Agradecemos Jakob Viel seus comentários úteis e sugestões. Agradecemos também a Anne de Jong por sua ajuda na análise bioinformática. Yanglei Yi e Zhibo Li são suportados pelo Conselho de bolsa da China (CSC). Agradecemos a NWO-TTW Perspectief Programma Back2Roots (TKI-AF-15510) pelo apoio financeiro ao OPK.

1. germinação de batata em condições esterilizadas
<ol><li>Enxague a superfície de batata com água estéril. Banhar-se a batata em etanol a 70% e, depois, em 3% de hipoclorito de sódio, por 5 min cada. Enxágue novamente com água esterilizada para remover qualquer restante hipoclorito de sódio.</li>
	<li>Preparar os materiais necessários para a germinação e crescimento de tubérculos de batata; esterilizar os potes plásticos, cestas de enxertia, vermiculita e regar em autoclave a 121 ° C por 20 min. Nota: Certifique-se que todos os materiais utilizados são autoclaváveis. Caso contrário, use outros métodos de esterilização.</li>
	<li>Colocar a batata superfície-esterilizados para o cesto de enxertia e colocar isso em um pote esterilizado contendo vermiculita úmida.</li>
	<li>Manter o pote em uma câmara climática a 24 ° C durante três semanas. Defini-lo em ciclos de 16 h de luz (120 µmol m-2s-1) e 8 h de escuridão. Para evitar contaminações microbianas, use uma caixa de fibra de vidro grande para cobrir o pote.</li>
</ol>2. recolha de exsudados de raiz de batata
<ol><li>Quando o broto de brotos, transferência a cesta com a batata inteira para um copo esterilizado preenchido com 150 mL de água destilada esterilizada, com a batata tubérculos colocados logo acima da superfície da água e as raízes submersas na água (ver Figura 1). Manter as mudas em câmara climática e usar as mesmas configurações como antes [24 ° C; ciclos de 16 h de luz (120 µmol m-2s-1), 8 h das trevas].</li>
	<li>A partir do segundo dia, recolher a água que contém o exsudado e volte a encher o copo com água desionizada estéril. Executar a amostragem todos os dias até o sétimo dia após a transferência o seedling.</li>
	<li>Armazenar cada amostra separadamente a 4 ° C. Para cada amostra, espalhar 100 µ l em um Luria-Bertani (LB; triptona 1%, extrato de levedura 0,5%, 0,5% NaCl) placa de ágar para verificar se há contaminação microbiana. Descarte as amostras contaminadas.</li>
	<li>Combinar as amostras coletadas de um seedling e concentrá-las por liofilização-los a-40 ° C, até um volume final de 150 mL.</li>
</ol>3. cultivo de bactérias
<ol><li>Marcam uma estirpe b. mycoides de um estoque de glicerol-80 ° C em uma placa de ágar LB e incubar a placa a 30 ° C durante a noite.</li>
	<li>Inocular um meio líquido de LB com uma única colônia da placa e crescer a cultura numa incubadora agitando a 200 rpm durante a noite a 30 ° C.</li>
</ol>4. tratamento e recolha de amostras de bactérias
<ol><li>Para comparar a curva de crescimento das bactérias tratadas com exsudados de raiz para um controlo negativo, prepare uma série de frascos contendo 50 mL de meio líquido LB. Adicionar 0,5 mL de cultura bacteriana durante a noite para todos os frascos e adicionar 0%, 5%, 10%, ou 15% (v/v) exsudado de raiz ou deionizada estéril para o meio, respectivamente.</li>
	<li>Use uma cultura com água desionizada, em vez de exsudados de raiz como um controle. Medir a densidade óptica em 600 nm (OD600) cada 1 h depois. Gera uma curva de crescimento plotando os valores de600 OD versus tempo. Nota: Nós vimos que 10% dos exsudados da raiz não afetou o crescimento de b. mycoides (ver Figura 2); Esta proporção foi usada para experimentos de RNA-seq.</li>
	<li>Diluir uma cultura de b. mycoides durante a noite com 90 mL de LB médio pré-aquecido e o OD inicial600 de ~ 0,05 num frasco de 300 mL. Adicionar 10 mL de exsudados de raiz para a cultura e incube a 30 ° C, durante 1 h.</li>
	<li>Use um tratamento de água desionizada estéril 10 mL como um controle. Coletar células de cultura de centrifugação a 9.000 x g por 2 min a 4 ° C. Desprezar o sobrenadante e imediatamente congelar a pelota em nitrogênio líquido, em seguida, guarde-a-80 ° C até o uso.</li>
</ol>5. isolamento de RNA
Nota: Antes de iniciar o isolamento, prepare a bancada, cremalheiras e pipetas, limpá-las com uma solução de descontaminação de RNase (ver Tabela de materiais). Usando luvas de todos os tempos e certifique-se de todos os tubos, dicas e soluções são livre de RNase. Mantenha sempre as amostras no gelo quando possível.
<ol><li>Adicionar 0,1% (v/v) de pyrocarbonate de dietilo (DEPC) para água ultrapura (100 µ l de DEPC por 100 mL de solução), misture bem e mantê-lo à temperatura ambiente durante a noite. Autoclave a mistura para inactivar o DEPC após o tratamento durante a noite. Use esta água ultrapura DEPC tratada para preparar o buffer TE [10 mM Tris-HCl (pH = 8); 1 mM EDTA] e uma solução de SDS (p/v) de 10%.</li>
	<li>Descongelar as pelotas de célula no gelo e suspender cada um em 400 µ l de tampão TE (DEPC). Transferi as suspensões para tubos de tampa de rosca de 2 mL.</li>
	<li>Premix 300 µ l de álcool de clorofórmio-isoamílico (24:1 v/v) e 300 µ l de fenol (ácido fenol, grau de RNA) e deixe a mistura repousar durante 5 min. Tome 500 µ l da fase orgânica superior para adicionar às células ressuspensa. Em seguida, adicione 50 µ l de SDS 10% e 0,5 g de grânulos de vidro (0,5 µm) no tubo.</li>
	<li>Feche a tampa firmemente e coloque o tubo em um homogeneizador de grânulo-moinho (ver Tabela de materiais). Execute uma homogeneização de pulso 3 × 45 s com um intervalo de 1 min no gelo.</li>
	<li>Centrifugar as amostras por 10 min a 11.000 x g (4 ° C) e transferir a fase superior para um novo tubo.</li>
	<li>Adicionar 500 µ l de álcool de clorofórmio-isoamílico (24:1) para a fase superior da etapa 5.5 e centrifugar a mistura por 5 min a 11.000 x g (4 ° C). Nota: As etapas a seguir foram modificadas desde as instruções do fabricante de um isolamento de RNA baseado em filtro comercial vidro fibra kit (veja a Tabela de materiais).</li>
	<li>Transferir 500 µ l da fase superior para um novo tubo, adicione 2 volumes (1 mL) de um tampão de Lise/vinculação e misture pipetando para cima e para baixo.</li>
	<li>Combinar os tubos do filtro e coleção (do kit de isolamento de RNA) e pipetar a mistura da etapa 5.7 no tubo do filtro. Centrifugar os tubos por 15 s em 8.000 x g e descarte o escoamento.</li>
	<li>Prepare-se 1,5 mL microcentrifuge tubos com 100 µ l de tampão de DNase, 10 µ l de DNase I e 5 µ l de um inibidor de RNase (ver Tabela de materiais). Adicione a mistura de solução preparada no filtro do tubo do filtro e incube-lo por 20-30 min em 15-25 ° C.</li>
	<li>Execute as etapas de lavagem de acordo com as instruções do fabricante. Use 50 µ l de um tampão de eluição para Eluir o RNA.</li>
	<li>Salve o RNA eluted a-80 ° C e coloque um filme plástico parafina em torno dele. Ao mesmo tempo, transferi alguns microlitros de amostra para um tubo de microcentrifugadora de 1,5 mL para executar uma verificação de qualidade.</li>
</ol>6. sequenciamento e a verificação da qualidade do RNA
<ol><li>Verifique o RNA com um espectrofotômetro microvolume. Nota: Rácios de absorvância no 260/280 devem estar acima de 1.8 e rácios no 260/230 deve ultrapassar pelo menos 1.8, de preferência acima de 2.0.</li>
	<li>Executar o RNA purificado em um instrumento de eletroforese automatizada utilizando a análise de RNA recomendo kit (veja a Tabela de materiais); o número de integridade do RNA (RIN) deve ser pelo menos acima de 7 para prosseguir.</li>
	<li>Usar um montante total de 3 µ g de RNA por amostra para gerar bibliotecas com uma preparação de biblioteca de RNA-Seq kit (veja a Tabela de materiais) seguindo as recomendações do fabricante. Nota: Os preparativos da biblioteca foram sequenciados em uma plataforma de sequenciamento de alta produtividade e leituras emparelhado-final foram geradas.</li>
</ol>7. dados análise usando o T-REx Pipeline baseado na Web
<ol><li>Realizar uma filtragem de qualidade usando o FASTX-kit de ferramentas versão 0.0.13 (PQV escores de qualidade de > 20). Apare as leituras de RNA-Seq crus de sequências de adaptador com a ferramenta Trimmomatic. Nota: Todas as análises a jusante foram baseadas em dados limpos com alta qualidade.</li>
	<li>Baixar o banco de dados do genoma de ATCC 6462 NCBI b. mycoides (adesão NCBI n º: CP009692.1) e usá-lo como um genoma de referência para mapear o lê limpo usando Bowtie2/2.2.3.</li>
	<li>V0.6.1 de HTSeq de uso para contar o número de leituras mapeados para cada gene e as leituras por quilobase de transcrição por leituras mapeadas milhões (RPKM) de cada gene baseiam no comprimento do gene e leituras Conde mapeado para este gene.</li>
	<li>Para a análise dos dados pelo pipeline T-REx, use a tabela RPKM como entrada, bem como três arquivos descritivos: (i) um arquivo de fatores para descrever a experiência em fatores, (ii) um arquivo de contrastes para definir quais fatores serão comparados para expressão gênica diferencial e (iii) um arquivo de classe para descrever grupos de genes de interesse. Nota: Os arquivos descritivos são formato de in.txt. Exemplos podem ser encontrados na página Web do T-REx (http://genome2d.molgenrug.nl/). Uma instrução detalhada para análise de dados usando o T-REx pode ser encontrada em uma publicação anterior por de Jong et al 14.</li>
</ol>

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Os autores declaram que eles têm não tem interesses financeiro concorrente.

Interações planta-micróbio tem sido a hipótese de ser determinado por um equilíbrio afinado entre bactérias e plantas. Tais interações são altamente complexas e difíceis de estudar em um sistema natural, que é composto por diversas espécies microbianas, potencialmente atuando como consórcios. Este artigo descreve um protocolo simplificado para estudar a resposta bacteriana exsudados de raiz em condições bem controladas. O perfil do transcriptoma de rizobactérias, após a exposição exsudados de raiz, for...

As plantas podem exsudato até 20% do carbono fixado durante a fotossíntese através de raízes para a rizosfera1, ou seja, a zona estreita de solo perto das raízes. Devido à maior disponibilidade de nutrientes, a rizosfera é um habitat adequado para diversos microorganismos, incluindo bactérias promotoras de crescimento vegetal. Os exsudados de raiz contêm uma variedade de compostos inorgânicos como íons, ácidos inorgânicos, oxigênio e água. No entanto, a maioria dos exsudados da raiz é formada por materiais orgânicos, que podem ser divididos em compostos de baixo peso molecular e alto peso molecular de compostos. Os compostos de baixo peso molecular incluem aminoácidos, ácidos orgânicos, açúcares, compostos fenólicos, ácidos graxos e uma matriz de metabólitos secundários. Os compostos de alto peso molecular consistem de mucilagem e proteínas de2,3. Microrganismos da rizosfera podem usar alguns destes compostos como fonte de energia para o crescimento e desenvolvimento. Os exsudados de raiz desempenham um papel importante na formação da comunidade de rhizobacterial, desde que os compostos vegetais produzidos no exsudado podem influenciar o comportamento das bactérias associadas a rizosfera afetando a expressão de genes específicos.
Compreender a resposta bacteriana exsudados de raiz é um passo fundamental em decifrar os mecanismos de interação planta-micróbio. Como a resposta bacteriana às interações planta-micróbio é o produto da expressão gênica diferencial, podem ser estudado pela análise do transcriptoma. Usando esse método, estudos anteriores identificaram vários importantes genes envolvidos em interações planta-micróbio. Em Pseudomonas aeruginosa, genes envolvidos no metabolismo, quimiotaxia e tipo de secreção II foram mostrados para responder a de exsudados de raiz de beterraba4. Ventilador et al. 5 estudou o perfil transcriptomic de b. amyloliquefaciens FZB42 em resposta exsudados de raiz de milho. Seus resultados mostram que, dos genes fortemente induzidos por exsudados da raiz, vários grupos estão envolvidos em vias metabólicas relacionadas com a utilização de nutrientes, quimiotaxia, motilidade e não-ribossomais síntese de peptídeos antimicrobianos e policetídeos.
A precisão desses estudos baseia-se na coleção de exsudados radiculares. Embora vários métodos descreveu a coleção de exsudados radiculares para finalidades diferentes, eles exigem instrumentos sofisticados ou não são executados em condições bem controladas6,7,8. Além disso, microrganismos inibindo a rizosfera podem influenciar a composição do exsudado de raiz, afetando a permeabilidade de membrana da célula de planta e danificar os tecidos da raiz, particularmente no caso dos consórcios de microorganismos9. Ao investigar a resposta microbiana exsudados de raiz, é importante usar bem definidas condições para evitar a alteração dos compostos por outros microorganismos10. Além disso, o RNA de alta qualidade é necessária para RNA-seq baseados em estudos de transcriptoma. No entanto, quando se lida com as estirpes não-modelo-bacteriana, os protocolos padrão ou jogos comerciais geralmente têm uma baixa eficiência devido a fatores desconhecidos ou propriedades especiais de crescimento.
O protocolo descrito aqui foi verificado usando b. mycoides, que é uma bactéria Gram-positiva, formadoras de esporos do filo Firmicute. É onipresente na rizosfera de várias espécies de plantas. Várias propriedades promover crescimento de planta têm sido relatadas para esta espécie, incluindo indução de resistência sistemática (ISR) na beterraba açucareira11, inibição do umedecimento-fora agente patogénico Pythium para pepino12, bem como nitrogênio fixação na rizosfera girassol13. No entanto, os mecanismos moleculares de sua interação com a planta hospedeira não são bem estudados.
O objetivo dos experimentos apresentados aqui é estudar a resposta de transcriptomic de endosphere-isolado b. mycoides exsudados de raiz de batata. Em suma, o protocolo consiste das seguintes etapas: primeiro, recolher exsudados de raiz de batata em condições estéreis. Em seguida, extrai o RNA de alta qualidade de células bacterianas tratadas com exsudados de raiz. A etapa final é a análise de dados usando o web-based T-REx gasoduto14. Este protocolo foi usado para identificar genes de b. mycoides que mostram uma mudança nos níveis de expressão em caso de contacto com exsudados de raiz e, portanto, podem desempenhar um papel importante nas interações planta-micróbio.

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			<td height="21" style="height:21px;width:260px;">sodium hypochlorite</td>
			<td style="width:236px;">Sigma</td>
			<td style="width:152px;">&#160;CAS: 7681-52-9&#160;</td>
			<td style="width:339px;">10-15%&#160; active chlorine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Luria-Bertani (LB) broth</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">incubater</td>
			<td style="width:236px;">New Brunswick Scientific</td>
			<td style="width:152px;">Innova 4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Genesys 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">liquid nitrogen</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">glass beads</td>
			<td style="width:236px;">Sigma</td>
			<td style="width:152px;">G8893</td>
			<td>0.5 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">2.0 ml tube with screw cap</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1.5 ml and 2.0 ml eppendorf tube</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead mill homogenizer</td>
			<td>BioSpec</td>
			<td style="width:152px;">607</td>
			<td>Mini_beadbeater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:152px;">5430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Diethyl pyrocarbonate (DEPC)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 1609-47-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 151-21-3&#160;</td>
			<td>10% solution prepared with DEPC treated MQ water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">TE buffer</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>10 mM Tris-HCl; 1 mM EDTA, pH=8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">phenol</td>
			<td>Sigma</td>
			<td style="width:152px;"></td>
			<td>RNA grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">chloroform-isoamyl alcohol&#160;</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>prepare 24:1 of chloroform:isoamyl alcohol, store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">High pure RNA isolation kit</td>
			<td style="width:236px;">Roche</td>
			<td style="width:152px;">11828665001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase Decontamination Solution</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:152px;">AM9780</td>
			<td>RNase-Zap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Automated electrophoresis instrument</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">2100</td>
			<td>Bioanalyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microvolume spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Nanodrop ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNA quality analysis kit</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">RNA 6000 Nano kit&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase inhibitor</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">RiboLock</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Directional RNA library Prep kit</td>
			<td style="width:236px;">NEB</td>
			<td style="width:152px;">Ultra</td>
			<td style="width:339px;">For Illumina</td>
		</tr>
	</tbody>
</table>

plant-microbe interaction: transcriptional response of bacillus mycoides to potato root exudates

Bactérias benéficas associadas a planta é um papel importante na promoção do crescimento e prevenção de doenças em plantas. A aplicação (PGPR) rizobactérias promotoras de crescimento da planta como agentes de biofertilizantes ou biocontrole tornou-se uma alternativa eficaz para o uso de fertilizantes convencionais e pode aumentar a produtividade das culturas a baixo custo. Interações planta-micróbio dependem de sinais de planta-secretada de acolhimento e uma reação diante de suas bactérias associadas. No entanto, os mecanismos moleculares de bactérias benéficas como respondem a seus associados de origem vegetal os sinais não são totalmente compreendidos. Avaliar a resposta de transcriptomic de bactérias exsudados de raiz é uma poderosa abordagem para determinar a expressão de gene bacteriano e regulamento sob condições rizosférico. Tal conhecimento é necessário compreender os mecanismos subjacentes envolvidos em interações planta-micróbio. Este artigo descreve um protocolo detalhado para estudar a resposta de transcriptomic de b. mycoides EC18, uma cepa isolada do endosphere a batata, exsudados de raiz de batata. Com a ajuda da recente tecnologia de sequenciamento de alta produtividade, este protocolo pode ser executado em várias semanas e produzem enormes conjuntos de dados. Primeiro, nós coletamos os exsudados de raiz em condições estéreis, após o qual eles são adicionados à b. mycoides culturas. O RNA destas culturas é isolado, usando um método de fenol/clorofórmio, combinado com um kit comercial e submetido a um controlo de qualidade por um instrumento automatizado de electroforese. Após o sequenciamento, análise de dados é executada com o pipeline de T-REx baseado na web e um grupo de genes diferencialmente expressos é identificado. Este método é uma ferramenta útil para facilitar novas descobertas sobre os genes de bactérias envolvidas em interações planta-micróbio.

Microorganismos associados a planta podem influenciar positivamente crescimento das plantas e a saúde. No entanto, os mecanismos das complexas interações entre plantas e seus simbiontes microbianos não são totalmente compreendidos. Exsudados de raiz desempenham um papel importante na regulação da atividade rhizobacterial e comportamento, e geralmente postula-se que a colonização microbiana de raízes se inicia com a atração de micróbios exsudados de raiz. O objetivo deste trab...

Watch this Scientific Journal Video about Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates at JoVE.com

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

O objetivo do protocolo aqui apresentado é estudar a resposta de transcriptomic de endosphere-isolado de Bacillus mycoides exsudados de raiz de batata. Este método facilita a identificação de genes de bactérias importantes envolvidos em interações planta-micróbio e em princípio aplicável a outros fungos endofíticos e plantas, com pequenos ajustes.

Interação planta-micróbio: Resposta transcricional de Bacillus Mycoides exsudados de raiz de batata

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

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Research

JoVE Journal

Biology

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

12.5K visualizações.  University of Groningen. O objetivo do protocolo aqui apresentado é estudar a resposta de transcriptomic de endosphere-isolado de Bacillus mycoides exsudados de raiz de batata. Este método facilita a identificação de genes de bactérias importantes envolvidos em interações planta-micróbio e em princípio aplicável a outros fungos endofíticos e plantas, com pequenos ajustes.

Vídeo: Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Induzida por vírus silenciamento gênico (VIGS) em Nicotiana benthamiana Tomate e

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Biologia

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Mismatch Repair imagem e respostas celulares a danos no DNA em Bacillus subtilis</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Usando a resposta optocinético para estudar a função visual de zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificação de γH2AX Focos em resposta à radiação ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Profiling única célula transcricional dos cardiomiócitos de rato adulto

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Usando scanners de mesa para coletar imagens de alta resolução tempo decorrido da Arabidopsis Root gravitrópico Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltração e Agroinfecção PVX em Batata e Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Coleta e análise de<em&gt; Arabidopsis</em&gt; Floema exsudados Usando o método EDTA facilitada

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Métodos para descobrir Uso Promotor Alternativa e regulação da transcrição de Murino Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimento CRISPR Vector construção e caracterização de DNA Modificações pela geração de raízes de tomateiro Cabeludo

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Interação planta-micróbio: Resposta transcricional de Bacillus Mycoides exsudados de raiz de batata

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