The largest group of plant disease resistance (R) genes that share similar structures contains a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers and homology searches from public databases. Multiple combinations of degenerate primers were designed from three conserved motifs (one motif was used for a subgroup-specific primer design) in the NBS regions of R genes of various plants. All combinations of primer pairs were used to amplify genomic DNA from sorghum. TIR-specific primer combinations showed no PCR amplification in sorghum. Homology searches identified many NBS-encoding sequences among the expressed or genomic molecular database entries for sorghum. Motif analysis of the sorghum NBS sequences that were identified in this study revealed eight major conserved motifs plus two additional highly conserved motifs, but no TIR-specific motifs. Phylogenetic analysis of sorghum NBS sequences showed tree topology typical of NBS-LRR genes, including clustered nodes and longbranch lengths. Eleven distinct families of NBS sequences, representing a highly diverse sample, were isolated from Sorghum bicolor. With two exceptions, sorghum RGA families appeared to be closely related in sequence to at least one R-gene cloned from other species. In addition, deduced amino acid sequences of sorghum RGAs showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)- type R-genes. Mapping with sorghum RGA markers revealed one linkage group containing four out of ten randomly selected markers, suggesting non-random distribution of NBS sequences in the sorghum genome. Rice sequences homologous to sorghum NBS sequences were found from two-way BLAST searches. Some of them were shown to be orthologs, when determined by using phylogenetic approaches which combined five different evolution models and tree-building methods.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-12T13:20:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_FlaviaAraujo_2015_VersãoFinal.pdf: 4388867 bytes, checksum: 1ebc175e90318442d9eb1b8a4a688b3c (MD5) / Made available in DSpace on 2016-07-12T13:20:39Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_FlaviaAraujo_2015_VersãoFinal.pdf: 4388867 bytes, checksum: 1ebc175e90318442d9eb1b8a4a688b3c (MD5) Previous issue date: 2015-03-02 / FACEPE / A cultura do feijão-caupi [Vigna unguiculata (L.) Walp.] apresenta importância econômica em nível internacional, entretanto ela é frequentemente acometida por uma diversidade de patógenos. Nesse contexto a família gênica NBS-LRR de genes de Resistência (R) se destaca devido ao seu papel fundamental na defesa das plantas contra o ataque de patógenos, sendo a maior e mais diversificada família desse grupo. O objetivo do presente estudo foi caracterizar genes NBS-LRR em feijão-caupi, desenvolver marcadores moleculares RGA (Resistance Gene Analogs) e validar genes diferencialmente expressos em uma interação planta-vírus. Inicialmente, sequências candidatas para genes NBS-LRR foram obtidas no banco de dados NordEST, procedendo-se com a anotação dos dados, tradução e identificação dos domínios conservados por meio de ferramentas in silico. Um total de 57 sequências NBS-LRR completas foi identificado em feijão-caupi. Como as proteínas codificadas pelos genes R apresentam domínios e motivos conservados, foi possível desenvolver marcadores RGAs usando as sequências de feijão-caupi como sonda contra o banco de Phaseolus vulgaris L. Foram desenhados 16 pares de iniciadores para P. vulgaris, identificando-se um percentual de 87,5% de transferibilidade para o feijão-caupi. Destes, dois foram polimórficos e apresentaram segregação mendeliana em uma população de mapeamento para o vírus do mosaico severo do feijão-caupi (CPSMV). Os 57 candidatos foram ancorados nos 20 pseudocromossomos de Glycine max (L.) Merr., verificando-se repetições in tandem deste grupo gênico. A análise de expressão gênica diferencial in silico foi realizada utilizando dados de RNAseq e SuperSAGE. A validação da expressão gênica via RT-qPCR foi através do desenho de primers, com os dados de SuperSAGE, onde três genes alvo apresentaram indução nos níveis de expressão após 16 horas da inoculação com o patógeno. Esses resultados mostram-se valiosos para o melhoramento genético do feijão-caupi. / The cowpea [Vigna unguiculata (L.) Walp.] culture has an international economic importance, however it is often affected by a diversity of pathogens. In this context the NBS-LRR family of resistance (R) genes stands out because of its key role in plant defense against pathogen attack, being the largest and most diverse family of this group. The present work aimed the characterization of NBS-LRR genes from cowpea, the development of RGA (resistance gene analogs) markers and the validation of differentially expressed genes in plant-virus interaction. Initially, NBS-LRR gene candidate sequences were obtained from NordEST database, proceeding with data annotation, translation and identification of conserved domains through in silico methods. A total of 57 NBS-LRR complete sequences were identified for cowpea. Since R-gene encoded proteins exhibit conserved domains and motifs, it was possible to develop RGA markers using cowpea sequences as probes against Phaseolus vulgaris L. Sixteen primer pairs of P. vulgaris were designed, from which 87.5% were transferable to cowpea. From those, two were polymorphic and showed Mendelian segregation in a mapping population for the cowpea severe mosaic virus (CPSMV). The 57 candidates were anchored in the 20 Glycine max (L.) Merr. pseudo-chromosomes, revealing in tandem repetitions for this group of gene. Differential gene expression analysis in silico was performed using data from RNAseq and SuperSAGE. The validation of gene expression by RT-qPCR was carried out after design of primers using SuperSAGE data, from which three target genes presented induction in expression levels at 16 hours after the pathogen inoculation. These results represent valuable data for the genetic improvement of cowpea.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-05T13:55:43Z No. of bitstreams: 1 joaosuendermoreira.pdf: 2727096 bytes, checksum: 15959d5ff7f375e41f8c2bc20e5308fe (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-05T13:58:13Z (GMT) No. of bitstreams: 1 joaosuendermoreira.pdf: 2727096 bytes, checksum: 15959d5ff7f375e41f8c2bc20e5308fe (MD5) / Made available in DSpace on 2016-10-05T13:58:13Z (GMT). No. of bitstreams: 1 joaosuendermoreira.pdf: 2727096 bytes, checksum: 15959d5ff7f375e41f8c2bc20e5308fe (MD5) Previous issue date: 2009-03-12 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Um dos principais problemas mundiais na agricultura está diretamente relacionado às enormes perdas na produção causada por fungos fitopatogenicos, onde sua infecção nas culturas se dá desde o plantio até a pós- colheita. O fungo Botrytis cinerea, causador do mofo cinzento em mais de 200 espécies de plantas é um grande problema no agronegócio em todo o mundo. O uso de fungicidas é o principal método de controle em plantas, enquanto os resultados obtidos de imediato e a facilidade de aplicação justificam o seu uso. No entanto, o uso contínuo de fungicidas pode promover a seleção de fungos resistentes além de causar a contaminação de ecossistemas. Com o objetivo de encontrar uma solução para esse problema, diversos estudos tem se concentrado na busca de novas alternativas de controle, como por exemplo, as proteínas de plantas com atividades antifúngicas (AFPs). Nesse sentido, uma seleção de peptídeos antimicrobianos de flores, folhas e sementes de espécies do gênero Lippia bem como o isolamento de peptídeos antifúngicos de flores de Lippia sidoides foi o objetivo desse trabalho. Neste trabalho, a purificação e identificação de dois novos peptídeos de aproximadamente 10 kDa e 15 kDa de flores de Alecrim-pimenta (Lippia sidoides) foi descrito. As flores de L. sidoides, depois de secadas em estufa a 25o por cinco dias, foram submetidas à extração de proteínas com solução de HCl 0,1% e NaCl 0,6M, seguido por precipitação com sulfato de amônia (100%). Após a precipitação, o extrato foi dialisado contra água destilada (cut off 1,0 kDa) e liofilizado. A fração rica foi aplicada em cromatografia hidrofóbica Octyl-sepharose, seguida por cromatografia de fase-reversa de HPLC (Vydac C18-TP). Bioensaios com EB, e fração PR in vitro indicaram a inibição do crescimento do fungo Botrytis cinerea. As sequências N-terminal, obtidas por degradação de Edman, seguidas por alinhamento indicam que esses dois peptídeos podem ser classificados com proteínas R NBS-LRR. Esta descoberta pode contribuir, futuramente, para o desenvolvimento de produtos biotecnológicos como drogas antifúngicas e plantas transgênicas com resistência elevada a fungos patogênicos. / One of the major global issues in agriculture could be directly related to the severe production crop losses caused by phytopathogenic fungi, especially when infection affects post-harvest cultures. In this view, the fungus Botrytis cinerea is able to cause gray mold in more than 200 species of plants, being considered a major problem for the agribusiness. The use of fungicides is a primary fungi control method in plants, due to velocity and facility of application. However, the continuous use of fungicides may promote the selection of resistant fungi and also the ecosystems contamination. Aiming to find different solutions to this problem, several studies have focused on the search for new alternatives to fungi control, such as plant proteins with antifungal activities (AFPs). In this view, a selection of antimicrobial peptides from flowers, leaves and seeds from Lippia genus and further isolation of antifungal peptides from Lippia sidoides flowers was focused in this work. In this work, the purification and identification of two novel peptides of approximately 10 kDa and 15 kDa from flowers of rosemary-pepper (L. sidoides) was described. L. sidoides flowers were oven dried at 25 oC for 5 days, following protein extraction with a solution containing 0.1% HCl 0.6 M NaCl, and further ammonium sulfate precipitation (100%). After precipitation the extract was dialyzed against distilled water (cut off 1.0 kDa) and lyophilized. The rich fraction was applied onto an Octyl-Sepharose hydrophobic chromatography, followed by HPLC reversed-phase chromatography (Vydac C18-TP). Bioassays using crude extract and in vitro PR fraction indicated the inhibition of Botrytis cinerea growth. N-termini sequences, obtained by the Edman degradation, followed by alignment indicate that these two peptides can be classified as NBS-LRR R proteins. This discovery may help in a near future to the development of biotechnology products such as antifungal drugs and transgenic plants with enhanced resistance to fungal pathogens.

Anthracnose, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. Cloning and characterization of the host resistance (R) genes against the pathogen will improve our knowledge of molecular mechanisms underlying host resistance and facilitate the development of resistant alfalfa cultivars. However, the intractable genetic system of cultivated alfalfa, owing to its tetrasomic inheritance and outcrossing nature, limits the ability to carry out genetic analysis in alfalfa. Nonetheless, the model legume Medicago truncatula, a close relative of alfalfa, provides a surrogate for cloning the counterparts of many agronomically important genes in alfalfa. In this study, we used genetic map-based approach to clone RCT1, a host resistance gene against C. trifolii race 1, in M. truncatula. The RCT1 locus was delimited within a physical interval spanning ~200 kilo-bases located on the top of M. truncatula linkage group 4. Complementation tests of three candidate genes on the susceptible alfalfa clones revealed that RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad spectrum anthracnose resistance. Thus, RCT1 offers a novel resource to develop anthracnose-resistant alfalfa cultivars. Furthermore, the cloning of RCT1 also makes a significant contribution to our understanding of host resistance against the fungal genus Colletotrichum.