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. P...

<ol>
	<li>Jiang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Wilt+in+China:+History,+Current+Status,+and+Future+Perspectives.">Bacterial Wilt in China: History, Current Status, and Future Perspectives.</a> Frontiers in Plant Science. 11 (8), 1549 (2017).</li><li>Mansfield, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top+10+plant+pathogenic+bacteria+in+molecular+plant+pathology.">Top 10 plant pathogenic bacteria in molecular plant pathology.</a> Molecular plant pathology. 13 (6), 614-629 (2012).</li><li>Elphinstone, J. G., Allen, C., Prior, P., Hayward, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The current bacterial wilt situation: a global overview. In: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. , 9-28 (2005).</li><li>Jones, J. B., Jones, J. P., Stall, R. E., Zitter, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Compendium of Tomato 1094 Diseases. , (1991).</li><li>Ho-Pl&#225;garo, T., Huertas, R., Tamayo-Navarrete, M. I., Ocampo, J. A., Garc&#237;a-Garrido, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+Agrobacterium+rhizogenes-mediated+transformation+of+tomato+suitable+for+the+study+of+arbuscular+mycorrhizal+symbiosis.">An improved method for Agrobacterium rhizogenes-mediated transformation of tomato suitable for the study of arbuscular mycorrhizal symbiosis.</a> Plant Methods. 14, 34 (2018).</li><li>Wydra, K., Beri, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+of+homogalacturonan,+rhamnogalacturonan+I+and+arabiogalactan+protein+in+xylem+cell+walls+of+tomato+gentoypes+in+reaction+to+Ralstonia+solanacearum.">Structural changes of homogalacturonan, rhamnogalacturonan I and arabiogalactan protein in xylem cell walls of tomato gentoypes in reaction to Ralstonia solanacearum.</a> Physiological and Molecular Plant Pathology. 68, 41-50 (2006).</li><li>Wydra, K., Beri, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+changes+in+methyl-ester+distribution+of+homogalacturonan+and+side+chain+composition+of+rhamnogalacturonan+I+as+possible+components+of+basal+resistance+in+tomato+inoculated+with+Ralstonia+solanacearum.">Immunohistochemical changes in methyl-ester distribution of homogalacturonan and side chain composition of rhamnogalacturonan I as possible components of basal resistance in tomato inoculated with Ralstonia solanacearum.</a> Physiological and Molecular Plant Pathology. 70, 13-24 (2007).</li><li>Hern&#225;ndez-Blanco, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+cellulose+synthases+required+for+Arabidopsis+secondary+cell+wall+formation+enhances+disease+resistance.">Impairment of cellulose synthases required for Arabidopsis secondary cell wall formation enhances disease resistance.</a> Plant Cell. 19 (3), 890-903 (2007).</li><li>Denanc&#233;, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+wat1+(walls+are+thin1)-mediated+resistance+to+the+bacterial+vascular+pathogen,+Ralstonia+solanacearum,+is+accompanied+by+cross-regulation+of+salicylic+acid+and+tryptophan+metabolism.">Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.</a> Plant Journal. 73 (2), 225-239 (2013).</li><li>Digonnet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+route+of+Ralstonia+solanacearum+colonization+in+Arabidopsis+thaliana+roots+during+a+compatible+interaction:+focus+at+the+plant+cell+wall.">Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall.</a> Planta. 236 (5), 1419-1431 (2012).</li><li>Sang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ralstonia+solanacearum+type+III+effector+RipAY+targets+plant+redox+regulators+to+suppress+immune+responses.">The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses.</a> Molecular Plant Pathology. 19 (1), 129-142 (2018).</li><li>Remigi, P., Anisimova, M., Guidot, A., Genin, S., Peeters, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversification+of+the+GALA+type+III+effector+family+contributes+to+Ralstonia+solanacearum+adaptation+on+different+plant+hosts.">Functional diversification of the GALA type III effector family contributes to Ralstonia solanacearum adaptation on different plant hosts.</a> New Phytologist. 192, 976-987 (2011).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assignment+to+positively+selected+sites+in+the+core+type+III+effector+RipG7+from+Ralstonia+solanacearum.">Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum.</a> Molecular Plant Pathology. 17, 553-564 (2016).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2-&#916;&#916;CT+method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2-&#916;&#916;CT method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Le&#243;n-Morcillo, R. J., Mart&#237;n-Rodr&#237;guez, J. A., Vierheilig, H., Ocampo, J. A., Garc&#237;a-Garrido, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+activation+of+the+9-oxylipin+pathway+during+arbuscular+mycorrhiza+formation+in+tomato+and+its+regulation+by+jasmonate+signalling.">Late activation of the 9-oxylipin pathway during arbuscular mycorrhiza formation in tomato and its regulation by jasmonate signalling.</a> Journal of Experimental Botany. 63 (10), 3545-3558 (2012).</li><li>Amrhein, V., Greenland, S., McShane, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retire+statistical+significance.">Retire statistical significance.</a> Nature. 567, 305-307 (2019).</li></ol>

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 ...

This protocol is significant because it allows scientists to perform straightforward genetic analysis of bacterial wilt disease in tomato. The main advantage of this technique is that the manipulation of gene expression and subsequent assays can be performed in a short time and with the small requirements of equipment and plant growth space. This is method is very versatile, and it may be applied to other crop plants to perform both pathogen inoculation and other physiological assays.To maintain the sterility during manipulation of seedlings in vitro, it is important to keep the plates closed when they are outside of the flow hood. This is an easy technique, but it requires multiple step for manipulation. The visualization of this method will help to see small tricks that are difficult to explain in the manuscript, helping other researchers to implement the method in their labs.Helping to demonstrate this procedure will be Achen Zhao, a graduate student. After sterilizing and washing the tomato seeds, transfer them to half-strength Murashige and Skoog medium without sucrose. Keep the seeds in the dark at a temperature between 25 and 28 degrees Celsius for three days.Place autoclaved 8.5-square-centimeter filters inside nine-square-centimeter Petri dishes containing half-strength Murashige and Skoog medium, and place six germinated tomato seeds on each plate. Seal the plate with Micropore tape, and incubate the germinated seeds at 25 to 28 degrees Celsius for three to four days. Grow the Agrobacterium rhizogenes MSU440 in solid LB medium with appropriate antibiotics for two days at 28 degrees Celsius before plant transformation.Using a sterile scalpel, cut the radicle and the bottom of the hypocotyl of the tomato seedlings. Use plastic tips or a scalpel blade to harvest A.rhizogenes biomass from the surface of the LB medium, and carefully dip the cut tomato seedlings in the bacterial biomass. After this, cover the tomato seedlings with a two-centimeter-by-four-centimeter semicircular filter paper in order to keep a high humidity and facilitate survival and new root development.It is important to keep the seedlings in high humidity in an environment that allows transpiration. To do this, cover the seedlings with filter paper, and close the plate with Micropore tape. Store the transformed tomato seedlings for six to seven days in a growth chamber at a temperature between 25 and 28 degrees Celsius.Then use a sterile scalpel to cut the new emerging hairy roots, and allow the seedlings to produce new hairy roots. Once the second generation of new hairy roots appears, remove the filter paper on top of the seedlings, and seal the plate with Micropore tape again. To visualize DsRed fluorescence or any other equipment for plant in vivo imaging, mark the positive transformed roots, which can be identified by the red fluorescence.Use a scalpel to remove the negative non-transformed roots, which can be identified by their lack of red fluorescence. Transfer the seedlings that show red fluorescence to a new plate containing half-strength Murashige and Skoog medium in order to facilitate the development of the transformed root as the main root. Keep the seedlings that do not show red fluorescence in the same plate to check the emergence of fluorescent roots in later time points.Cover the seedlings with a two-centimeter-by-four-centimeter semicircle filter paper, seal the plate, and incubate the seedlings to let them develop new hairy roots. Prepare the inoculation pots where the surface of the roots will be exposed to the bacterial inoculum by first soaking the inoculation pots with water, pouring off any excess water, and then placing them in a plastic planting tray. Using tweezers, transfer the selected seedlings with transformed roots to the inoculation pots.Cover the tray with plastic wrap or a transparent lid, and keep them at 25 to 28 degrees Celsius with 65%humidity. Remove the cover after five or six days. Grow Ralstonia in five LB liquid medium in an orbital shaker at 28 degrees Celsius, with shaking at 200 rpm, until the stationary phase.Measure the optical density at 600 nanometers to determine the bacterial numbers. Dilute the bacterial culture with water to an OD600 of 1. Place 16 to 20 inoculation pots that contain transformed tomato plants into an inoculation tray.Then pour 300 milliliters of diluted bacterial inoculum into the tray, and allow the plants to soak in the inoculum for 20 minutes. After this, prepare a new tray with a layer of potting soil. Move the inoculated pots into the new tray, and place the trays in a growth chamber with 75%humidity, a temperature between 26 and 28 degrees Celsius, and a photoperiod of 12 hours of light and 12 hours of darkness.Here, tomato roots are transformed and inoculated with Ralstonia to perform straightforward genetic analysis for the study of bacterial wilt disease. The development of disease symptoms is tracked in tomato plants with roots transformed with an empty vector and those transformed with an RAI construct targeting tomato CESA6. The disease index data are collected from the same experimental unit over time according to an arbitrary scale from zero to four and do not follow a Gaussian distribution, ruling out the use of standard tests for parametric data.As a standard approach, a U Mann-Whitney, two-tailed, non-parametric test to compare both the control and infection curves is used. According with this analysis, the difference between the medians of both curves appears to be non-significant. It is also possible to quantify the area under the disease progress curve, which allows combining multiple observations of disease progress into a single value.The area under the disease progress curve shows a higher value for control plants compared to tomato CESA6 RNAi plants at the end of the infection process, indicating that tomato CESA6-silenced plants are more resistant to Ralstonia infection than control plants. Confidence intervals offer a way of estimating, with high probability, a range of values in which the population value of a given variable is found. As seen here, the area of the 95%confidence interval for the control and infection curves estimate a higher chance of resistance when CESA6 is silenced.Disease index values can be transformed into binary data, considering a disease index lower than two corresponding to zero and a disease index equal or higher than two corresponding to one. This allows the representation of a survival curve after Ralstonia inoculation. The differences in the survival rate between the control and the infected plants are not statistically significant according to the Gehan-Breslow-Wilcoxon statistical test.The expression of CESA6 is then analyzed in two randomly selected transformed roots before the inoculation step, showing that the enhanced resistance to Ralstonia correlates with the reduced expression of CESA6. After two rounds of selection, a transformation rate of 35 to 40%can be obtained, and this value can be increased by performing additional selection rounds. While performing this procedure, it is important to keep the seedlings covered with a piece of filter paper to maintain high humidity and to seal the plates with Micropore to ensure gas exchange.After the root transformation, many of the treatments can be applied to study the plant response by observation of root physiology or using molecular biology, cell biology, or biochemistry. Mainly, this technique will allow researchers with limited resources to perform genetic analysis of bacterial infection in tomato roots.

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11.3K visualizações.  Chinese Academy of Sciences. Este protocolo é significativo porque permite que os cientistas realizem uma análise genética simples da doença bacteriana do murcho no tomate. A principal vantagem dessa técnica é que a manipulação da expressão genética e dos ensaios subsequentes pode ser realizada em pouco tempo e com os pequenos requisitos de equipamentos e espaço de crescimento da planta. Este método é muito versátil, e pode ser aplicado a outras plantas agrícolas para realizar tanto a inoculação de pató...