Validação funcional de genes envolvidos com estresse de Meloidogyne incognita via RNA interferente in planta

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2014.O fitonematoide Meloidogyne incognita é considerado um dos patógenos de plantas mais relevantes devido a sua distribuição mundial e os danos severos causados a várias culturas importantes agronomicamente. Práticas agronômicas têm tido geralmente pouco sucesso e alto custo, sendo o cultivo de variedades resistentes, quando existentes, a forma mais eficiente de controle. Uma estratégia alternativa promissora é a transformação genética de plantas para expressão de moléculas que afetem o metabolismo e desenvolvimento do nematoide. No presente trabalho foram selecionados dois genes-alvo homólogos a genes essenciais do nematoide modelo Caenorhabditis elegans para silenciamento gênico de M. incognita in planta: Isocitrato liase e hsp90. Visando a validação desses genes, para o controle deste fitonematoide, os fragmentos gênicos foram isolados e clonados em um vetor binário para expressão de dsRNA in planta. Plantas transgênicas de Nicotiana tabacum contendo as construções de RNAi foram obtidas através de transformação via Agrobacterium tumefaciens. As plantas expressando dsRNA de hsp90 apresentaram uma atraso na formação de galhas e menor número de células gigantes, observado aos 14 DAI. Os bioensaios com as plantas de tabaco GM nas gerações T1 e T2 mostraram os efeitos do silenciamento dos genes-alvo na reprodução do nematoide. Os testes de resistência mostraram, 45 DAI, uma redução média de 65% nos ovos por grama de raiz nas plantas expressando dsRNA de Isocitrato liase e redução média de 30% nas plantas expressando dsRNA de hsp90. A análise do silenciamento de hsp90 em ovos coletados das plantas expressando dsRNA foi confirmada por qRT-PCR. Em conclusão, genes-alvo com efeitos essenciais foram encontrados, exercendo papel importante no desenvolvimento e reprodução do nematoide. Portanto, essa metodologia pode ser utilizada como uma ferramenta biotecnológica para indução de resistência ao fitonematoide M. incognita em grandes culturas de interesse comercial. _________________________________________________________________________________ ABSTRACTThe phytonematode Meloidogyne incognita is considered the most important plant pathogen, because its worldwide distribution and the severe damage caused to a large variety of agronomically important crops. The phytopathogen has a life cycle that is divided in six developmental stages (Egg, Juvenile 1-4, Female) that can persist for 6 to 8 weeks. The Juvenile 2 enters the root using mechanical force and enzymatic degradation to establish the plant-pathogen interaction that finally differentiates in female that deposit 2,000 eggs. Current agronomic practices have usually been unsuccessful and expensive, so the cultivation of resistant varieties is actually the most efficient way to control nematodes. In this work, we selected target genes for silencing homologues lethal genes of the model nematode Caenorhabditis elegans. Aiming validation of these target genes, the fragments were isolated, cloned in RNAi binary vector for tobacco transformation via Agrobacterium tumefaciens. After this, tobacco seeds were collected and genotyped to select the transformed ones. Bioassays on GM tobacco showed the effects of silencing in the nematode reproduction. The resistance tests conducted with GM tobacco at 45 DAI showed a reduction of 65% in eggs per root gram in transgenic lines of Isocitrate lyase and 30% in transgenic lines of hsp90. In conclusion, we found new target genes that showed deleterious phenotype in nematodes. Therefore, this methodology can be used as a promising biotechnological tool to induce nematode resistance in GM crops

Isolamento de genes e construção de vetores para o uso no silenciamento gênico de Meloidogyne incognita

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2008.Fitonematóides são responsáveis por grandes perdas econômicas na agricultura mundial estimadas em US 125 bilhões anuais. Esses fitopatógenos têm seu ciclo de vida dividido em seis estádios de desenvolvimento (ovo, juvenil 1-4 e adulto), durando em torno de 28 dias. O juvenil 2 penetra na raiz de planta hospedeira por força mecânica e degradação enzimática para estabelecer a interação planta-patógeno e se diferenciar em fêmea adulta apomítica, depositando em torno de 2000 ovos. Práticas agronômicas têm tido geralmente pouco sucesso e alto custo, sendo o cultivo de variedades resistentes, quando existentes, a forma mais eficiente de controle. Uma estratégia alternativa promissora é a transformação genética de plantas para expressão de moléculas que afetem o parasitismo. A metodologia de RNA interferente tem revolucionado a pesquisa experimental e muitas aplicações biotecnológicas estão sendo desenvolvidas. A abordagem planejada é a transformação genética de icotiana tabacum com construções plasmidiais para expressão de RNA dupla fita, tendo como propósito o silenciamento dos genes-alvo Isocitrato Liase, Arginina Quinase, Proteína 14-3-3 e Proteína de choque térmico 90 de Meloidogyne incognita. Desses genes-alvo, quatro fragmentos gênicos foram selecionados, isolados, subclonados em vetor para RNAi e estão em fase de transformação de planta, para futura realização de bioensaios avaliação de resistência a fitonematóides. Adicionalmente, foi realizada uma análise da expressão dos quatro genes-alvo, normalizados com os genes constitutivos de β- actina e 18S rRNA, por PCR quantitativo em tempo real. Observou-se que alguns genes-alvo são diferencialmente expressos quando comparados as fases de ovo, juvenil 2 e fêmea. A maior diferença de expressão foi do gene codificador de Isocitrato Liase, que é 100 vezes mais expresso em ovo e fêmeas, comparado com J2. _________________________________________________________________________________ ABSTRACTPlant parasitic nematodes are responsible for huge economic losses in world agriculture that reaches US 125 billion yearly. The most important of these phytopatogens, Meloidogyne incognita, has six developmental stages during the life cycle (egg, four juveniles and female). The juvenile 2 enters the host root via mechanical force and enzymatic degradation to establish the host-pathogen interaction and differentiate in apomitic adult female, which deposits 2000 eggs. Current agronomic practices have usually been unsuccessful and expensive, so the cultivation of resistant varieties is actually the most efficient way to control nematodes. In this work, it was chosen to transform icotiana tabacum using plasmidial constructions for double strand RNA expression, aiming silencing target genes Isocitrate Lyase, Arginine Kinase, 14-3-3 Protein and Heat Shock Protein 90 of Meloidogyne incognita. Four regions of target genes were selected, isolated, subcloned in RNAi vector and are being used for plant transformation. In addition, it was done an expression analysis of the four target genes, normalized with housekeeping genes β- actin e 18S rRNA, using quantitative real time PCR. This experiment showed that these genes are differentially expressed when compared during egg, J2 and female phases. The major expression difference observed was the Isocitrate Lyase gene, which is a hundred times more expressed in egg or female when compared with J2

Método e composições para controle genético de insetos-praga em plantas de algodão através do silenciamento de genes da família da lacase

Universidade Federal do Rio Grande do SulEmpresa Brasileira de Pesquisa AgropecuáriaDepositad

Método e composições para controle genético de insetos-praga em plantas de algodão através do silenciamento de genes da família da lacase

Universidade Federal do Rio Grande do SulEmpresa Brasileira de Pesquisa AgropecuáriaDepositad

Meloidogyne incognita: molecular cloning and characterization of a cDNA encoding a cathepsin D-like aspartic proteinase

Herein we describe the cloning and characterization of a cDNA encoding an aspartic proteinase from the root-knot nematode Meloidogyne incognita. Using PCR techniques, a 1471-bp cDNA fragment encoding a cathepsin D-like (Mi-asp1) transcript was isolated from second- stage larvae mRNA. Its predicted amino acid sequence comprises a pro-region of 71 amino acid residues and a mature protease of 378 amino acid residues with a predicted molecular mass of 41.502 kDa. Protein sequence comparisons of Mi-asp1 with GenBank ™ (DQ360827) sequences showed 59–71% identity with nematode- specific cathepsin D-like aspartic proteinases. Southern blot analysis, RT-PCR amplification and EST mining suggest the existence of a developmentally expressed gene family encoding aspartic proteinases in M. incognita. Mi-asp1 may represent a potential target for molecular intervention for the purposes of plant–parasitic nematode control

Quantitative RT-PCR showing the transcript levels of proteases in eggs exposed to dsRNAs.

<p>Eggs were collected from <i>M. incognita</i> females that infected transgenic tobacco lines expressing dsRNA for Mi-SER, Mi-CPL and Mi-ASP, Mi-SER and Mi-CPL fused (fusion). (A) Analysis of <i>Mi-asp-1</i>; in <i>M. incognita</i> (Mi) eggs of 35S-dsCPL and 35S-dsSER plants <i>Mi-asp-1</i> was not exposed to a specific dsRNA. (B) Analysis of <i>Mi-cpl-1</i> gene; in Mi eggs of 35S-dsSER plants <i>Mi-cpl-1</i> was not exposed to a specific dsRNA. (C) Analysis of <i>Mi-ser-1</i> gene; in Mi eggs from 35S-dsCPL plants <i>Mi-ser-1</i> was not exposed to a specific dsRNA. Significant differences were assessed by Iteration test (REST Software) where proteases gene expression in nematodes eggs from different plants lines were compared to control plants (*, **, *** = P ≤ 0.05, 0.01 and 0.001, respectively). </p

Gene cloning and transgenic tobacco plant generation for host-derived RNA-interference of <i>Meloidogyne incognita</i> proteases.

<p>(A) Regions of proteinases genes used in RNAi experiments. Numbers indicate nucleotide positions. (B) Schematic representation of the pK7GWIWG2(I) (Karimi et al. 2002) hairpin double-stranded RNA (dsRNA) constructs containing the sense and antisense coding regions fragments of <i>Mi-asp-1</i>, <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> separately and together. (C) Characterization of RNAi lines for silencing of <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> and the fragments in tandem of <i>Mi-asp-1</i>, <i>Mi-ser-1</i> and <i>Mi-cpl-1</i> (Fusion), by PCR. Attempts for generate ds-<i>Mi-asp-1</i> lines were not successful. Sense (S) fragment, anti-sense (AS) fragment, undistinguishable fragment (Sense or Anti-sense) (F). (D) RT-PCR of the single-stranded pK7GWIWG2(I) intron (spacer) of the hairpin dsRNA was used to confirm the expression of <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> and fusion dsRNAs in seedlings of independent transgenic tobacco lines at 15 d post-germination.</p

Knock-down of <i>Meloidogyne incognita</i> proteases affects nematode reproduction and egg viability (after 45 DAI).

<p>(A) Number of eggs per gram of root. (B) Total egg hatching ratio. Experiments were repeated twice. Different letters mean statistical significance through one-way ANOVA and Tukey test (<i>p</i>≤0.05).</p

Relative abundance of specific protease gene transcripts in <i>Meloidogyne incognita</i>.

<p>Real-time qRT-PCR analysis of <i>M. incognita</i> proteases transcript levels at different stages of the nematode life cycle. (A) Cathepsin D-like aspartic proteinase (<i>Mi-asp-1</i>, Accession: DQ360827). (B) Chymotrypsin-like serine proteinase (<i>Mi-ser-1</i>, AY714229). (C) Cathepsin L cystein proteinase (<i>Mi-cpl-1</i>, AJ557572). Each bar represents the mean of duplicate assays repeated twice. Standard errors are shown. Different letters mean statistical difference (<i>p</i>≤0.05) according to the iteration test (Rest 2009 Software). The results are presented as fold change in comparison to the stage that had the smaller relative expression value that was arbitrarily designed as 1.</p

<i>In</i><i>silico</i> analyses of all <i>Meloidogyne incognita</i> aspartic, serine and cysteine proteases ESTs present in EST data bank dbEST.

<p>Representation of <i>M. incognita</i> proteases expressed sequence tags (ESTs) in databanks. Bars show the percentage of proteases EST number relative to the total number of EST available for each developmental stage. ESTs from proteases were retrieved from NCBI-dbEST (<a href="http://www.ncbi.nlm.nih.gov/dbest/index.html" target="_blank">http://www.ncbi.nlm.nih.gov/dbEST/index.html</a>) and their representation was assessed by the number of ESTs relative to the total number of ESTs available for the developmental stage considered. The developmental stages considered were; eggs (14,671 ESTs), freshly hatched J2s (33,835 ESTs), mixed parasitic stages (3,133 ESTs) and females (4,427 ESTs). The distribution of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ESTs is indicated for comparison. </p