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><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 ...

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11.5K Views.  Chinese Academy of Sciences. Dit protocol is belangrijk omdat het wetenschappers in staat stelt om eenvoudige genetische analyse van bacteriële verwelking ziekte in tomaat uit te voeren. Het belangrijkste voordeel van deze techniek is dat de manipulatie van genexpressie en daaropvolgende testen in korte tijd en met de kleine eisen van apparatuur en plantengroeiruimte kan worden uitgevoerd. Deze methode is zeer veelzijdig, en het kan worden toegepast op andere gewasplanten om zowel pathogenenenting als andere fysiologisc...

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

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Environment

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 ...

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Bioengineering

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Biology

High-Efficiency Production of Transgenic Soybean Hairy Roots

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

Author Spotlight: Soybean Hairy Root Transformation for the Analysis of Gene Function  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

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.

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

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

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

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

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

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

One-Step Hairy Root Transformation Method for Advancing Root-Related Biology

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and&#160;then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

Author Spotlight: Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation 

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

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

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