Expresión de la resistencia en plantas de tomate a nematodos formadores de nódulos (Meloidogyne javanica)

La resistencia genética mediada por los genes R es uno de los sistemas de defensa de las plantas frente a patógenos y se activa una vez que los patógenos han superado la defensa basal que otorgan la cutícula y pared celular. Los mecanismos de resistencia genética se inician a su vez, por el reconocimiento de productos derivados de genes de avirulencia de los patógenos (avr) por parte de las proteínas R. Tanto la respuesta de defensa basal como la respuesta de defensa por genes R están influenciadas por patrones de regulación hormonal, que incluye a las principales hormonas vegetales ácido salicílico (SA), ácido jasmónico (JA) y etileno (ET). En tomate (Solanum lycopersicum) uno de los genes R es el gen MiG1, que confiere resistencia a nematodos formadores de nódulos (Meloidogyne javanica, M. incognita y M. arenaria). Uno de los eventos más importantes que caracterizan a la respuesta de resistencia es la reacción hipersensible (HR), que está mediada por la activación temprana de una serie de sistemas enzimáticos, entre los que destaca el de las peroxidasas (PRXs) Clase III. Su función es importante tanto para limitar el establecimiento y expansión del nematodo, al generar ambientes altamente tóxicos por su contribución en la producción masiva de ROS, como por su implicación en la síntesis y depósito de lignina generando barreras estructurales en el sitio de infección. Además de estos mecanismos de defensa asociados a la resistencia constitutiva, las plantas pueden desarrollar resistencia sistémica adquirida (SAR) que en la naturaleza ocurre, en ocasiones, en una fase posterior a que la planta haya sufrido el ataque de un patógeno. Así mismo hay diferentes productos de origen químico como el benzotiadiazol o BTH (ácido S-metil benzol-(1,2,3)-tiadiozole-7-carbónico ester) que pueden generar esta misma respuesta SAR. Como resultado, la planta adquiere resistencia sistémica frente a nuevos ataques de patógenos. En este contexto, el presente trabajo aborda en primer lugar el análisis comparativo, mediante microarrays de oligonucleótidos, de los transcriptomas de los sistemas radicales de plantas de tomate de 8 semanas de edad de dos variedades, una portadora del gen de resistencia MiG1 (Motelle) y otra carente del mismo y, por tanto, susceptible (Moneymaker), antes y después de la infección por M. javanica. Previo a la infección se observó que la expresión de un gran número de transcritos era más acusada en la variedad resistente que en la susceptible, entre ellos el propio gen MiG1 o los genes PrG1 (o P4), LEJA1 y ER24, lo que indica que, en ausencia de infección, las rutas hormonales del SA, JA y ET están más activas en la raíz de la variedad resistente. Por el contrario, un número mucho menor de transcritos presentaban su expresión más reducida en Motelle que en Moneymaker, destacando un gen de señalización para sintetizar la hormona giberelina (GA). La infección por M. javanica causa importantes cambios transcripcionales en todo el sistema radical que modifican sustancialmente las diferencias basales entre plantas Motelle y Moneymaker, incluida la sobreexpresión en la variedad resistente de los transcritos de MiG1, que se reduce parcialmente, mientras que las rutas hormonales del SA y el JA continuan más activas que en la susceptible (evidente por los genes PrG1 y LEJA1). Además, los cambios asociados a la infección del nematodo se evidencian por las grandes diferencias entre los dos tiempos post-infección considerados, de tal forma que en la fase temprana (2 dpi) de la interacción compatible predomina la sobreexpresión de genes de pared celular y en la tardía (12 dpi) los relacionados con el ARN. En el análisis de la interacción incompatible, aunque también hay muchas diferencias entre ambas fases, hay que destacar la expresión diferencial común de los genes loxA y mcpi (sobrexpresados) y del gen loxD (reprimido) por su implicación en defensa en otras interacciones planta-patógeno. Cabe destacar que entre las interacciones compatible e incompatible hubo muy pocos genes en común. En la etapa temprana de la interacción compatible destacó la activación de genes de pared celular y la represión de la señalización; en cambio, en la interacción incompatible hubo proteínas principalmente implicadas en defensa. A los 12 días, en la interacción compatible los genes relacionados con el ARN y la pared celular se sobreexpresaban principalmente, y se reprimían los de proteínas y transporte, mientras que en la incompatible se sobreexpresaron los relacionados con el estrés, el metabolismo secundario y el de hormonas y se reprimieron los de ARN, señalización, metabolismo de hormonas y proteínas. Por otra parte, la técnica de silenciamiento génico VIGS reveló que el gen TGA 1a está implicado en la resistencia mediada por el gen MiG1a M. javanica. Así mismo se evaluó el transcriptoma de todo el sistema radical de la variedad susceptible tras la aplicación del inductor BTH, y se comparó con el transcriptoma de la resistente. Los resultados obtenidos revelan que el tratamiento con BTH en hojas de Moneymaker ejerce notables cambios transcripcionales en la raíz; entre otros, la activación de factores de transcripción Myb (THM16 y THM 27) y del gen ACC oxidasa. Las respuestas inducidas por el BTH parecen ser de corta duración ya que no hubo transcritos diferenciales comunes a las dos fases temporales de la infección comparadas (2 y 12 dpi). El transcriptoma de Moneymaker tratada con BTH resultó ser muy diferente al de la variedad resistente Motelle, ambas sin infectar, destacando la mayor expresión en el primero del gen LeEXP2, una expansina relacionada con defensa frente a nematodos. Las respuestas inducidas por los nematodos en Moneymaker-BTH también fueron muy distintas a las observadas previamente en la interacción incompatible mediada por MiG1, pues sólo se detectaron 2 genes sobreexpresados comunes a ambos eventos. Finalmente, se abordó el estudio de la expresión diferencial de genes que codifican PRXs y su relación con la resistencia en la interacción tomate/M. javanica. Para ello, se realizó en primer lugar el estudio del análisis del transcriptoma de tomate de la interacción compatible, obtenido en un estudio previo a partir de tejido radical infectado en distintos tiempos de infección. Se han identificado 16 unigenes de PRXs con expresión diferencial de los cuales 15 se relacionan por primera vez con la respuesta a la infección de nematodos. La mayoría de los genes de PRXs identificados, 11, aparecen fuertemente reprimidos en el sitio de alimentación, en las células gigantes (CG). Dada la implicación directa de las PRXs en la activación del mecanismo de producción de ROS, la supresión de la expresión génica local de genes de PRXs en el sitio de establecimiento y alimentación pone de manifiesto la capacidad del nematodo para modular y superar la respuesta de defensa de la planta de tomate en la interacción compatible. Posteriormente, de estos genes identificados se han elegido 4: SGN-U143455, SGN-U143841 y SGN-U144042 reprimidos en el sitio de infección y SGN-U144671 inducido, cuyos cambios de expresión se han determinado mediante análisis por qRT-PCR y de hibridación in situ en dos tiempos de infección (2 dpi y 4 dpi) y en distintos tejidos radicales de tomate resistente y susceptible. Los patrones de expresión obtenidos demuestran que en la interacción incompatible la transcripción global de los 4 genes estudiados se dispara en la etapa más temprana en el sitio de infección, detectándose la localización in situ de transcritos en el citoplasma de las células corticales de la zona meristemática afectadas por el nematodo. A 4 dpi se observó que los niveles de expresión en el sitio de infección cambian de tendencia y los genes SGN-U144671 y SGN-U144042 se reprimen significativamente. Los diferentes perfiles de expresión de los genes PRXs en los dos tiempos de infección sugieren que su inducción en las primeras 48 horas es crucial para la respuesta de defensa relacionada con la resistencia frente a la invasión del nematodo. Por último, al analizar el tejido radical sistémico, se detectó una inducción significativa de la expresión en la fase más tardía de la infección del gen SGN-U144042 en el genotipo susceptible y del SGN-U143841 en ambos genotipos. En este estudio se describe por primera vez la inducción de la expresión sistémica de genes de PRXs en tomate durante la interacción compatible e incompatible con M. javanica lo que sugiere su posible implicación funcional en la respuesta de defensa SAR activada por la infección previa del nematodo. ABSTRACT Plants defend themselves from pathogens by constitutive and/or induced defenses. A common type of induced defense involves plant resistance genes (R), which are normally activated in response to attack by specific pathogen species. Typically, a specific plant R protein recognizes a specific pathogen avirulence (avr) compound. This initiates a complex biochemical cascade inside the plant that results in synthesis of antipathogen compounds. This response can involve chemical signaling, transcription, translation, enzymes and metabolism, and numerous plant hormones such as salicylic acid (SA), jasmonates (JA) and ethylene (ET). Induced plant defense can also activate Class III peroxidases (PRXs), which produce reactive oxygen species (ROS), regulate extracellular H2O2, and play additional roles in plant defense. R-gene activation and the resulting induced defense often remain localized in the specific tissues invaded by the plant pathogen. In other cases, the plant responds by signaling the entire plant to produce defense compounds (systemic induction). Plant defense can also be induced by the exogenous application of natural or synthetic elicitors, such as benzol-(1,2,3)-thiadiazole-7-carbothionic acid. There is much current scientific interest in R-genes and elicitors, because they might be manipulated to increase agricultural yield. Scientists also are interested in systemic induction, because this allows the entire plant to be defended. In this context, one of the aims of this investigation was the transcriptoma analysis of the root systems of two varieties of tomato, the resistant variety (Motelle) that carrier MiG1 and the susceptible (Moneymaker) without MiG1, before and after infection with M. javanica. The overexpression was more pronounced in the transcriptoma of the resistant variety compared with susceptible, before infection, including the MiG1 gene, PrG1 (or P4) genes, LEJA1 and ER24, indicating that hormone SA, JA and ET are active in the resistant variety. Moreover, GA hormone presents an opposite behavior. M. javanica infection causes significant transcriptional changes in both compatible (Moneymaker-M. javanica) and incompatible (Motelle-M. javanica) interaction. In the incompatible transcriptome root system, was notably reduced the expression of the MiG1 gene, and a continuity in the expression of the hormonal pathways of SA and JA. In other hand, transcriptional profile changes during compatible interaction were associated with nematode infection. The large differences between the two times point infection considered (2 dpi and 12 dpi) indicates an overexpression of cell wall related genes in the first phase, and conversely an overexpression of RNA genes in the late phase. Transcriptoma analysis of incompatible interaction, although there were differences between the two phases, should be highlighted the common differential gene expression: loxA and mcpi (overexpressed) and loxD gene (suppressed), as they are involved in defenses in other plant-pathogen interactions. The VIGS tool has provided evidence that TGA 1a is involved in MiG1 mediated resistance to M. javanica. Likewise, the systemic application of BTH was assessed and compared with susceptible and resistant variety. Root system transcriptoma of BTH treatment on leaves showed the activation of Myb transcription factors (THM16 and THM27), the ACC oxidase gene. and the LeEXP2 gene, encoding for an expansin enzyme, related with defense against nematodes. The activation appears to be reduced by subsequent infection and establishment of nematodes. To assist in elucidate the role of tomato PRXs in plant defence against M. javanica, the transcriptome obtained previously from isolated giant cells (GC) and galls at 3 and 7 dpi from the compatible interaction was analysed. A total of 18 different probes corresponding to 16 PRX encoding genes were differentially expressed in infection site compared to the control uninfected root tissues. Most part of them (11) was down-regulated. These results yielded a first insight on 15 of the PRX genes responding to tomato–Meloidogyne interaction and confirm that repression of PRX genes might be crucial for feeding site formation at the initial stages of infection. To study the involvement of PRX genes in resistance response, four genes have been selected: SGN-U143455, SGN-U143841 and SGN-U144042 consistently down-regulated and SGN-U144671 consistently up-regulated at infection site in compatible interaction. The expression changes were determined by qRT-PCR and in situ location at 2 dpi and 4 dpi, and in different root tissues of resistant and susceptible plants. Early upon infection (2 dpi), the transcripts levels of the four genes were strongly increased in infected tissue of resistant genotype. In situ hybridization showed transcript accumulation of them in meristem cortical cells, where the nematode made injury. The results obtained provide strong evidence that early induction of PRX genes is important for defence response of the resistance against nematode invasion. Moreover, the induction patterns of SGN-U144042 gene observed at 4 dpi in distal noninfected root tissue into the susceptible genotype and of SGN-U143841 gene in both genotypes suggest a potential involvement of PRX in the systemic defence response.

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Islanding detection in Microgrids using Harmonic Signature

In recent years, there has been a growing interest in incorporating microgrids in electrical power networks. This is due to various advantages they present, particularly the possibility of working in either autonomous mode or grid connected, which makes them highly versatile structures for incorporating intermittent generation and energy storage. However, they pose safety issues in being able to support a local island in case of utility disconnection. Thus, in the event of an unintentional island situation, they should be able to detect the loss of mains and disconnect for self-protection and safety reasons. Most of the anti-islanding schemes are implemented within control of single generation devices, such as dc-ac inverters used with solar electric systems being incompatible with the concept of microgrids due to the variety and multiplicity of sources within the microgrid. In this paper, a passive islanding detection method based on the change of the 5th harmonic voltage magnitude at the point of common coupling between grid-connected and islanded modes of operation is presented. Hardware test results from the application of this approach to a laboratory scale microgrid are shown. The experimental results demonstrate the validity of the proposed method, in meeting the requirements of IEEE 1547 standards.

Exploring new roles for the rpoS gene in the survival and virulence of the fire blight pathogen Erwinia amylovora

Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits.

Paisaje de mujer: sistematización contemporánea de variables dispersas

Vivimos en las escuelas de ayer una vida de ciencia ficción. La tecnología se ha superpuesto a unos escenarios vigentes desde ha ce siglos, ocultando una realidad, que se ve superada por la cotidianidad . Lo habitual, como extremo de la ignorancia, se refugia en la seguridad que garantiza la cercanía de las cosas, las personas y las acciones, tergiversando la memoria. La supresión d e lo habitual puede ser un momento poderoso en el conocimiento y es posible mediante el extrañamiento . Es necesario definir nuevas reglas del juego, trasgrediendo todas las leyes creadas para controlar las formas y las personas. Es necesario rastrear y vis ibilizar todos los mecanismos ocultos destinados a trasmitir aquellos contenidos cuyo fin es perpetuar un sistema de valores incompatible con la dignidad y la justicia para todos los se res que habitamos en este mundo ( currículum oculto ). La asignatura “pa isaje de mujer” pretende colonizar el territorio de lo habitual – donde todos somos culpables - intentando fabricar un atlas de género mediante la cartografía de todas las representaciones existentes en las distintas esferas de la cotidianidad, para pode r coronar la cima del grado cero

Propuesta de enseñanza-aprendizaje de un modelo de juego en fútbol de alto rendimiento

Introducción: En el entrenamiento de los deportes de equipo, está generalizada la descripción de la periodización del entrenamiento en función de las capacidades físicas trabajadas en cada microciclo. Este proceso de planificación y programación tiene un fundamento científico y es necesario, pero no es incompatible con un registro y una programación de los contenidos tácticos a trabajar. Para ello, debe existir un modelo de juego basado en las características de los jugadores y un registro detallado de cada partido, en función de unos indicadores de rendimiento que permitan evaluar la asimilación de dicho modelo de juego. Objetivo: Realizar una revisión de la literatura más reciente acerca de planificación en fútbol y a partir de esta definir unas tareas para desarrollar un modelo de juego concreto, además de un método de registro de datos y de evaluación del proceso enseñanza-aprendizaje táctico-estratégico en el fútbol de alto rendimiento. Material y métodos: Mediante la diversa bibliografía, la información sobre el modelo de juego y los vídeos de partidos completos proporcionados por el cuerpo técnico del Club Deportivo Leganés; he desarrollado la propuesta de una metodología de entrenamiento, y unas tareas como base de la enseñanza-aprendizaje del modelo de juego Resultados: El resultado de este trabajo es una propuesta metodológica que puede ser adaptable a otros entrenadores, modelos de juego e incluso deportes, ya que es una herramienta flexible. Conclusiones: El registro detallado de un modelo de juego, de unas tareas para desarrollar cada uno de sus aspectos y su evaluación mediante unos indicadores de rendimiento diseñados por el entrenador, forman en conjunto una herramienta práctica para el desarrollo de dicho modelo de juego y por lo tanto aumentar el rendimiento del equipo.

Trabalho de equipa entre o jardim-de-Infância e a escola

Relatório de estágio apresentado à Escola Superior de Educação de Santarém para obtenção do grau de Mestre em Educação pré-escolar e ensino do 1º ciclo do ensino básico

Clearing Up Confusions about Religion and Evolution

A number of confusions plague the debate concerning religion and evolution. Included here are the belief that religion and science must be incompatible and the thesis that evolution is evidence for atheism. Clearing up these confusions helps remove obstacles to genuine dialogue, while acknowledging that difficulties remain.

Molinism, Open Theism, and Moral Luck

It is sometimes thought that the choice between Molinism and open theism involves a trade-off in values: Molinism asserts that God has providential power but allows God indirectly to manipulate that in virtue of which human beings are to be judged; while open theism grants human beings more power over that in virtue of which they are tp be judged, but at the price of giving up providence. I argue here that this picture is misconstrued---that Molinism gives human agents more power over that in virtue of which they may be judged than does open theism. Since open theism confines the possible avenues for evaluating agents to their behavior in the actual world, open theism is incompatible with any solution to the problem of moral luck which appeals to counterfactual behavior, and so (I argue) is impugned by the problem,. Molinists, by contrast, have a promising solution to that problem.

DETERMINAÇÃO DA POSIÇÃO DE REPOUSO DA LÍNGUA EM INDIVÍDUOS COM OCLUSÃO DENTÁRIA NORMAL

O propósito neste estudo foi determinar a posição de repouso da língua em indivíduos com oclusão dentária normal e respiração nasal, por meio de telerradiografias em norma lateral realizadas após a ingestão de bário. A amostra foi composta por 66 radiografias de indivíduos brancos com oclusão dentária normal, sendo 26 do sexo masculino e 40 do sexo feminino, na faixa etária de 12 a 21 anos de idade, procedentes de escolas da região do Grande ABC Paulista. O critério utilizado para diagnóstico da oclusão normal foi As Seis Chaves para a Oclusão Normal preconizadas por Andrews (1972), devendo estar presentes no mínimo quatro das seis chaves, sendo obrigatória a presença da primeira chave de oclusão que é a da relação interarcos. As radiografias foram obtidas com o indivíduo em posição natural da cabeça após a ingestão de contraste de sulfato de bário para evidenciar o controle da língua. Posteriormente foi feito o desenho anatômico das estruturas pesquisadas, marcados os pontos cefalométricos, traçadas as linhas e os planos, e por último obtidas as seguintes medidas lineares: comprimento e altura da língua, distância do dorso da língua na sua porção média até o palato duro e a distância entre a ponta da língua e a incisal do incisivo inferior. Por meio dos resultados encontrados verificou-se que não existe um padrão único de posicionamento de repouso da língua dentro da cavidade oral, em pacientes respiradores nasais, variando muito sua distância até a incisal dos incisivos inferiores, bem como até o palato duro, havendo uniformidade apenas no fato da língua tocar o palato mole em todos os indivíduos da amostra. Não houve relação estatisticamente significante entre a posição de repouso da língua e os biotipos faciais e nem dimorfismo sexual.

DETERMINAÇÃO DA POSIÇÃO DE REPOUSO DA LÍNGUA EM INDIVÍDUOS COM OCLUSÃO DENTÁRIA NORMAL

O propósito neste estudo foi determinar a posição de repouso da língua em indivíduos com oclusão dentária normal e respiração nasal, por meio de telerradiografias em norma lateral realizadas após a ingestão de bário. A amostra foi composta por 66 radiografias de indivíduos brancos com oclusão dentária normal, sendo 26 do sexo masculino e 40 do sexo feminino, na faixa etária de 12 a 21 anos de idade, procedentes de escolas da região do Grande ABC Paulista. O critério utilizado para diagnóstico da oclusão normal foi As Seis Chaves para a Oclusão Normal preconizadas por Andrews (1972), devendo estar presentes no mínimo quatro das seis chaves, sendo obrigatória a presença da primeira chave de oclusão que é a da relação interarcos. As radiografias foram obtidas com o indivíduo em posição natural da cabeça após a ingestão de contraste de sulfato de bário para evidenciar o controle da língua. Posteriormente foi feito o desenho anatômico das estruturas pesquisadas, marcados os pontos cefalométricos, traçadas as linhas e os planos, e por último obtidas as seguintes medidas lineares: comprimento e altura da língua, distância do dorso da língua na sua porção média até o palato duro e a distância entre a ponta da língua e a incisal do incisivo inferior. Por meio dos resultados encontrados verificou-se que não existe um padrão único de posicionamento de repouso da língua dentro da cavidade oral, em pacientes respiradores nasais, variando muito sua distância até a incisal dos incisivos inferiores, bem como até o palato duro, havendo uniformidade apenas no fato da língua tocar o palato mole em todos os indivíduos da amostra. Não houve relação estatisticamente significante entre a posição de repouso da língua e os biotipos faciais e nem dimorfismo sexual.

CTXφ immunity: Application in the development of cholera vaccines

CTXφ is a filamentous bacteriophage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae. CTXφ is unusual among filamentous phages because it encodes a repressor and forms lysogens. CTXφ can infect the existing live-attenuated V. cholerae vaccine strains derived from either the El Tor or classical V. cholerae biotypes and result in vaccine reversion to toxinogenicity. Intraintestinal CTXφ transduction assays were used to demonstrate that El Tor biotype strains of V. cholerae are immune to infection with the El Tor-derived CTXφ, whereas classical strains are not. The El Tor CTXφ repressor, RstR, was sufficient to render classical strains immune to infection with the El Tor CTXφ. The DNA sequences of the classical and El Tor CTXφ repressors and their presumed cognate operators are highly diverged, whereas the sequences that surround this “immunity” region are nearly identical. Transcriptional fusion studies revealed that the El Tor RstR mediated repression of an El Tor rstA-lacZ fusion but did not repress a classical rstA-lacZ fusion. Likewise, the classical RstR only repressed a classical rstA-lacZ fusion. Thus, similar to the mechanistic basis for heteroimmunity among lambdoid phages, the specificity of CTXφ immunity is based on the divergence of the sequences of repressors and their operators. Expression of the El Tor rstR in either El Tor or classical live-attenuated V. cholerae vaccine strains effectively protected these vaccines from CTXφ infection. Introduction of rstR into V. cholerae vaccine strains should enhance their biosafety.

Similarities in normal and binocularly rivalrous viewing

We report here a series of observations—most of which the reader can experience directly—showing that distinct components of patterned visual stimuli (orthogonal lines of a different hue) vary in perception as sets. Although less frequent and often less complete, these perceptual fluctuations in normal viewing are otherwise similar to the binocular rivalry experienced when incompatible scenes are presented dichoptically.

Directed evolution of a thermostable esterase

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14°C increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind SC10-RNase in SI N. alata SC10SC10 and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia × SI N. alata SC10SC10) hybrids with reduced levels of HT-protein continued to express SC10-RNase but failed to reject SC10-pollen. Control hybrids expressing both SC10-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.