Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests

Potential Use of a Serpin from Arabidopsis for Pest Control

Although genetically modified (GM) plants expressing toxins from Bacillus thuringiensis (Bt) protect agricultural crops against lepidopteran and coleopteran pests, field-evolved resistance to Bt toxins has been reported for populations of several lepidopteran species. Moreover, some important agricultural pests, like phloem-feeding insects, are not susceptible to Bt crops. Complementary pest control strategies are therefore necessary to assure that the benefits provided by those insect-resistant transgenic plants are not compromised and to target those pests that are not susceptible. Experimental GM plants producing plant protease inhibitors have been shown to confer resistance against a wide range of agricultural pests. In this study we assessed the potential of AtSerpin1, a serpin from Arabidopsis thaliana (L). Heynh., for pest control. In vitro assays were conducted with a wide range of pests that rely mainly on either serine or cysteine proteases for digestion and also with three non-target organisms occurring in agricultural crops. AtSerpin1 inhibited proteases from all pest and non-target species assayed. Subsequently, the cotton leafworm Spodoptera littoralis Boisduval and the pea aphid Acyrthosiphon pisum (Harris) were fed on artificial diets containing AtSerpin1, and S. littoralis was also fed on transgenic Arabidopsis plants overproducing AtSerpin1. AtSerpin1 supplied in the artificial diet or by transgenic plants reduced the growth of S. littoralis larvae by 65% and 38%, respectively, relative to controls. Nymphs of A. pisum exposed to diets containing AtSerpin1 suffered high mortality levels (LC50 = 637 µg ml−1). The results indicate that AtSerpin1 is a good candidate for exploitation in pest control

Competitive Performance of Transgenic Wheat Resistant to Powdery Mildew

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes

Evaluación pronóstica de las neumonías en pacientes con EPOC

Objetivo: Estudiar la incidencia, gravedad y mortalidad de las neumonías ocurridas en una cohorte de pacientes con enfermedad pulmonar obstructiva crónica (EPOC) seguidos durante 3 años. Pacientes y métodos: Se incluyó en el estudio a 596 pacientes con diagnóstico espirométrico de EPOC. Los parámetros a evaluar fueron la mortalidad y la gravedad valorada de acuerdo con el Pneumonia Severity Index (PSI) para la neumonía adquirida en la comunidad (NAC). Resultados: De 596 pacientes incluidos en el estudio, 75 (12,6%) desarrollaron al menos un episodio de neumonía durante el seguimiento. La incidencia global de neumonía fue de 55,1 por 1.000 personas-año. Hubo 88 episodios en 75 pacientes. El grado de la EPOC, valorado según el FEV1 como porcentaje del teórico, era en 9 pacientes leve, en 24 moderado y en 42 grave. De los episodios de neumonía, 76 (86,3%) fueron adquiridos en la comunidad y 12 (13,6%) en el hospital. Al valorar la gravedad de la NAC, 14 episodios correspondían al grupo V, 28 al grupo IV, 20 al grupo III y 14 a los grupos I y II. La mortalidad global fue del 12,5% (11/88). La mortalidad en las neumonías nosocomiales fue del 41,7% (5/12) y la mortalidad en las NAC fue del 7,8% (6/76) (OR: 6,67; intervalo de confianza del 95%, 1,65-26,93). Al valorar la mortalidad en las NAC según la gravedad, se encontró que la mortalidad en el grupo V fue de un 35,7% (5/14), en el grupo IV del 3,5% (1/28) y nula en el resto de los grupos. Conclusiones: Hay una elevada incidencia de neumonía en los pacientes con EPOC. Más de la mitad de las NAC (55,2%) ocurridas en nuestros pacientes con EPOC están dentro de los grupos de riesgo del PSI IV y V

Barley cystain variants against phytopathogenic fungi pests and their impact on natural enemies

The goal of this study has been to know the effects of the barley cystatin Hv-CPI and seven derived variants generated by direct-mutagenesis, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata and to assess the potential impact of these proteins on the spiner soldier bug (SSB), Podisus maculiventris, a generalist hemipteran predator. Among the different cystatins tested, the variant C68 G, in which the only cysteine residue was changed to glycine, showed the highest inhibitory activity when tested in vitro against commercial cysteine proteases and CPB digestive enzymes. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing this variant, resulted in significant lower weight gains compared to those fed on non-transformed plants. No effects on survival, development, and weight, were observed when SSB nymphs fed on prey CPB reared with transgenic potato plants. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays. The effects of the barley inhibitor Hv-CPI and its variants on the growth of the the necrotrophic fungus Fusarium oxysporum have been also analysed. The cystatin Hv-CPI inhibited fungal spore germination by 25%, the five point mutations inhibited spore germination by 18 to 40%, while the two truncated forms had no antifungal effect

Barley cystain variants against phytopathogenic fungi pests and their impact on natural enemies

The goal of this study has been to know the effects of the barley cystatin Hv-CPI and seven derived variants generated by direct-mutagenesis, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata and to assess the potential impact of these proteins on the spiner soldier bug (SSB), Podisus maculiventris, a generalist hemipteran predator. Among the different cystatins tested, the variant C68 G, in which the only cysteine residue was changed to glycine, showed the highest inhibitory activity when tested in vitro against commercial cysteine proteases and CPB digestive enzymes. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing this variant, resulted in significant lower weight gains compared to those fed on non-transformed plants. No effects on survival, development, and weight, were observed when SSB nymphs fed on prey CPB reared with transgenic potato plants. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays. The effects of the barley inhibitor Hv-CPI and its variants on the growth of the the necrotrophic fungus Fusarium oxysporum have been also analysed. The cystatin Hv-CPI inhibited fungal spore germination by 25%, the five point mutations inhibited spore germination by 18 to 40%, while the two truncated forms had no antifungal effect

Mejoras en la preservación pulmonar: tres años de experiencia con una solución de dextrano bajo en potasio

Objetivo: La calidad de la preservación pulmonar es uno de los aspectos más determinantes en el éxito del trasplante pulmonar. En octubre del año 2000 modificamos nuestra solución de preservación pulmonar, que hasta entonces era el Euro-Collins (EC), y comenzamos a utilizar una solución de dextrano bajo en potasio, comercializada como Perfadex (PER). El objetivo de este estudio es analizar los resultados de ambos métodos. Material y métodos: Hemos analizado los resultados de 68 trasplantes pulmonares con PER y los hemos comparado con los de una serie retrospectiva del mismo número de trasplantes realizados con EC. Resultados: No existen diferencias significativas respecto a la edad o el diagnóstico de los receptores entre ambos grupos. El tiempo en lista de espera fue mayor en el grupo de PER. La causa de muerte del donante más frecuente del grupo EC fue el traumatismo craneoencefálico (62%), mientras que en el grupo de PER fue la hemorragia cerebral (54%). En el grupo de PER se realizaron más trasplantes bipulmonares que en el de EC (el 78 y el 53%, respectivamente; p = 0,002). No hay diferencias en la indicación de circulación extracorpórea o tiempos de isquemia entre ambos grupos. Se evaluó la función pulmonar temprana a través del índice de oxigenación (PaO2/FiO2) a la llegada a la unidad de cuidados intensivos, que fue comparable entre ambos grupos. La incidencia de disfunción grave del injerto (PaO2/FiO2 < 150 mmHg) fue significativamente inferior en el grupo de PER frente al de EC (el 16 y el 37%, respectivamente; p = 0,01). No encontramos diferencias significativas respecto a las horas de ventilación mecánica ni en cuanto a la mortalidad postoperatoria entre las 2 series. Conclusiones: Con la aplicación clínica de esta nueva solución de preservación pulmonar se obtiene una reducción del 50% en la incidencia de la lesión de isquemia-reperfusión grave del injerto en el postoperatorio inmediato del trasplante pulmonar