The sunflower transcription factor HaWRKY76 confers drought and flood tolerance to Arabidopsis thaliana plants without yield penalty

Key message: Arabidopsis transgenic plants expressing the sunflower transcription factor HaWRKY76 exhibit increased yield and tolerance to drought and flood stresses. The genetic construct containing HaWRKY76 is proposed as a potential biotechnological tool to improve crops. Abstract: Water deficit and water excess are abiotic stress factors that seriously affect crops worldwide. To increase the tolerance to such stresses without causing yield penalty constitutes a major goal for biotechnologists. In this survey, we report that HaWRKY76, a divergent sunflower WRKY transcription factor, is able to confer both dehydration and submergence tolerance to Arabidopsis transgenic plants without yield penalty. The expression pattern of HaWRKY76 was analyzed in plants grown in standard conditions and under different watering regimes indicating a regulation by water availability. The corresponding cDNA was isolated and cloned under the control of a constitutive promoter and Arabidopsis plants were transformed with this construct. These transgenic plants presented higher biomass, seed production and sucrose content than controls in standard growth conditions. Moreover, they exhibited tolerance to mild drought or flood (complete submergence/waterlogging) stresses as well as the same or increased yield, depending on the stress severity and plant developmental stage, compared with controls. Drought tolerance occurred via an ABA-independent mechanism and induction of stomatal closure. Submergence tolerance can be explained by the carbohydrate (sucrose and starch) preservation achieved through the repression of fermentation pathways. Higher cell membrane stability and chlorenchyma maintenance could be the nexus between tolerance responses in front of both stresses. Altogether, the obtained results indicated that HaWRKY76 can be a potential biotechnological tool to improve crops yield as well as drought and flood tolerances.Fil: Raineri, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ribichich, Karina Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Fusarium head blight of wheat: evaluation of morphological characters associated with desease resistance

El agente causal de la fusariosis de la espiga de trigo aislado más frecuentemente en la Argentina es Fusarium graminearum Schwabe. El patógeno ingresa principalmente por las anteras, o bien por penetración directa o por estomas, expandiéndose luego al raquis y a otras espiguillas. Los mecanismos de resistencia principales son: a la penetración (Tipo I), y a la propagación (Tipo II). En estudios previos, se han postulado características exomorfológicas asociadas con la resistencia Tipo I. En este estudio se proponen cuatro caracteres exomorfológicos que pueden considerarse asociados a susceptibilidad o resistencia: ángulo mayor de apertura de antecios, ángulo entre lemma1 y gluma1, longitud de raquilla y de raquis. Se inocularon espigas en espigazón y floración del cultivar susceptible PROINTA Oasis y del resistente Sumai 3, y a diferentes tiempos post-inoculación se midieron los caracteres mencionados. Se determinaron las diferencias para cada variedad y entre variedades para cada caracter y se compararon con los valores de severidad de la enfermedad. En ambas variedades los caracteres se mantuvieron constantes en función del tiempo. Una apertura mayor de antecios durante la floración en relación con la espigazón podría relacionarse con una mayor severidad en la variedad resistente y una superficie menor de lemma expuesta para ambas variedades, podría señalar la entrada al patógeno durante la espigazón. Por el contrario, las longitudes de raquilla y de raquis fueron mayores en la variedad resistente que en la susceptible mientras que fue menor la severidad de la enfermedad, lo cual apoya la existencia de una forma de resistencia Tipo II en Sumai 3.The most frequent pathogenic species producing head blight of wheat (Triticum aestivum L.) isolated in Argentina is Fusarium graminearum Schwabe. The fungus invades anthers, or may penetrate directly or by natural openings. Then, colonizes the rachis and the other spikelets. The main resistance mechanisms described are: to penetration (Type I), and to the spread (Type II). Previous studies have proposed morphological characteristics as associated with resistance Type I. We propose four morphological characters as associated to resistance or susceptibility. Spikes at heading and flowering stages of the susceptible cultivar PROINTA Oasis and the resistant Sumai 3, were inoculated and assesed at six times post inoculation. Disease severity was calculated, and the following morphological characters were measured: opening angle of anthecia, angle between lemma 1 and glume 1, rachilla and rachis lengths. Differences among these characters in each cultivar and between cultivars were evaluated, and were compared with the severity values. In both cultivars, the characters were constants over the time. The opening angle of anthecia was bigger at flowering than at heading and could be related with the increased severity in the resistant at that stage compared with the susceptible cultivar. The exposed lemma area was smaller at heading than at flowering in both cultivars which could represent the entry for the pathogen at the first stage. On the other hand, the rachilla and the rachis lengths were bigger in the resistant than the susceptible cultivar, and the severity was the opposite, which support the existence of a kind of resistance type II in the resistant cultivar.Facultad de Ciencias Agrarias y Forestale

Fusarium head blight of wheat: evaluation of morphological characters associated with desease resistance

El agente causal de la fusariosis de la espiga de trigo aislado más frecuentemente en la Argentina es Fusarium graminearum Schwabe. El patógeno ingresa principalmente por las anteras, o bien por penetración directa o por estomas, expandiéndose luego al raquis y a otras espiguillas. Los mecanismos de resistencia principales son: a la penetración (Tipo I), y a la propagación (Tipo II). En estudios previos, se han postulado características exomorfológicas asociadas con la resistencia Tipo I. En este estudio se proponen cuatro caracteres exomorfológicos que pueden considerarse asociados a susceptibilidad o resistencia: ángulo mayor de apertura de antecios, ángulo entre lemma1 y gluma1, longitud de raquilla y de raquis. Se inocularon espigas en espigazón y floración del cultivar susceptible PROINTA Oasis y del resistente Sumai 3, y a diferentes tiempos post-inoculación se midieron los caracteres mencionados. Se determinaron las diferencias para cada variedad y entre variedades para cada caracter y se compararon con los valores de severidad de la enfermedad. En ambas variedades los caracteres se mantuvieron constantes en función del tiempo. Una apertura mayor de antecios durante la floración en relación con la espigazón podría relacionarse con una mayor severidad en la variedad resistente y una superficie menor de lemma expuesta para ambas variedades, podría señalar la entrada al patógeno durante la espigazón. Por el contrario, las longitudes de raquilla y de raquis fueron mayores en la variedad resistente que en la susceptible mientras que fue menor la severidad de la enfermedad, lo cual apoya la existencia de una forma de resistencia Tipo II en Sumai 3.The most frequent pathogenic species producing head blight of wheat (Triticum aestivum L.) isolated in Argentina is Fusarium graminearum Schwabe. The fungus invades anthers, or may penetrate directly or by natural openings. Then, colonizes the rachis and the other spikelets. The main resistance mechanisms described are: to penetration (Type I), and to the spread (Type II). Previous studies have proposed morphological characteristics as associated with resistance Type I. We propose four morphological characters as associated to resistance or susceptibility. Spikes at heading and flowering stages of the susceptible cultivar PROINTA Oasis and the resistant Sumai 3, were inoculated and assesed at six times post inoculation. Disease severity was calculated, and the following morphological characters were measured: opening angle of anthecia, angle between lemma 1 and glume 1, rachilla and rachis lengths. Differences among these characters in each cultivar and between cultivars were evaluated, and were compared with the severity values. In both cultivars, the characters were constants over the time. The opening angle of anthecia was bigger at flowering than at heading and could be related with the increased severity in the resistant at that stage compared with the susceptible cultivar. The exposed lemma area was smaller at heading than at flowering in both cultivars which could represent the entry for the pathogen at the first stage. On the other hand, the rachilla and the rachis lengths were bigger in the resistant than the susceptible cultivar, and the severity was the opposite, which support the existence of a kind of resistance type II in the resistant cultivar.Facultad de Ciencias Agrarias y Forestale

Comparative EST analysis provides insights into the basal aquatic fungus Blastocladiella emersonii

BACKGROUND: Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. In this sense, some ancestral characteristics of fungi and animals or fungi and plants could have been retained in this aquatic fungus and lost in members of late-diverging fungal species. To identify in B. emersonii sequences associated with these ancestral characteristics two approaches were followed: (1) a large-scale comparative analysis between putative unigene sequences (uniseqs) from B. emersonii and three databases constructed ad hoc with fungal proteins, animal proteins and plant unigenes deposited in Genbank, and (2) a pairwise comparison between B. emersonii full-length cDNA sequences and their putative orthologues in the ascomycete Neurospora crassa and the basidiomycete Ustilago maydis. RESULTS: Comparative analyses of B. emersonii uniseqs with fungi, animal and plant databases through the two approaches mentioned above produced 166 B. emersonii sequences, which were identified as putatively absent from other fungi or not previously described. Through these approaches we found: (1) possible orthologues of genes previously identified as specific to animals and/or plants, and (2) genes conserved in fungi, but with a large difference in divergence rate in B. emersonii. Among these sequences, we observed cDNAs encoding enzymes from coenzyme B(12)-dependent propionyl-CoA pathway, a metabolic route not previously described in fungi, and validated their expression in Northern blots. CONCLUSION: Using two different approaches involving comparative sequence analyses, we could identify sequences from the early-diverging fungus B. emersonii previously considered specific to animals or plants, and highly divergent sequences from the same fungus relative to other fungi

Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly outyields the wild type

HaHB4 is a sunflower transcription factor belonging to the homeodomain-leucine zipper I family whose ectopic expression in Arabidopsis triggers drought tolerance. The use of PCR to clone the HaHB4 coding sequence for wheat transformation caused unprogrammed mutations producing subtle differences in its activation ability in yeast. Transgenic wheat plants carrying a mutated version of HaHB4 were tested in 37 field experiments. A selected transgenic line yielded 6% more (P<0.001) and had 9.4% larger water use efficiency (P<0.02) than its control across the evaluated environments. Differences in grain yield between cultivars were explained by the 8% improvement in grain number per square meter (P<0.0001), and were more pronounced in stress (16% benefit) than in non-stress conditions (3% benefit), reaching a maximum of 97% in one of the driest environments. Increased grain number per square meter of transgenic plants was accompanied by positive trends in spikelet numbers per spike, tillers per plant, and fertile florets per plant. The gene transcripts associated with abiotic stress showed that HaHB4’s action was not dependent on the response triggered either by RD19 or by DREB1a, traditional candidates related to water deficit responses. HaHB4 enabled wheat to show some of the benefits of a species highly adapted to water scarcity, especially in marginal regions characterized by frequent droughts.EEA PergaminoFil: González, Fernanda Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Departamento Ecofisiología; Argentina.CONICET-UNNOBA.CITNOBA; ArgentinaFil: Capella, Matías. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ribichich, Karina Fabiana. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Curín, Facundo. CONICET-UNNOBA. CITNOBA, (Pergamino); ArgentinaFil: Giacomelli, Jorge Ignacio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ayala, Francisco. INDER/BIOCRES (Rosario); ArgentinaFil: Watson, Gerónimo. INDEAR/BIOCERES (Rosario); ArgentinaFil: Otegui, María Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Ecofisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Lía Chan, Raquel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral; Argentin

An Interdisciplinary Approach to Study the Performance of Second-generation Genetically Modified Crops in Field Trials: A Case Study With Soybean and Wheat Carrying the Sunflower HaHB4 Transcription Factor

Research, production, and use of genetically modified (GM) crops have split the world between supporters and opponents. Up to now, this technology has been limited to the control of weeds and pests, whereas the second generation of GM crops is expected to assist farmers in abiotic stress tolerance or improved nutritional features. Aiming to analyze this subject holistically, in this presentation we address an advanced technology for drought-tolerant GM crops, upscaling from molecular details obtained in the laboratory to an extensive network of field trials as well as the impact of the introduction of this innovation into the market. Sunflower has divergent transcription factors, which could be key actors in the drought response orchestrating several signal transduction pathways, generating an improved performance to deal with water deficit. One of such factors, HaHB4, belongs to the homeodomain-leucine zipper family and was first introduced in Arabidopsis. Transformed plants had improved tolerance to water deficits, through the inhibition of ethylene sensitivity and not by stomata closure. Wheat and soybean plants expressing the HaHB4 gene were obtained and cropped across a wide range of growing conditions exhibiting enhanced adaptation to drought-prone environments, the most important constraint affecting crop yield worldwide. The performance of wheat and soybean, however, differed slightly across mentioned environments; whereas the improved behavior of GM wheat respect to controls was less dependent on the temperature regime (cool or warm), differences between GM and wild-type soybeans were remarkably larger in warmer compared to cooler conditions. In both species, these GM crops are good candidates to become market products in the near future. In anticipation of consumers’ and other stakeholders’ interest, spectral analyses of field crops have been conducted to differentiate these GM crops from wild type and commercial cultivars. In this paper, the potential impact of the release of such market products is discussed, considering the perspectives of different stakeholders.Fil: González, Fernanda Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Norte. Estación Experimental Agropecuaria Pergamino; ArgentinaFil: Rigalli, Nicolas Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas. Universidad Nacional de Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; ArgentinaFil: Miranda, Patricia Vivian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Romagnoli, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas. Universidad Nacional de Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; ArgentinaFil: Ribichich, Karina Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Trucco, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Portapila, Margarita Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas. Universidad Nacional de Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; ArgentinaFil: Otegui, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Successful field performance in warm and dry environments of soybean expressing the sunflower transcription factor HB4

Ribichich, Karina Fabiana. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral Santa Fe, Argentina.Chiozza, Mariana. INDEAR/BIOCERES. Rosario, Argentina.Ávalos Britez, Selva. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino (EEA Pergamino). Pergamino, Buenos Aires, Argentina.Cabello, Julieta V. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral Santa Fe, Argentina.Arce, Augustin L. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral Santa Fe, Argentina.Watson, Gerónimo. INDEAR/BIOCERES. Rosario, Argentina.Arias, Claudia. Universidad Nacional de Rosario. CIFASIS. Rosario, Argentina.Portapila, Margarita. Universidad Nacional de Rosario. CIFASIS. Rosario, Argentina.Trucco, Federico. INDEAR/BIOCERES. Rosario, Argentina.Otegui, María Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino (EEA Pergamino). Pergamino, Buenos Aires, Argentina.Chan, Raquel Lía. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral Santa Fe, Argentina.3142–3156Soybean yield is limited primarily by abiotic constraints. No transgenic soybean with improved abiotic stress tolerance is commercially available. We transformed soybean plants with genetic constructs able to express the sunflower transcription factor HaHB4, which confers drought tolerance to Arabidopsis and wheat. One line (b10H) carrying the sunflower promoter was chosen among three independent lines because it exhibited the best performance in seed yield, and was evaluated in the greenhouse and in 27 field trials in different environments in Argentina. In greenhouse experiments, transgenic plants showed increased seed yield under stress conditions together with greater epicotyl diameter, larger xylem area, and increased water use efficiency compared with controls. They also exhibited enhanced seed yield in warm and dry field conditions. This response was accompanied by an increase in seed number that was not compensated by a decrease in individual seed weight. Transcriptome analysis of plants from a field trial with maximum difference in seed yield between genotypes indicated the induction of genes encoding redox and heat shock proteins in b10H. Collectively, our results indicate that soybeans transformed with HaHB4 are expected to have a reduced seed yield penalty when cultivated in warm and dry conditions, which constitute the best target environments for this technology

Sequence analysis expressed during the sporulation phase of the aquatic fungus Blastocladiella emersonii

Blastocladiella emersonii é um fungo aquático da classe dos quitridiomicetos, notável pelas mudanças morfogenéticas que ocorrem durante o seu ciclo de vida. Neste trabalho isolamos 8.495 seqüências expressas (ESTs) deste fungo, que representam 3.226 seqüências únicas putativas. Destas seqüências, 37% foram classificadas segundo o processo biológico onde estariam envolvidas, de acordo com o sistema de anotação do Gene Ontology (GO). Analisamos os perfis de expressão in silico das ESTs usando estatística Bayesiana e os resultados obtidos foram validados por Northern blot para sete perfis de expressão selecionados. Pudemos encontrar boa correlação entre vários padrões de expressão e determinados processos biológicos. Foram selecionadas algumas seqüências potencialmente envolvidas com a esporulação do fungo para melhor caracterização. Analisamos a expressão de dois genes codificando centrinas (BeCenl e BeCen2) pertencentes a subfamílias distintas. Centrinas são proteínas ligantes de cálcio envolvidas em diferentes processos como o direcionamento do aparelho flagelar e a duplicação dos centros organizadores de microtúbulos (MTOCs). Observamos que os níveis da proteína BeCenl, que não havia sido descrita em fungos, apresentam um máximo aos 150 min da esporulação, defasado do pico de expressão do seu mRNA que ocorre aos 90 min deste estágio. A proteína BeCen2 está presente em níveis constantes durante todo a ciclo de vida do fungo, embora o seu mRNA apresente um pico de expressão aos 120 min da esporulação. Experimentos de imunofluorescência localizaram a proteína BeCenl no citoplasma e no corpo basal do zoósporo. Estes dados sugerem que BeCenl atue na re-orientação e movimento dos corpos basais e BeCen2 na duplicação dos MTOCs. Investigamos também a expressão de dois genes codificando proteína-quinases dependentes de ciclina putativas (BeCdkl e BeCdk2). Apenas uma Cdk (Cdkl) foi descrita em fungos como diretamente envolvida no controle do ciclo celular. Ambos os genes apresentam expressão diferencial, com níveis máximos de mRNA para os dois casos aos 90 min da esporulação. Por outro lado, a proteína BeCdkl está presente durante todo o ciclo de vida do fungo e foi localizada no núcleo e no capacete nuclear dos zoósporos. Um transportador putativo de hexose (Bemst) foi também analisado, com base na regulação por glicose de genes envolvidos com o ciclo celular observada em eucariotos. Verificou-se que os níveis do mRNA de Bemst diminuem drasticamente durante a esporulação, mas glicose ou outras hexoses não afetaram a expressão de Bemst.Blastocladiella emersonii is an aquatic fungus that belongs to the class of chytridiomycetes, notable for the morphogenetic processes which occur during its life cycle. In this work we have isolated 8,495 expressed sequence tags (ESTs) from this fungus, representing 3,226 putative unigenes. From these unigenes, 37% were classified into a biological process, as a result of Gene Ontology (GO) annotation. Furthermore, we analyzed the expression profile in silico of each transcript using Bayesian Statistics and seven of these profiles were validated by Northern blot analysis. In addition, we found a good correlation between several of these expression patterns and certain biological processes. Some ESTs potentially involved in the sporulation of the fungus were selected to be further characterized. We analyzed the expression of two genes encoding two centrins (BeCenl and BeCen2) of distinct subfamilies. Centrins are calcium-binding proteins involved in different processes such as basal body orientation and duplication of the microtubuleorganizing centers (MTOCs). The amount of BeCenl, a centrin ortholog not previously described in fungi, presents a maximum at 150 min of sporulation, whereas the peak of its mRNA occurs at 90 min of this stage. Protein BeCen2 presents constant levels during the entire life cycle of the fungus, even though its mRNA shows a peak of expression at 120 min of sporulation. In addition, immunofluorescent studies localized BeCenl in the cytoplasm and the basal body of zoospores. These results suggest that BeCenl plays a role in re-orientation and movement of basal bodies and BeCen2 in MTOCs duplication. We also investigated the expression of two genes encoding putative cyclin-dependent protein kinases (BeCdkl and BeCdk2). Only one type of Cdk (Cdkl) directly involved in cell cycle control has been described in other fungi. Both genes were found to be differentially expressed, with maximum mRNA levels being detected in either case at 90 min of sporulation. In contrast, BeCdk1 is present throughout the life cycle of the fungus and was immunolocalized in the nuc1eus and the nuclear cap of zoospores. A putative hexose transporter (Bemst) was also investigated, taking into account the regulation by glucose of cell cycle controlled genes in eukaryotes. We found that Bemst mRNA levels decrease drastically during sporulation, but glucose and other hexoses had no effect on Bemst expression

Sequence analysis expressed during the sporulation phase of the aquatic fungus Blastocladiella emersonii

Blastocladiella emersonii é um fungo aquático da classe dos quitridiomicetos, notável pelas mudanças morfogenéticas que ocorrem durante o seu ciclo de vida. Neste trabalho isolamos 8.495 seqüências expressas (ESTs) deste fungo, que representam 3.226 seqüências únicas putativas. Destas seqüências, 37% foram classificadas segundo o processo biológico onde estariam envolvidas, de acordo com o sistema de anotação do Gene Ontology (GO). Analisamos os perfis de expressão in silico das ESTs usando estatística Bayesiana e os resultados obtidos foram validados por Northern blot para sete perfis de expressão selecionados. Pudemos encontrar boa correlação entre vários padrões de expressão e determinados processos biológicos. Foram selecionadas algumas seqüências potencialmente envolvidas com a esporulação do fungo para melhor caracterização. Analisamos a expressão de dois genes codificando centrinas (BeCenl e BeCen2) pertencentes a subfamílias distintas. Centrinas são proteínas ligantes de cálcio envolvidas em diferentes processos como o direcionamento do aparelho flagelar e a duplicação dos centros organizadores de microtúbulos (MTOCs). Observamos que os níveis da proteína BeCenl, que não havia sido descrita em fungos, apresentam um máximo aos 150 min da esporulação, defasado do pico de expressão do seu mRNA que ocorre aos 90 min deste estágio. A proteína BeCen2 está presente em níveis constantes durante todo a ciclo de vida do fungo, embora o seu mRNA apresente um pico de expressão aos 120 min da esporulação. Experimentos de imunofluorescência localizaram a proteína BeCenl no citoplasma e no corpo basal do zoósporo. Estes dados sugerem que BeCenl atue na re-orientação e movimento dos corpos basais e BeCen2 na duplicação dos MTOCs. Investigamos também a expressão de dois genes codificando proteína-quinases dependentes de ciclina putativas (BeCdkl e BeCdk2). Apenas uma Cdk (Cdkl) foi descrita em fungos como diretamente envolvida no controle do ciclo celular. Ambos os genes apresentam expressão diferencial, com níveis máximos de mRNA para os dois casos aos 90 min da esporulação. Por outro lado, a proteína BeCdkl está presente durante todo o ciclo de vida do fungo e foi localizada no núcleo e no capacete nuclear dos zoósporos. Um transportador putativo de hexose (Bemst) foi também analisado, com base na regulação por glicose de genes envolvidos com o ciclo celular observada em eucariotos. Verificou-se que os níveis do mRNA de Bemst diminuem drasticamente durante a esporulação, mas glicose ou outras hexoses não afetaram a expressão de Bemst.Blastocladiella emersonii is an aquatic fungus that belongs to the class of chytridiomycetes, notable for the morphogenetic processes which occur during its life cycle. In this work we have isolated 8,495 expressed sequence tags (ESTs) from this fungus, representing 3,226 putative unigenes. From these unigenes, 37% were classified into a biological process, as a result of Gene Ontology (GO) annotation. Furthermore, we analyzed the expression profile in silico of each transcript using Bayesian Statistics and seven of these profiles were validated by Northern blot analysis. In addition, we found a good correlation between several of these expression patterns and certain biological processes. Some ESTs potentially involved in the sporulation of the fungus were selected to be further characterized. We analyzed the expression of two genes encoding two centrins (BeCenl and BeCen2) of distinct subfamilies. Centrins are calcium-binding proteins involved in different processes such as basal body orientation and duplication of the microtubuleorganizing centers (MTOCs). The amount of BeCenl, a centrin ortholog not previously described in fungi, presents a maximum at 150 min of sporulation, whereas the peak of its mRNA occurs at 90 min of this stage. Protein BeCen2 presents constant levels during the entire life cycle of the fungus, even though its mRNA shows a peak of expression at 120 min of sporulation. In addition, immunofluorescent studies localized BeCenl in the cytoplasm and the basal body of zoospores. These results suggest that BeCenl plays a role in re-orientation and movement of basal bodies and BeCen2 in MTOCs duplication. We also investigated the expression of two genes encoding putative cyclin-dependent protein kinases (BeCdkl and BeCdk2). Only one type of Cdk (Cdkl) directly involved in cell cycle control has been described in other fungi. Both genes were found to be differentially expressed, with maximum mRNA levels being detected in either case at 90 min of sporulation. In contrast, BeCdk1 is present throughout the life cycle of the fungus and was immunolocalized in the nuc1eus and the nuclear cap of zoospores. A putative hexose transporter (Bemst) was also investigated, taking into account the regulation by glucose of cell cycle controlled genes in eukaryotes. We found that Bemst mRNA levels decrease drastically during sporulation, but glucose and other hexoses had no effect on Bemst expression