like motifs. / Ph. D.

Salmonella is a facultative intracellular bacteria which can grow and replicate within the infected host cells as well as extracellulary. The use of intracellular bacteria that have access to the host cell cytosol may allow a more specific targeting of DNA vaccine vectors to professional Antigen presenting cells (APC). The strategy of using live attenuated Salmonella to deliver plasmid-encoded antigens under the transcriptional control of eukaryotic promoters has been used successfully in vaccination. Another strategy, heterologous antigens can be expressed in Salmonella as fusions with recombinant or native proteins. This approach has been used mainly to direct the expression of the desired antigen to a particular location of the bacterial cell and increase the immunogenicity of foreign antigens by fusing them to proteins that could exert a carrier effect. Salmonella type III secretion system (TTSS)-mediated translocation can be used for efficient delivery of heterologous antigens to the cytosol of antigen-presenting cells leading to prominent both CD4 and CD8 T-cells. In this work we tried SPI2 membranal translocated effectors antigen fusions such as SseJ, SifA, SseL and SteC. Our In-vitro and in-vivo experiments prove that SseJ effector fusion is the best candidate for vaccination. In previous work it was shown that SifB promoter was the most efficient in-vivo inducible promoter. Here we show that SseJ antigen fusion protein under control of SifB is the most efficient in comparison to other effector fusions under control of other invivo inducible promoters. htrA/purD douple mutant S.typhimurium was used as attenuated carrier for vaccination, in this study we find that delta SifA mutant can stimulate in-vitro T-cell proliferation to same level.

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