Name: tomato root transformation followed by inoculation with ralstonia solanacearum for straightforward genetic analysis of bacterial wilt disease
Uploaded: 2020-03-11T13:00:00
Description: 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

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.
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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 ...

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