Transcriptomic analysis of the putative endophyte Cupriavidus metallidurans CH34 exposed to Nicotiana plumbaginifolia

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@phdthesis{96635ced0bed4f618efc4f1829ec7796,
title = "Transcriptomic analysis of the putative endophyte Cupriavidus metallidurans CH34 exposed to Nicotiana plumbaginifolia",
abstract = "Plant-bacteria relationships, and more specifically endophytic relationships, were and are still broadly studied because of their potential agricultural benefits. This Master Thesis aimed at investigate the poorly explored relation between the plant Nicotiana plumbaginifolia and the bacterial strain Cupriavidus metallidurans CH34. This plant was chosen because it belongs to the family Solanaceae, where plant-bacteria relationships are deeply known, whereas this bacterium was chosen for its metal-resistance and putative endophytism but these aspects are not highlighted in this Master Thesis as it is focused on the exploration of genes implied in the plant-bacteria interaction. Transcriptomic analysis was chosen to identify the bacterial genes that are differentially expressed when bacteria are in contact with plants, supposedly responding to root exudates. To perform this analysis and to study this relationship, a protocol was developed based on the related literature and optimized based on the main results obtained during this Master Thesis. Several trials were performed in order to get better plant and bacterial growth, by optimizing both plant and bacterial culture and co-culture time and conditions. To selectthe best culture and co-culture conditions, root architecture and plant weight were followed. Longer primary and lateral roots, higher lateral roots number and better leaves development were targeted. Bacterial density was also measured. Different plant culture methods and media were tested. In vitro culture in square Petri boxes was preferred to vessels containing vermiculite supplied with Hoagland solution. Four different mineral solutions were tested and 10-time diluted Murashige and Skoog (MS/10) medium was selected because it provided better plant root and bacterial growth. Co-culture conditions were also worked out. 12 ml tubes were preferred to 2 ml tubes.Co-culture was performed at 20C and boxes were slightly shaken to allow both bacterial and plant growth. Finally, plants were grown during 17 days at 20C on MS/10 agar medium and then incubated during 1 week into 12 ml tubes containing 1.6 ml liquid MS/10. 0.4 ml bacteria of an overnight culture were added during 10 h and 24 h co-culture before performing RNA extraction. The RNA extraction protocol was also adapted by adjusting the amount of treated bacterial pellets, by adding a lysozyme pre-treatment and by doing a TURBOTM DNase incubation which is more effective than DNase I in removing DNA contamination. High RNA concentrations, 260/230 and 280/230 ratios were finally obtained. However, obtained RIN (RNA Integrity Number) values were lower than the RIN threshold to perform high-quality microarray analysis. These low values, which measure the RNA purity and quality, could be explained by RNA degradation. This Master Thesis shows the difficulty and the time-consuming aspect of obtaining and optimizing a protocol, especially when working with 1-month experiments and living organisms.",
keywords = "Cupriavidus, endophytes, RNA extraction",
author = "{Vanden Balck}, Laetitia and {Van Houdt}, Rob",
note = "Score=10",
year = "2016",
month = "8",
day = "31",
language = "English",
school = "VUB - Vrije Universiteit Brussel",

}

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TY - THES

T1 - Transcriptomic analysis of the putative endophyte Cupriavidus metallidurans CH34 exposed to Nicotiana plumbaginifolia

AU - Vanden Balck, Laetitia

A2 - Van Houdt, Rob

N1 - Score=10

PY - 2016/8/31

Y1 - 2016/8/31

N2 - Plant-bacteria relationships, and more specifically endophytic relationships, were and are still broadly studied because of their potential agricultural benefits. This Master Thesis aimed at investigate the poorly explored relation between the plant Nicotiana plumbaginifolia and the bacterial strain Cupriavidus metallidurans CH34. This plant was chosen because it belongs to the family Solanaceae, where plant-bacteria relationships are deeply known, whereas this bacterium was chosen for its metal-resistance and putative endophytism but these aspects are not highlighted in this Master Thesis as it is focused on the exploration of genes implied in the plant-bacteria interaction. Transcriptomic analysis was chosen to identify the bacterial genes that are differentially expressed when bacteria are in contact with plants, supposedly responding to root exudates. To perform this analysis and to study this relationship, a protocol was developed based on the related literature and optimized based on the main results obtained during this Master Thesis. Several trials were performed in order to get better plant and bacterial growth, by optimizing both plant and bacterial culture and co-culture time and conditions. To selectthe best culture and co-culture conditions, root architecture and plant weight were followed. Longer primary and lateral roots, higher lateral roots number and better leaves development were targeted. Bacterial density was also measured. Different plant culture methods and media were tested. In vitro culture in square Petri boxes was preferred to vessels containing vermiculite supplied with Hoagland solution. Four different mineral solutions were tested and 10-time diluted Murashige and Skoog (MS/10) medium was selected because it provided better plant root and bacterial growth. Co-culture conditions were also worked out. 12 ml tubes were preferred to 2 ml tubes.Co-culture was performed at 20C and boxes were slightly shaken to allow both bacterial and plant growth. Finally, plants were grown during 17 days at 20C on MS/10 agar medium and then incubated during 1 week into 12 ml tubes containing 1.6 ml liquid MS/10. 0.4 ml bacteria of an overnight culture were added during 10 h and 24 h co-culture before performing RNA extraction. The RNA extraction protocol was also adapted by adjusting the amount of treated bacterial pellets, by adding a lysozyme pre-treatment and by doing a TURBOTM DNase incubation which is more effective than DNase I in removing DNA contamination. High RNA concentrations, 260/230 and 280/230 ratios were finally obtained. However, obtained RIN (RNA Integrity Number) values were lower than the RIN threshold to perform high-quality microarray analysis. These low values, which measure the RNA purity and quality, could be explained by RNA degradation. This Master Thesis shows the difficulty and the time-consuming aspect of obtaining and optimizing a protocol, especially when working with 1-month experiments and living organisms.

AB - Plant-bacteria relationships, and more specifically endophytic relationships, were and are still broadly studied because of their potential agricultural benefits. This Master Thesis aimed at investigate the poorly explored relation between the plant Nicotiana plumbaginifolia and the bacterial strain Cupriavidus metallidurans CH34. This plant was chosen because it belongs to the family Solanaceae, where plant-bacteria relationships are deeply known, whereas this bacterium was chosen for its metal-resistance and putative endophytism but these aspects are not highlighted in this Master Thesis as it is focused on the exploration of genes implied in the plant-bacteria interaction. Transcriptomic analysis was chosen to identify the bacterial genes that are differentially expressed when bacteria are in contact with plants, supposedly responding to root exudates. To perform this analysis and to study this relationship, a protocol was developed based on the related literature and optimized based on the main results obtained during this Master Thesis. Several trials were performed in order to get better plant and bacterial growth, by optimizing both plant and bacterial culture and co-culture time and conditions. To selectthe best culture and co-culture conditions, root architecture and plant weight were followed. Longer primary and lateral roots, higher lateral roots number and better leaves development were targeted. Bacterial density was also measured. Different plant culture methods and media were tested. In vitro culture in square Petri boxes was preferred to vessels containing vermiculite supplied with Hoagland solution. Four different mineral solutions were tested and 10-time diluted Murashige and Skoog (MS/10) medium was selected because it provided better plant root and bacterial growth. Co-culture conditions were also worked out. 12 ml tubes were preferred to 2 ml tubes.Co-culture was performed at 20C and boxes were slightly shaken to allow both bacterial and plant growth. Finally, plants were grown during 17 days at 20C on MS/10 agar medium and then incubated during 1 week into 12 ml tubes containing 1.6 ml liquid MS/10. 0.4 ml bacteria of an overnight culture were added during 10 h and 24 h co-culture before performing RNA extraction. The RNA extraction protocol was also adapted by adjusting the amount of treated bacterial pellets, by adding a lysozyme pre-treatment and by doing a TURBOTM DNase incubation which is more effective than DNase I in removing DNA contamination. High RNA concentrations, 260/230 and 280/230 ratios were finally obtained. However, obtained RIN (RNA Integrity Number) values were lower than the RIN threshold to perform high-quality microarray analysis. These low values, which measure the RNA purity and quality, could be explained by RNA degradation. This Master Thesis shows the difficulty and the time-consuming aspect of obtaining and optimizing a protocol, especially when working with 1-month experiments and living organisms.

KW - Cupriavidus

KW - endophytes

KW - RNA extraction

UR - http://ecm.sckcen.be/OTCS/llisapi.dll/open/20239916

M3 - Master's thesis

ER -

ID: 1637095