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

ABSTRACT: Effective management practices are essential for controlling rust outbreaks. The main control method used is the application of fungicides, which increases substantially the cost of production and is harmful to the environment. 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 varieties is 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 conditions. RESUMO: Avanços dos estudos moleculares da interação da soja - ferrugem asiática. Práticas efetivas são necessárias para o controle da ferrugem. O principal método de controle utilizado é a aplicação de fungicidas, o que aumentará substancialmente o custo de produção e são prejudiciais ao meio ambiente. A prevenção ainda é a melhor maneira de evitar mais perdas significativas na produção de soja. Alternativas, como o plantio de variedades resistentes ao fungo, também são importantes. O uso de variedades resistentes ou tolerantes é o método mais promissor para o controle da ferrugem asiática da soja. Recentemente, cinco genes de resistência a ferrugem da soja foram descritos Rpp1, Rpp2, Rpp3, Rpp4 e Rpp5. No entanto, pouco se sabe sobre a interação molecular entre a planta e ferrugem da soja e as rotas desencadeadas na planta pelo reconhecimento do patógeno. Compreender os mecanismos moleculares envolvidos nas respostas de defesa é de primordial importância no planejamento de estratégias para controle do estresse e, consequentemente, para aumentar a adaptação das plantas a condições limitantes

GENOSOJA - The Brazilian Soybean Genome Consortium: high throughput omics and beyond.

Plant genomes are among the most complex and large ones of our planet, with high levels of redundancy when compared to other eukaryotic groups, leading to intricate processes for gene regulation and evolution. Such a complexity demands interdisciplinary and multidimensional approaches in order to allow a better understanding of the processes able to exploit the whole potential of the existing genes in different species, including crop plants. Among cultivated plants, soybean [Glycine max (L.) Merr.] occupies an outstanding position due to its importance as source of protein and oil that may also be converted into biodiesel. The seeds are rarely consumed in natura, but many traditional food products have been consumed, as soymilk, and tofu, as well as fermented products as soy sauce, and soy paste among others, besides its wide use for animal feed. Soybean cultivation has been highly concentrated geographically, with only four countries (USA, Brazil, Argentina and China) accounting for almost 90% of world output. Asia (excluding China) and Africa, the two regions where most of the food insecure countries are located, account for only 5% of production. Among countries classified as 'undernourished', only India and Bolivia are significant producers of soybeans (FAO, 2009). Available evidences indicate that the cultivated soybean was domesticated from its wild relative Glycine soja (Sieb. and Zucc.) in China about 5,000 years ago (Carter et al., 2004). Since then, soybean has been grown primarily in temperate regions for thousands of years, first in Northern Asia and in more recent years in North America and countries of the Southern Cone of Latin America (FAO, 2009). The remarkable success of this crop in temperate zones is well known, but the crop presents also an important role in cropping systems of the tropics and subtropics, also in low fertile regions, as the Brazilian cerrado savannah (Spehar, 1995). The actual area cultivated worldwide with soybean is estimated to cover 103.5 millions of hectares, from which 24.2 only in Brazil, with considerable increases in the production achieved without significant increase in the cultivated area (Embrapa Soybean, 2011). As a legume, soybean is able to develop symbiotic interactions with rhizobia, allowing the fixation and assimilation of atmospheric N2, bearing quite specific mechanisms to coordinate this complex interaction (Oldroyd and Downie, 2008), absent in most angiosperm groups. Besides this peculiarity, soybean presents 2n = 40 chromosomes and was early characterized as an ancient polyploid (paleopolyploid) through genetic mapping studies that identified homeologous chromosome regions based upon duplicate RFLP markers (Shoemaker et al., 1996; Lee et al., 1999; 2001). Due to its allopolyploid nature, the first approaches regarded the generation of expressed sequences from different library tissues and conditions, including mainly ESTs (Expressed Sequence Tags; Nelson et al., 2005) partially in annotated databases, including ca. 40.000 full length cDNA clones available (Umezawa et al., 2008, see also RIKEN Soybean Full-Length cDNA Database), besides analyses regarding RNAseq under different tissues and development stages, as well as under different stressing situations (e.g. Libault et al., 2010; Severin et al., 2010). Also a complete shotgun genome sequence of the soybean cultivar Williams 82 was released, comprising 1.1-gigabase genome size allowing the integration of physical and high-density genetic maps, including 46,430 predicted protein-coding genes (Schmutz et al., 2010). The total amount of data publicly available at GenBank (NCBI) includes more than 120,000 nucleotide sequences (mainly mRNA), ~1,460,000 ESTs, ~368,000 genome sequences, ~80,000 proteins, 118 deposited structures and more than 6,2 million SNPs. Such numbers show that working with soybean is a very challenging task. By the other hand, despite of the wide data availability, most data regard cultivars from temperate regions (as Williams 82), not adapted for cultivation under tropical conditions, as it is the case of central Brazil and many other tropical countries that are subjected to distinct environmental stresses. The proposition of creating the GENOSOJA consortium was submitted in 2007 to the National Council for Scientific and Technological Development (CNPq), an agency linked to the Brazilian Ministry of Science and Technology (MCT), starting its activity in March 2008 with the participation of nine Brazilian institutions from different regions (Figure 1). The proposal aimed to study the soybean genome from its organization into the structural level, seeking to characterize and sequence important genomic regions and their products, thus contributing to the identification of genes using transcriptional and proteomic methods, especially considering the plant response to different biotic and abiotic stresses that affect the culture in the Southern hemisphere. Still, the GENOSOJA network aimed to approach not only whether a gene is induced or suppressed under a given condition, but also to determine the levels at which it is expressed, the interactions with other genes, their physical location and products, allowing the identification of important genes and metabolic pathways, vital for the development and study of plants tolerant to challenging situations. The GENOSOJA project is still in course and is structured into six Project Components (Figure 2), including management and addressing of different aspects of the soybean genome: I. Project management - responsible for the project administration, organization of meetings, group integration and research reports, among others. II. Structural Genomics - includes research activities related to the genomic physical architecture, including BAC anchoring (in the cultivar Conquista), promoter analysis and sequencing of gene-rich regions, also in comparison with other wild relatives of the genus Glycine, allowing studies of synteny and indication of regions important for ressequencing. This component is also responsible for the identification of single base polymorphisms (SNPs), very important for mapping purposes and marker assisted selection. III. Transcriptomics -comprises the largest research group, responsible for various expression profiling approaches using different strategies to access transcripts generated under different biotic (Asian rust: Phakopsora pachyrhizi, CPMMV: Cowpea mild mottle virus, nematodes: Meloydogyne javanica and Pratylenchus brachyurus) and abiotic (water deficit) stresses. In this workgroup different strategies were used, including: a) Subtractive cDNA libraries (76 bp tags, Solexa Illumina® sequencing) using contrasting materials submitted to biotic interactions, including diseases (~40 million tags; Asian rust and virus inoculation) and beneficial interactions (~10 million tags; inoculation with Bradyrhizobium japonicum), as well as water deficit (~42 million tags, comparing tolerant and susceptible accessions). b) SuperSAGE comprising ~3,2 Solexa Illumina® 26-bp tags distributed in six libraries generated under biotic (water deficit) and abiotic (Asian rust) stress comparisons. c) MicroRNA libraries (Solexa Illumina®, 1924 bp) including four libraries regarding water deficit ( 4,8 millions miRNAs) and other four regarding Asian rust (~7,9 million miRNAs). d) cDNA sequences (2,112 sequences, Sanger method) from roots infested with the nematode M. javanica compared with non stressed control. The three first above mentioned experiments were carried out using the same experimental conditions, generating an extensive comparable dataset to allow the understanding of the gene expression dynamic (Subtractive cDNA and SuperSAGE libraries), including biotic and abiotic cross-talk responses as well as the post transcriptional control (miRNA). IV. Proteomics -aimed to study the protein profile of soybean plants, low-mass protein and peptides identification and protein-protein interactions, using the same accessions and biological conditions established for the transcriptomic analyses to ensure complementarity and reduction of experimental variability, and thus, allowing the integration of both datasets in the functional characterization of the soybean genome. V. Expression Assays (transgenesis) -considering the results of transcriptomics and proteomics, most valuable gene candidates are being transformed in order to infer about their effects or biological function. Members of this group are also evaluating the vicinity of genes (UTRs) for the identification of regulatory regions (promoters, enhancers, cis-elements, etc.) that control their expression. VI. Bioinformatics -this workgroup developed the GENOSOJA database (see web resource) that includes a set of tools integrating the entire project data as compared with available sequences from other public data banks. The present issue represents the starting point of an extensive catalogue of products generated by the GENO-SOJA consortium, since all members agree that many additional inferences will be soon mature for publication and application to breeding projects. Thousands of candidate genes differentially expressed have been identified and are being validated using quantitative real time PCR, many regarding strongly induced genes in contrasting libraries (e.g. stressed against control or tolerant against sensible in the same condition). Many of them refer to uncharacterized genes, with no given function, representing relevant data to be worked out in future functional studies, since they may represent not yet described genes, some possibly unique to legumes and important for plant breeding. Finally, the present volume does not represent a milestone for completion of the GENOSOJA project, but an announcement of its birth, crowned with solid growth, integration and consolidation prospects. The data generated by the GENOSOJA consortium will also join the worldwide effort to study the soybean genome through the participation in the International Soybean Genome Consortium (ISGC). In this sense, the next step involves the public release of the generated data, which shall be available for the world community, allowing the effective integration with other networks throughout the world

Expression analyses of candidate resistance genes in the Rpp4 Asian Soybean Rust resistance locus.

Asian Soybean Rust (ASR), caused Phakopsora pachyrhizi, is considered the most severe soybean disease around the world. Infection of susceptible genotypes leads to early defoliation, incomplete seed development, and yield losses as high as 80%. Five ASR resistance genes have been identified in soybean: Rpp1, Rpp2, Rpp3, Rpp4 and Rpp5. Of particular interest is Rpp4, which has remained stable and confers resistance against P. pachyrhizi isolates from around the world. Rpp4 was mapped to soybean linkage group G (chromosome 18), 1.9 cM from simple sequence repeat (SSR) marker Satt288. Sequencing of this region in the susceptible genotype Williams 82 identified a cluster of three CC-NBS-LRR resistance genes. Virus Induced Gene Silencing was used to demonstrate that orthologous genes were responsible for resistance. We have now sequenced a >460 kb region of the Rpp4 locus in the resistant mapping parent PI459025B. Eight CC-NBS-LRR resistance genes have been identified in this region. In order to obtain more information about Rpp4 function, we are using real time quantitative PCR (qRT-PCR) to analyze the expression of all eight 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 monitors the expression of all eight genes. Direct sequencing of the RT-PCR product differentiates between the eight genes. Detailed sequence analyses of the Rpp4 locus suggest that intra- and intergenic duplications and recombination have played an important role in creating genetic diversity. Alternative splicing of intragenic duplications may create additional sequence diversity at an RNA level. We are developing primers that will allow us to monitor alternative splicing events. Sequencing of the RT-PCR products will determine if alternative splicing plays a role in generating additional sequence diversity at the Rpp4 locus.Edição de Poster da 11. Annual National Outreach Scholarship Conference, Raleigh

Identificação de genes diferencialmente expressos em soja em resposta à Phakopsora pachyrhizi pela metodologia ACP.

A ferrugem asiática da soja (FAS), causada pelo fungo Phakopsora pachyrhizi, é uma doença foliar destrutiva em quase todos os países produtores de soja. Ferramentas biotecnológicas podem auxiliar no entendimento dos mecanismos de resposta de defesa a este fungo em nível molecular e consequentemente no controle da doença. Para identificar genes envolvidos em resposta à infecção com FAS, RNA de folhas infectadas e não infectadas de genótipos de soja resistente (PI561356) e suscetível (BRS 184), foram analisadas pela metodologia ACP (Annealing Control Primer). Quarenta ACPs foram utilizados para identificar e sequenciar 59 genes diferencialmente expressos (DEGs) e 44 destes genes mostraram homologia com proteínas conhecidas e foram identificados como envolvidos principalmente em fotossíntese, síntese e degradação de proteínas. A maioria dos DEGs que foi induzida nas plantas resistentes estava envolvida na categoria funcional de defesa, energia, atividade antioxidante e transporte celular. Quatro genes com diferentes padrões de expressão foram selecionados e caracterizados por meio de análises de RT-qPCR para obter um perfil de expressão específico nos genótipos de soja resistente (PI561356), tolerante (BRS 231) e suscetível (BRS 184). Análises de RT-qPCR revelaram que as respostas iniciais são mais intensas nas plantas resistentes. Adicionalmente, foi possível identificar o gene tiazol como candidato para estudos mais detalhados de seu envolvimento com a resistência a FAS

Mapeamento de QTLs de características sob influência da ferrugem asiática da soja.

A ferrugem asiática da soja, causada pelo fungo Phakopsora pachyrhizi (Syd. & P.Syd.), é um dos principais fatores bióticos causadores de prejuízos de redução do potencial sobre a cultura da soja, podendo causar redução de produtividade superiores a 75%. Hoje, sabe-se que uma das formas mais eficazes de controle da doença é através da resistência genética. Devido à baixa durabilidade de genes de resistência vertical, estudos para o desenvolvimento de linhagens com genes de resistência horizontal são de extrema importância. Assim, objetivou-se nesse trabalho identificar QTLs em soja para características agronômicas sob influência da ferrugem asiática. Métodos: 83 marcadores de microssatélites e 2 marcadores morfológicos foram utilizados para a construção de um mapa genético em uma população de linhagens endogâmicas recombinantes (RILs) Ao mesmo tempo, foram feitas avaliações fenotípicas para várias características correlacionadas a doença nas RILs sob ação do patógeno em campo experimental da Embrapa Soja e em fitotron no Japão, a fim de buscar QTLs relacionados à resistência horizontal ao patógeno, e também para verificar a existência de linhagens com maior nível de resistência à doença. Resultados: Uma cobertura parcial de 1.023,5 cM do genoma da soja foi obtida em 19 grupos de ligação. Foram detectados 17 QTLs que possam estar contribuindo para a resistência horizontal à doença, sendo a maior parte deles localizados no grupo C2 e L. Grande parte dos QTLs observados possui efeito pleiotrópico para mais de uma característica analisada, fato constatado através de análises de correlação entre as características estudadas. Foram selecionadas 16 linhagens que apresentaram as melhores características sob ação da ferrugem asiática da soja. Para algumas características, verificou-se a existência de segregação transgressiva, já que neste caso os genes estavam dispersos entre os parentais. Conclusão: Os dados gerados poderão contribuir para o programa de melhoramento contra a doença, O mapa gerado também pode servir para detecção de outros QTLs relacionados a outras características de grande interesse agronômico, já que ambas as cultivares que foram cruzadas possuem resistência a várias outras doenças