HD‑Zip I subfamily gene expression in soybean subjected to drought

O objetivo deste trabalho foi identificar genes candidatos da subfamília de fatores transcricionais HD‑Zip I que contribuem para a tolerância à seca em soja. Foram avaliados trifólios de soja de cultivar tolerante (Embrapa  48) e suscetível à seca (BR  16), sob três níveis de deficit hídrico: ausência, moderado (‑1,5 MPa) e severo (‑3,0 MPa). Pela análise dos promotores, foi identificada a presença de possíveis elementos cis-regulatórios relacionados à resposta à seca, nos três genes avaliados (GmHB6, GmHB13 e GmHB21). No entanto, não houve padrão de distribuição específico associado à maior tolerância do genótipo à seca. Com a análise comparativa, foram identificados seis elementos cis‑regulatórios potencialmente envolvidos na indução da expressão gênica sob seca. O gene GmHB13 foi exclusivamente induzido pela seca no genótipo tolerante, e o gene GmHB6 apresentou redução da expressão somente no genótipo suscetível. Já o gene GmHB21, apresentou aumento da expressão em ambos os genótipos. O gene GmHB13 é um importante elemento na regulação do mecanismo de tolerância à seca em soja, na cultivar tolerante Embrapa 48.The objective of this work was to identify candidate genes of the HD‑Zip I transcription factors subfamily that contribute to drought tolerance in soybean. Trifoliate soybean leaves of drought tolerant (Embrapa 48) and susceptible (BR 16) cultivars were evaluated under three levels of drought: absence, moderate (-1.5 MPa), and severe (-3.0 MPa). Promoter analysis indicated the presence of putative cis-regulatory elements related to water stress in the three genes evaluated (GmHB6, GmHB13, and GmHB21). However, there was no specific pattern of distribution associated with the higher drought tolerance of the genotype. By comparative analysis, six cis-regulatory elements potentially involved in the induction of gene expression under drought were identified. Gene GmHB13 was exclusively induced by drought in the tolerant cultivar, and GmHB6 had its expression reduced only in the susceptible cultivar. Gene GmHB21 showed increased expression in both genotypes. Gene GmHB13 is an important element in the regulation of the drought tolerance mechanism in soybean, in the tolerant cultivar Embrapa 48

Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4 -mediated Asian soybean rust resistance pathway

Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to Phakopsora pachyrhizi Sydow, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression in mock-inoculated and P. pachyrhizi-infected leaves of resistant soybean accession PI459025B (Rpp4) and the susceptible cultivar (Williams 82) across a 12-day time course. Unexpectedly, two biphasic responses were identified. In the incompatible reaction, genes induced at 12 h after infection (hai) were not differentially expressed at 24 hai, but were induced at 72 hai. In contrast, genes repressed at 12 hai were not differentially expressed from 24 to 144 hai, but were repressed 216 hai and later. To differentiate between basal and resistance-gene (R-gene) mediated defence responses, we compared gene expression in Rpp4-silenced and empty vector-treated PI459025B plants 14 days after infection (dai) with P. pachyrhizi. This identified genes, including transcription factors, whose differential expression is dependent upon Rpp4. To identify differentially expressed genes conserved across multiple P. pachyrhizi resistance pathways, Rpp4 expression datasets were compared with microarray data previously generated for Rpp2 and Rpp3-mediated defence responses. Fourteen transcription factors common to all resistant and susceptible responses were identified, as well as fourteen transcription factors unique to R-gene-mediated resistance responses. These genes are targets for future P. pachyrhizi resistance research

Molecular characterization of resistance to Asian soybean rust mediated by Rpp4

Práticas de gerenciamento são essenciais para o controle da ferrugem. O principal método de controle utilizado é a aplicação de fungicida, o qual aumenta substancialmente o custo de produção e são prejudiciais ao ambiente. A prevenção ainda é a melhor maneira de evitar perdas na produção de soja. Alternativas como plantar cultivares resistentes ao fungo também são importantes. O uso de variedades resistentes ou tolerantes é o método mais promissor para o controle da ferrugem asiática, cinco locus dominantes de resistência tem sido descrito na literatura: Rpp 1, Rpp2, Rpp3, Rpp4 e Rpp5. Entretanto pouco se sabe sobre a interação molecular desencadeada pela reconhecimento do patógeno pela planta em relação a ferrugem asiática. Entender os mecanismos moleculares envolvidos na resposta de defesa é de primária importância no planejamento de estratégias de controle do estresse e para consequente aumento da adaptação da planta. O Rpp4 foi mapeado no grupo de ligação G da soja (cromossomo 18) e o sequenciamento desta região no genótipo suscetível Williams82 (Wm82) e resistente (Pl459025B) identificou um cluster de genes de resistência CC-NBS- LRR. Meyers et al., (2009) desenvolveu construções para silenciamento induzido por vírus a partir das regiões NBD e LRR dos genes candidatos Rpp4 no genótipo Wm82 para testar se os genes parãlogos são responsáveis pela resistência no genótipo resistente (Pl459025B). Neste estudo, o RNA foi extraído de plantas silenciadas Rpp4LRR e de plantas controle (Vetor vazio). Análises transcricional de 3 replicatas biológicas foi feita utilizando GeneChip® Soybean Genome Array (Affymetrix®). Um total de 383 genes foram encontrados ser diferencialmente expressos entre plantas Rpp4 silenciadas e plantas não silenciadas (controle) quando infectadas com P. pachyrhizi. Dos 383 genes diferencialmente expressos, 22 foram induzidos e 361 foram reprimidos. Além disso, utilizando a ferramenta Clover (cis-element over representation) e TRANSFAC (transcription factor database) identificamos 33 sitios para fatores de transcrição presentes nos promotores dos genes diferencialmente expressos. Finalmente, para elucidar quais os genes são exclusivamentes mediados pela sinalização do Rpp4, nós comparamos os resultados de nosso experimento com os resultados de microarranjos oriundos de Rpp2, Rpp3 e Rpp4 resistente e suscetível. Nós identificamos 101 genes exclusivos. Além disso, com o objetivo de se obter maior informação sobre a função do Rpp4 nós utilizamos PCR quantitativo em tempo real (RT-qPCR) para analisar a expressão de todos os genes Rpp4 em diferentes tecidos da planta, diferentes estágios de desenvolvimento e depois da inoculação com P. pachyrhizi. Nós desenvolvemos um par de primers no domínio NBD que nós permitiu monitorar a expressão de todos os genes. O sequenciamento direto dos produtos originados no RT-qPCR nos permitiu diferenciar entre os 10 genes. Além disso nós examinamos a ocorrência de splice alternativo do gene Rpp4 na soja sob efeito da inoculação.The main control method used is the application of fungicides, which substantially increase the cost of production and are harmful to the environment. Prevention is still the best way to avoid more significant losses in soybean production. Alternatives, such as planting resistant varieties to the fungus, are also important. The use of resistant or tolerant varieties is the most promising method for control of Asian soybean rust. Recently, five single dominant genes to specific soybean rust isolates were described: Rpp1, Rpp2, Rpp3, Rpp4 and Rpp5. However, little is known about the molecular interaction between soybean and soybean rust and on the molecular pathway triggered by pathogen recognition. Understanding the molecular mechanisms involved in defense responses is of primary importance in planning strategies for controlling stress and consequently increasing plant adaptation to limiting conditions. Previously, Rpp4 was mapped to soybean linkage group G (chromosome 18), and the sequencing of this region in the susceptible genotype Williams 82 (Wm82) and resistant (Pl459025B) identified a cluster of CC- NBS-LRR resistance genes. Meyers et al. (2009) developed Virus lnduced Gene Silencing constructs from the NBD and LRR regions of the Wm82 Rpp4 Candidate genes to test whether paralogous genes were responsible for resistance in the resistant parent (Pl459025B). ln this study, RNA extracted from the same Rpp4 LRR silenced and empty vector treated plants described by Meyer et al. (2009) was compared. Transcriptome analyses of three independent biological replicates was performed using the GeneChip® Soybean Genome Array (Affymetrix®). A total of 383 genes were found to be significantly differentially expressed between Rpp4 silenced and no silenced plants infected with P. pachyrhizi. Of the 383 differentially expressed genes, 22 were induced, and 361 were suppressed. Using Clover (cis-element over representation) software and the TRANSFAC (transcription factor database we identified 33 transcription factor-binding sites significantly over represented in our differentially expressed genes when compared to all genes in the soybean genome, several with known roles in defense. Finally, to elucidate which genes are exclusively related to Rpp4-mediated signaling, we have compared the results of our experiment with microarray results from Rpp2, Rpp3 and Rpp4 resistant and susceptible reactions. We have identified 101 genes unique to the Rpp4-signaling pathway. ln addition, in order to obtain more information about Rpp4 function, we used real time quantitative PCR (RT-qPCR) to analyze the expression of all Rpp4 genes in different plant tissues, in different stages of development and after inoculation with P. pachyrhizi. We have developed a single pair of primers from the NBD domain that allow us to monitor the expression of all ten genes. Direct sequencing of the RT-qPCR product differentiates between the ten genes. ln addition we examined the occurrence of alternative splice Rpp4 gene under inoculation effect.Conselho Nacional de Desenvolvimento Científico e Tecnológic

REVIEW - Advances on molecular studies of the interaction soybean - Asian rust

Effective management practices are essential for controlling rust outbreaks. The main control methodused is the application of fungicides, which increases substantially the cost of production and is harmful to theenvironment. Prevention is still the best way to avoid more significant losses in soybean yields. Alternatives,such as planting resistant varieties to the fungus, are also important. The use of resistant or tolerant varietiesis the most promising method for controlling Asian soybean rust. Recently, five dominant genes resistant to soybean rust were described: Rpp1, Rpp2, Rpp3, Rpp4 and Rpp5. However, little is known about the molecular interaction among soybean plant and soybean rust and on the molecular pathway triggered by pathogen recognition. Understanding the molecular mechanisms involved in defense responses is of primary importance for planning strategies to control stress and, consequently, to increase plant adaptation to limiting condition

Expressão de genes da subfamília HD-Zip I em soja submetida à seca

O objetivo deste trabalho foi identificar genes candidatos da subfamília de fatores transcricionais HD-Zip I que contribuem para a tolerância à seca em soja. Foram avaliados trifólios de soja de cultivar tolerante (Embrapa 48) e suscetível à seca (BR 16), sob três níveis de deficit hídrico: ausência, moderado (-1,5 MPa) e severo (-3,0 MPa). Pela análise dos promotores, foi identificada a presença de possíveis elementos cis-regulatórios relacionados à resposta à seca, nos três genes avaliados (GmHB6, GmHB13 e GmHB21). No entanto, não houve padrão de distribuição específico associado à maior tolerância do genótipo à seca. Com a análise comparativa, foram identificados seis elementos cis-regulatórios potencialmente envolvidos na indução da expressão gênica sob seca. O gene GmHB13 foi exclusivamente induzido pela seca no genótipo tolerante, e o gene GmHB6 apresentou redução da expressão somente no genótipo suscetível. Já o gene GmHB21, apresentou aumento da expressão em ambos os genótipos. O gene GmHB13 é um importante elemento na regulação do mecanismo de tolerância à seca em soja, na cultivar tolerante Embrapa 48.The objective of this work was to identify candidate genes of the HD-Zip I transcription factors subfamily that contribute to drought tolerance in soybean. Trifoliate soybean leaves of drought tolerant (Embrapa 48) and susceptible (BR 16) cultivars were evaluated under three levels of drought: absence, moderate (-1.5 MPa), and severe (-3.0 MPa). Promoter analysis indicated the presence of putative cis-regulatory elements related to water stress in the three genes evaluated (GmHB6, GmHB13, and GmHB21). However, there was no specific pattern of distribution associated with the higher drought tolerance of the genotype. By comparative analysis, six cis-regulatory elements potentially involved in the induction of gene expression under drought were identified. Gene GmHB13 was exclusively induced by drought in the tolerant cultivar, and GmHB6 had its expression reduced only in the susceptible cultivar. Gene GmHB21 showed increased expression in both genotypes. Gene GmHB13 is an important element in the regulation of the drought tolerance mechanism in soybean, in the tolerant cultivar Embrapa 48

Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4 -mediated Asian soybean rust resistance pathway

Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to Phakopsora pachyrhizi Sydow, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression in mock-inoculated and P. pachyrhizi-infected leaves of resistant soybean accession PI459025B (Rpp4) and the susceptible cultivar (Williams 82) across a 12-day time course. Unexpectedly, two biphasic responses were identified. In the incompatible reaction, genes induced at 12 h after infection (hai) were not differentially expressed at 24 hai, but were induced at 72 hai. In contrast, genes repressed at 12 hai were not differentially expressed from 24 to 144 hai, but were repressed 216 hai and later. To differentiate between basal and resistance-gene (R-gene) mediated defence responses, we compared gene expression in Rpp4-silenced and empty vector-treated PI459025B plants 14 days after infection (dai) with P. pachyrhizi. This identified genes, including transcription factors, whose differential expression is dependent upon Rpp4. To identify differentially expressed genes conserved across multiple P. pachyrhizi resistance pathways, Rpp4 expression datasets were compared with microarray data previously generated for Rpp2 and Rpp3-mediated defence responses. Fourteen transcription factors common to all resistant and susceptible responses were identified, as well as fourteen transcription factors unique to R-gene-mediated resistance responses. These genes are targets for future P. pachyrhizi resistance research.This article is from Functional Plant Biology 40 (2013): 1029, doi:10.1071/FP12296. Posted with permission.</p