We thank all lab members of the Macho laboratory for helpful discussions, Alvaro L&#243;pez-Garc&#237;a for statistical advice, and Xinyu Jian for technical and administrative assistance during this work. We thank the PSC Cell Biology core facility for assistance with fluorescence imaging This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB27040204), the Shanghai Center for Plant Stress Biology (Chinese Academy of Sciences) and the Chinese 1000 Talents program.

NOTE: Important parts of this method involve handling plant materials in vitro, and therefore it is important to keep sterile conditions during all these procedures, including the visualization of DsRed fluorescence. During all the transformation process, tomato seedlings grow at 25&#8722;28 &#176;C and 16 h/8 h light/dark (130 &#181;mol photons m-2s-1 light). Plates are sealed with micropore tape in order to facilitate gas exchange and transpiration.
1. Preparation of tomato plants and Agrobacterium rhizogenes
<ol>
	<li>Sterilize tomato seeds (Solanum lycopersicum cv. Moneymaker, LA2706, Tomato Genetics Resource Center, TGRC) with 5% (v/v) sodium hypochlorite for 5 min. Wash 4-5 times with distilled sterile water and keep the seeds shaking slowly in sterile water over-night to facilitate germination.</li>
	<li>Transfer the tomato seeds to half-strength Murashige and Skoog (1/2 MS) medium without sucrose (2.21 g/L MS, 8% w/v agar). Keep the seeds in the dark at 25&#8722;28 &#176;C for three days (Figure 1A). 
	NOTE: Around 40 seeds are usually needed for each construct.</li>
	<li>Autoclave 8.5 cm2 square filter papers. Place the square filter papers inside 9 cm2 square petri dishes containing 1/2 MS medium (place the paper on top of the agar) and place six germinated tomato seeds on each plate (Figure 1B). Seal the plate with micropore tape and incubate the germinated seeds at 25&#8722;28 &#176;C for 3&#8722;4 days.</li>
	<li>Grow Agrobacterium rhizogenes MSU440 in solid LB medium (with appropriate antibiotics) at 28 &#176;C two days before plant transformation. 
	NOTE: A. rhizogenes is provided by Dr. Juan Antonio L&#243;pez R&#225;ez, EEZ-CSIC, Granada, Spain. In the experiment described in this article, A. rhizogenes containing pK7GWIWG2_II-RedRoot::CESA6 or an empty vector, as control, were used. pK7GWIWG2_II-RedRoot contains a reporter gene, DsRed, driven by the Arabidopsis thaliana ubiquitin promoter (pAtUBQ10), and confers resistance to spectinomycin (50 &#181;g/mL). It is possible to use other vectors for gene overexpression, as reported before5. In this work, the amplification of the specific fragment for SlCESA6 silencing was obtained by reverse transcription (RT) PCR using RNA isolated from tomato (S. lycopersicum cv. Moneymaker) and ligated into p-ENTR/D-TOPO vector (Table 1). Subsequently, the SlCESA6-RNAi fragment was cloned into the pK7GWIWG2_II-RedRoot binary vector.</li>
</ol>
2. Plant transformation and selection
<ol>
	<li>Using a sterile scalpel, cut the radicle and the bottom of the hypocotyl of tomato seedlings (Figure 1C,D).</li>
	<li>Harvest A. rhizogenes biomass from the surface of the LB medium using plastic tips or a scalpel blade and carefully dip the cut tomato seedlings in the bacterial biomass (Figure 1E).</li>
	<li>After inoculation with A. rhizogenes, cover the tomato seedlings with a 2 cm x 4 cm semi-circular filter paper (Figure 1F), in order to keep a high humidity and facilitate survival and new root development.</li>
	<li>Store the transformed tomato seedlings for 6-7 days. Then, use a sterile scalpel to cut the new emerging hairy roots (Figure 1G,H; at this step, the roots are not transformed yet) and allow the seedlings to produce new hairy roots.</li>
	<li>Once the second generation of new hairy roots appear (Figure 1I), remove the filter paper on top of the seedlings, and seal the plate with micropore tape again. 
	NOTE: At this point, the presence of the DsRed fluorescent marker in the transformation vector allows to visualize the transformation efficiency in the new roots.</li>
	<li>To visualize DsRed fluorescence, use a stereomicroscope or any other equipment for plant in vivo imaging (Figure 1J). Mark the positive (red fluorescence) transformed roots and remove the negative non-transformed roots (no red fluorescence) using a sterile scalpel.</li>
	<li>Transfer the seedlings showing red fluorescence to a new plate containing 1/2 MS medium, in order to facilitate the development of the transformed root as the main root (Figure 1K). Keep the seedlings that do not show red fluorescence in the same plate to check the emergence of fluorescent roots (Figure 1L) in later time points.</li>
	<li>Alternative method based on antibiotic selection
	<ol>
		<li>Alternative to step 2.5, prepare half-filled 9 cm2 square plates containing 1/2 MS medium with the appropriate antibiotic. Incline the plates approximately 5&#176; during the preparation process to generate an empty space without medium (Figure 2A), allowing the shoots to grow avoiding contact with the antibiotic.</li>
		<li>Alternative to step 2.6, after cutting the first non-transformed hairy roots emerged (step 2.4), transfer the seedlings without roots to the half-filled plates using filter papers with the appropriate size (Figure 2B). 
		NOTE: As positive control, it is recommended to transform several seedlings with a plasmid containing the same antibiotic resistance and a reporter gene (in this protocol, pK7GWIWG2_II-RedRoot; kanamycin 50 &#181;g/mL).</li>
		<li>Alternative to step 2.7, let the seedlings develop new hairy roots and cut those roots that are not in direct contact with the surface of the filter sandwich papers, since these roots may avoid antibiotic selection. 
		NOTE: Due to the antibiotic effect, root development may be slower than in plates without antibiotics. New transformed hairy roots appear within 14&#8722;18 days after transferring the seedlings without roots to the half-filled plates (step 2.8.2) (Figure 2C-E).</li>
	</ol>
	</li>
	<li>Cover the seedlings with a 2 cm x 4 cm semi-circle filter paper, seal the plate and incubate the seedlings to let them develop new hairy roots. 
	NOTE: It is not necessary to eliminate the A. rhizogenes using antibiotics. The filter paper on top of the MS medium and the lack of sucrose inhibit the spread of the A. rhizogenes on the plate5.</li>
	<li>Five-to-seven days after the first root selection, repeat the selection process to select new transformed roots and remove non-transformed roots. Transfer the seedlings containing transformed roots to a new plate containing 1/2 MS medium.</li>
</ol>
3. Transfer to inoculation pots
<ol>
	<li>Prepare inoculation pots where the surface of the roots will be exposed to the bacterial inoculum: soak the inoculation pots with water, pour off any excess water, and place them in a plastic planting tray. Transfer the selected seedlings with transformed roots to inoculation pots using tweezers (Figure 3A).</li>
	<li>Cover the tray with plastic wrap or a transparent lid and keep them at 25&#8722;28 &#176;C and 65% humidity (16 h/8 h light/dark; 130 &#181;mol photons m-2s-1 light), to maintain a high level of humidity Figure 3B). Remove the cover after five or six days. 
	NOTE: The transformed tomato plants are ready for inoculation two-to-three weeks after transferring them to soil (Figure 3C). During the growth on soil, tomato plants may produce new roots that are not transformed. To determine the extent of this phenomenon, red fluorescence was visualized in roots of 3-week-old transformed tomato plants. Most roots (80%-100%) showed red fluorescence, indicating that they are indeed transformed (Figure 3D,E).</li>
</ol>
4. Soil-drenching inoculation
<ol>
	<li>Grow R. solancearum (strain GMI1000 in this protocol) in phi liquid medium (Table 211) in an orbital shaker (200 rpm) at 28 &#176;C until stationary phase.</li>
	<li>Determine bacterial numbers by measuring the optical density of the bacterial culture at 600 nm (OD600). Dilute the bacterial culture with water to an OD600 of 0.1 (in conditions used here, this corresponds to approximately 108 colony-forming units [CFU]/mL).</li>
	<li>Place 16&#8722;20 inoculation pots containing transformed tomato plants in an inoculation tray (29 cm x 20 cm; Figure 4A).</li>
	<li>Pour 300 mL (~15 mL per plant) of bacterial inoculum (OD600 of 0.1) into the tray containing the inoculation pots. Let them soak in the inoculum for 20 min (Figure 4B).</li>
	<li>Prepare a new tray with a layer of potting soil. Move the inoculated pots into the new tray (Figure 4C) and place the trays in a growth chamber with 75% humidity, 26&#8722;28 &#176;C, and a photoperiod of 12 h light and 12 h darkness (130 &#181;mol photons m-2s-1 light).</li>
</ol>
5. Determination of infection parameters and statistical analysis
<ol>
	<li>Score disease symptoms as previously described12,13, using a scale ranging from 0 (no symptoms) to 4 (complete wilting) (Figure 4D-H), each day after Ralstonia inoculation for two weeks. 
	NOTE: The disease index data are collected from the same experimental unit (each plant) over time.</li>
</ol>
6. Gene expression analysis
NOTE: The expression of the transgene or the silencing of the target gene can be determined by RT-PCR or by quantitative RT-PCR (qRT-PCR).
<ol>
	<li>Collect samples and extract RNA from a representative part of the transformed root system (to evaluate the effect on the target gene) and leaves (as internal control).</li>
	<li>Synthesize cDNA using 1 &#181;g of total DNase-treated RNA.</li>
	<li>Analyze gene expression of the target genes by qRT-PCR. Calculate the relative transcription levels using the 2-&#916;&#916;CT method14, using SlEF&#945;-1 as housekeeping gene15. 
	NOTE: Primer sequences are listed in Table 1.</li>
</ol>

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The authors have nothing to disclose.

Ralstonia solanacearum poses an important threat to agriculture; however, its interaction with natural hosts of agricultural importance is still poorly understood compared with other bacterial pathogens, especially in crop plant species. In most cases, genetic analysis is hindered by the time and expenses required to genetically modify host plants. To address this problem and facilitate genetic analysis of R. solanacearum infection in tomato, we have developed an easy method based on Agrobacterium r...

Ralstonia solanacearum, the causal agent of bacterial wilt disease, is a devastating soil borne vascular pathogen with a worldwide distribution that can infect a large range of plant species, including potato, tomato, tobacco, banana, pepper and eggplant, among others1,2. Yield losses caused by Ralstonia can reach 80-90% of production in tomato, potato or banana, depending on cultivar, climate, soil and other factors3. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae or Xanthomonas spp. Additionally, most studies in plant-microbe interactions are focused on the model plant Arabidopsis thaliana. Although research using these models has largely contributed to our understanding of plant-bacteria interactions, they do not address the current necessity to understand these interactions in crop plants. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction. Particularly, tomato, a natural host for Ralstonia, is the second most important vegetable crop worldwide and is affected by a plethora of diseases4, including bacterial wilt disease. In this work, we have developed an easy method to perform genetic analysis of Ralstonia infection of tomato. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, using DsRed fluorescence as selection marker5, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi.
A potential limitation of this method consists on the residual growth of non-transformed roots. This is particularly important in the cases where the plasmid used lacks a reporter gene that allows the selection of transformed roots. To solve this problem, we have developed an alternative method based on antibiotic selection, which inhibits the growth of non-transformed roots while allowing the growth of healthy antibiotic-resistant transformed roots. Since A. rhizogenes does not induce the transformation of shoots, they are susceptible to the antibiotic, and, therefore, they should be kept separated from the antibiotic-containing medium.
Although plant resistance against Ralstonia is not well understood, several reports have associated cell wall alterations to enhanced resistance to bacterial wilt6,7,8,9. It has been suggested that these cell wall alterations affect vascular development, an essential aspect for the lifestyle of Ralstonia inside the plant10. Mutations in genes encoding the cellulose synthases CESA4, CESA7 and CESA8 in Arabidopsis thaliana have been shown to impair secondary cell wall integrity, causing enhanced resistance to Ralstonia, which appears to be linked to ABA signalling8. Therefore, as a proof of concept for our method, we performed RNAi-mediated gene silencing of SlCESA6 (Solyc02g072240), a secondary cell-wall cellulose synthase, and ortholog of AtCESA8 (At4g18780). Subsequent soil-drenching inoculation with Ralstonia showed that silencing SlCESA6 enhanced resistance to bacterial wilt symptoms, suggesting that cell wall-mediated resistance to Ralstonia is likely conserved in tomato, and validating our method to carry out genetic analysis of bacterial wilt resistance in tomato roots. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>90 mm square Petri-dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD (Becton and Dickinson)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Casamino acids</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>In Vivo Plant Imaging System NightShade LB 985</td>
			<td>Berthold Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jiffy pots</td>
			<td>Jiffy Products International A.S.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore tape</td>
			<td>3M</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog medium (M519)</td>
			<td>Phytotechlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pindstrup substrate</td>
			<td>Pindstrup Mosebrug A/S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel and blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile clean bench</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wahtman paper</td>
			<td>Wahtman International Ltd. Maldstone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>OXOID</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

tomato root transformation followed by inoculation with ralstonia solanacearum for straightforward genetic analysis of bacterial wilt disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Figure 5 shows the development of disease symptoms of tomato plants with roots transformed with an empty vector (EV), and plants with roots transformed with an RNAi construct targeting SlCESA6 (Solyc02g072240). The disease index data (Figure 5A) are collected from the same experimental unit (each plant) over time according to an arbitrary scale from 0 to 4, and do not follow a Gaussian distribut...

Watch this Scientific Journal Video about Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease at JoVE.com

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

tomato-root-transformation-followed-inoculation-with-ralstonia

Research

JoVE Journal

Genetics

11.4K Views.  Chinese Academy of Sciences. Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Video: Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7&#8211;10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p &#60; 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant ...

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome&#8211;negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified.

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA ...