Characterizing Bacillus amyloliquefaciens UCMB5113 on a Plant Model Arabidopsis thaliana

MASTER’S  THESIS  IN  

MOLECULAR  MEDICAL  BIOLOGY  

45  hp  

HT2012-­‐VT2013  

Characterizing  Bacillus  amyloliquefaciens  UCMB5113  

on  a  plant  model  Arabidopsis  thaliana

PETER  VIJAY  METTAPALLY  

petmeh102@studentmail.oru.se

Örebro  University  2013  

Abstract:

Organic farming is gaining importance and acceptance worldwide due to its beneficial effects in agriculture and standing against losses caused by chemical fertilizers, pesticides and fungicides. Plant growth promoting bacteria (PGPB) plays an important role in organic farming by fixing atmospheric nitrogen, chelate iron, solubilizing phosphorous, producing and modulating phytohormones, providing antibiotics against pathogens. Understanding interaction mechanisms between PGPB and plant will be helpful in developing new formulations to form a strong symbiotic relationship between plant and bacteria. Bacillus

amyloliquefaciens UCMB5113 is a red pigmented, rod shaped Gram positive bacteria which

has been isolated from fields of the Ukraine. In the present study UCMB5113 and its interactions with the plant has been characterized. There was a significant promotion of plant root growth and protection against biotic stress with  the application of  10µl of 1x107_{/ml CFU}

UCMB5113 culture in Arabidopsis. The UCMB5113 can significantly withstand plant antimicrobial activity to stimulate plant root growth, but needs root hair defective RHD proteins to stimulate root hair elongation. UCMB5113 has significantly inhibited primary root elongation and developed number of lateral roots and root hairs in ethylene over expressed mutant, which suggests that it may be affecting ethylene signaling pathway in plants. UCMB5113 has a distinct red pigmentation which is a 38.5kDa water soluble protein with maximum absorbance at 422nm. These features are similar to the Orange Carotenoid Protein (OCP) of Synechocystis PCC 6803. This red pigmented protein has no significant effect on plant root growth promotion. Further biochemical and molecular studies are required to characterize and confirm the mechanisms of interaction.

Introduction

Plants either directly or indirectly are the main source of food for all organisms. There is a high demand for agricultural production like food grains, feed for cattle, fiber and biofuel for energy production which is needed to be increased by 1.1% per year from 2005/07 to 2050 (Tilman et al., 2011) (Alexandratos and Bruinsma, 2012). Chemical fertilizers cannot increase agriculture production and moreover, they cause environmental pollution. Beside this, there is a huge loss of agriculture products due to biotic and abiotic stresses. These effects of environmental pollution, biotic and abiotic stress to agricultural products can be minimized by organic farming by using Plant Growth Promoting Bacteria (PGPB) (Bashan and Holguin, 1998) PGPB is gaining importance and acceptance worldwide as these organisms are ecofriendly and provide beneficial effects on agriculture.

Fixing atmospheric nitrogen, chelating iron by siderophores, solubilizing phosphorous, producing and modulating phytohormones, producing antibiotics against pathogens and enhancing plant immune system to develop resistance against pests and diseases (Glick, 1995) are some of the beneficial effects provided by PGPB. Azoarcus, Azospirillum, Azotobacter, Arthrobacter, Bacillus, Clostridium, Enterobacter, Gluconacetobacter, Pseudomonas, and Serratia are some of the microorganisms which show plant growth promotion activity (Hurek and Reinhold-Hurek, 2003). Characterizing and understanding PGPB-plant interactions is important to develop formulations for strong symbiotic interactions between PGPB and plant to improve agriculture production. Among PGPB, Bacillus species are gaining more importance due to its capability to form endospores which can tolerate adverse conditions and produce different antibiotic compounds which stimulate the plant immune system  (Reva et al., 2004). These microbes are easy to grow and maintain and can be easily formulated to liquid form to spray in the fields.

Bacillus amyloliquefaciens UCMB5113 (Ukranian Collection of Microorganism Bacillus

strain 5113) from here on mentioned as UCMB5113. UCMB5113 has been isolated from the fields of the Ukraine. This organism has been identified as a red pigmented, Gram positive rod shaped bacteria which has a capability to colonize plant roots by withstanding antibacterial effects of root exudates (Reva et al., 2004).

The present study aims to know at what volume, UCMB5113 can promote root growth and protect plants against pathogens to know its ability to withstand high pressure of plant defensive mechanism and stimulate root growth. It is also aimed to understand the factors

responsible for stimulation and modification of root architecture. For these studies

Arabidopsis thaliana was used as a plant model system as it has a short life cycle, availability

Materials and methods:

Arabidopsis thaliana mutants (CYP79A1.6.3.1.1, CYP79A2.30.6, CYP79D2.28) (Bejai et al.,

2012), N2259, N2260, N2261 (Schiefelbein and Somerville, 1990), N8059 (López-Bucio et al., 2007) and Col-0 wild type were obtained from the Nottingham Arabidopsis Stock Centre (NASC) United Kingdom. Murashige and Skoog (MS) media, Lysogeny Broth (LB) media, Potato Dextrose Agar, Glucose and Gelrite were obtained from Duchefa Biochemie, Netherlands. 10 x 10 cms square and Petri plates were purchased from Greiner Bio-one, Germany. NuPAGE® pre cast Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gels, Lithium Dodecyl Sulfate (LDS) Sample buffer, Tris-Acetate SDS running buffer were obtained from NuPAGE®, Invitrogen Inc. (Lidingo, Sweden). Brilliant Blue R staining solution and Potassium ferricyanide were purchased from Sigma-Aldrich. Protein Assay Dye Reagent Concentrate was purchased from Bio-Rad. FLUOstar Omega Multiwell plate reader was purchased from BMG Labtech Company with version 1.10 OMEGA control software.

Seed sterilization and plant alignment:

Seeds were sterilized in 10% bleach added with a few drops of Tween 20 for 10 min and then sterilized with 70% ethanol for exactly 1 min, and sterilized with distilled water for three times for 5 min each. These sterilized seeds were mixed with 0.4% agar (Bactoagar) and placed on MS-media Petri plates (0.5% MS, 2% sucrose and 1% Gelrite with pH 5.8) and were incubated in growth chambers at temperature 22°C day and 18°C night, 16 h/8 h (day/night), with a fluorescent light intensity of 200 µmol m-2s-2, 70 % Relative Humidity (RH) for 5 days. After 5 days, 4 plants were aligned on 10 x 10 cm square plates with MS media without sucrose. Sucrose was not added in experimental plates to make bacteria depend on plants for the source of energy (Thoudal, 2012).

UCMB5113 culture:

A single pure colony was inoculated in 10 ml LB-media and incubated at 28°C overnight, this primary culture was transferred to 100 ml of LB-media and incubated at 28°C overnight and check the growth of culture by using a spectrophotometer at Optical Density (OD) at 600 nm. Culture was either diluted if the culture OD value is more than 0.4 at 600 nm or centrifuged to concentrate to get OD value to 0.4 at 600 nm. Culture at OD 0.4 has a concentration of 1 x

107CFU/ml. Culture was inoculated 3cm below the root tip (Palmqvist, 2011) and (Thoudal, 2012)

Alternaria brassicicola culture:

Alternaria brassicicola stock inoculum (20 µl) was spread on potato dextrose agar medium

and incubated in dark at room temperature for 15 days. The surface of the plates was sterilized with sterile water containing 0.02% Tween 20 and fungal hyphae were removed from the suspension by filtering through four layered autoclave sterilized cheesecloth. Concentration of spores was determined by using a hemocytometer and was adjusted to 1 × 105 spores/ml. Spore suspension of 5 µl was applied on to the wild type Col-0 plants for infection. After 4 days of infestation, damage score of leaves was given following the scale from 1-5 (1-10 to 20% severity, 2- 30 to 40% severity, 3- 50 to 60% severity, 4- 70 to 80% severity and 5- 90 to 100% severity) based on the severity (Botanga et al., 2012)

Red pigment extraction:

The UCMB5113 was grown in 100 ml of liquid LB-media as mentioned above. Culture was taken in 50 ml falcon centrifuge tubes and centrifuged at 5000 rpm for 10 min at room temperature. The cell pellet was collected separately and treated each with 50% Hexane, 50% Chloroform and distilled water respectively, and sonicated for 20 sec for 3 times. After sonication samples were centrifuged at 13000 rpm for 20 min at 20C. Compared with three solvents, red pigment was extracted completely in water solvent.

Water extracted pigment was processed further by collecting the supernatant. Supernatant was filtered through 0.45µ membrane filter, to filter out bacteria cell wall debris from the sample. This filtrate was scanned with a wavelength range of 330-600 nm in FLUOstar omega Multiwell plate reader to know the physical properties of the pigment.

To separate pigment from proteins, water extracted red pigment was treated with 100% Trichloroacetic acid (TCA) at a ratio of 1:4 TCA to sample concentration and incubated at 40C for 10 min and centrifuged at 14000 rpm for 5 min.

TCA precipitated red pigment indicates that the red pigment was a protein. Red pigmented protein (RPP) was resuspended in Phosphate buffered saline (PBS), pH 7.0 and total protein concentration were estimated by Bradford method using Bovine Serum Albumin (BSA) as

standard (following the protocol given by Bio-Rad manufacturer’s instructions) and subjected to electrophoresis.

After electrophoresis, gels were stained with Coomassie Brilliant Blue by washing gel with deionized water and stained with coomassie brilliant blue solution (0.1% coomassie R-250 in 40% ethanol and 10% acetic acid) for an hour and destained with 10% ethanol in 75% acetic acid solution on orbital shaker at room temperature until protein bands are visible. Potassium ferricyanide staining (Leong et al., 1992) was done by immersing gel immediately after electrophoresis in a potassium ferricyanide staining solution (100 mM Potassium ferricyanide mixed in 50 mM Tris-HCl and 100 mM NaCl pH 7.5) for 10 min in the dark and transferred to TCA/methanol solution to identify non-heme proteins.

Root analysis and statistics:

All the experiments were repeated at least twice and data were represented as mean ± s.e.m. Total root length, number of lateral roots and primary root length of plants was measured by using SmartRoot v3.6 software. All data were analyzed by using Graphpad Prism v5.03 for two tailed unpaired student t-test. (*p<0.05, ***p<0.001 represents statistically significant.)

Results:

The morphological difference between UCMB5113 treated and untreated plants was that the treated plants displayed number of lateral roots and decrease in primary root length compared with untreated plants.

Optimizing volume of UCMB5113 culture effecting root architecture:

The optimum volume of bacteria was important for effective plant growth and protection. For optimizing beneficial volume, 5 µl, 10 µl, 15 µl, 20 µl and 25 µl volumes of UCMB5113 (1x107CFU/ml) was taken and tested on wild type Col-0. The UCMB5113 treatment has restricted primary root length and stimulated lateral root elongation in treated plants compared with control plants (Fig 1B and 1C). There was a significant increase in total root length (Fig 1A) and number (no.) of lateral roots (Fig 1B) at 10 µl volume compared with control (p<0.001 versus control). There was a negative significant primary root growth was observed at the inoculation point with volumes 15 µl, 20 µl and 25 µl of UCMB5113 (Fig 1C p<0.001 mean values of 15 µl, 20 µl and 25 µl volume versus control), and positively significant growth was observed in the primary root through the inoculation point with volumes 10 µl (Fig 1C p<0.05 mean values of 10 µl volumes versus control). From these results, 10 µl volume of UCMB5113 (Fig 1A, 1B, and 1C) observed to be an optimal volume for effective growth, as this volume significantly increased total root length, number of lateral roots with more density and primary root was able to cross the inoculation point and moreover plant aerial parts observed to be healthier than control plants.

The effect of Alternaria brassicicola on UCMB5113 different volume treated plants:

The above experiment was continued to observe, at which volume of UCMB5113 induces recovery and resistance against A. brassicicola pathogen. UCMB5113 10 µl volume induces recovery and resistance against A. brassicicola pathogen (Fig 2 p<0.001 mean values of 10 µl volume versus control). Plants were more susceptible to A. brassicicola pathogen at volumes 15 µl, 20 µl and 25 µl of UCMB5113 culture as A. brassicicola damaging score was more than the control and 5 µl volume induced less damage (Fig 2). From this experiment it has been observed that 10 µl volume of UCMB5113 was optimum for plant protection to induce systemic resistance against pathogens.

Effect of glucosinolates on UCMB5113 root interactions:

Glucosinolates are one of the antimicrobial compounds produced by plant defensive system under biotic stress. To evaluate UCMB5113 interaction with the plant root system under high activation of plant defensive mechanism, different glucosinolates like (p-hydroxybenzyl glucosinolate, Benzyl glucosinolate and Isopropyl glucosinolate) over expressed transgenics, which induce these compounds in root exudates under infestation. Effect of UCMB5113 on glucosinolate over expressed transgenics was similar to wild type Col-0 by a significant increase in total root length, number of lateral roots and decrease in primary root length (Fig 3A, 3B and 3C p<0.05 and p<0.001 mean values of UCMB5113 treated versus untreated) was observed compared with the untreated transgenics. UCMB5113 treated plants induced a number of root hairs (data not shown) with high density compared with untreated transgenics was observed. These observations indicate that UCMB5113 can withstand high pressure of antimicrobial activity of plant defensive system for symbiotic association.

Effect of UCMB5113 on root hair defective (rhd) mutants:

Root hair defective (rhd) genes are a group of genes which are involved in root hair elongation (Schiefelbein and Somerville, 1990). Stimulation and regulation of rhd genes are interlinked and also depends on general factors like availability of calcium ions and phytohormones auxin and ethylene (Schiefelbein and Somerville, 1990). The rhd2, rhd3 and

rhd4 mutants were used to understand the mechanism of root hair stimulation by UCMB5113.

There was no significant difference observed in total root length, number of lateral roots and primary root length between UCMB5113 treated and untreated rhd mutants compare with wild type Col-0 (Fig 4A, 4B and 4C p<0.001 mean values of UCMB5113 treated versus untreated). These results indicate that UCMB5113 can stimulate root hair elongation under presence of rhd genes.

Effect of UCMB5113 on ethylene over expressing (eto2) mutant:

Most of the reported PGPB modify root architecture by effecting plant ethylene signaling pathway. To know if this mechanism is present in UCMB5113, eto2 mutant was used. UCMB5113 treatment has shown significant increase of total root length, increased number of lateral roots and decreased primary root length among wild type Col-0 and eto2 mutant of 5 days old plants (Fig 5A, 5B and 5C p<0.05 and p<0.001 mean values of UCMB5113 treated versus untreated), a similar pattern was followed till 14 days in eto2 mutant (Fig 5D, 5E and

5F p<0.001 mean values of UCMB5113 treated versus untreated). Roots at the age of 14 days old eto2 mutant treated with UCMB5113 were unable to grow beyond the UCMB5113 inoculation compared with control and untreated eto2 mutant, which indicates that UCMB5113 may regulate ethylene signaling pathway of plants. Further studies are required to understand the mechanisms.

Determination of red pigment of UCMB5113:

UCMB5113 is a red pigmented bacterium (Fig 6A) which is distinct from other B.

amyloliquefaciens strains. Crude red pigment has been extracted from UCMB5113 culture.

Red pigment was completely extracted in water solvent compared to 50% hexane and 50% chloroform solvents. Therefore, these results indicate that the red pigment of UCMB5113 is a water soluble pigment.

Water extracted red pigment filtrate has a maximum absorbance at 422nm (Fig 6B). Water extracted red pigment, treated with TCA revealed that red pigment of UCMB5113 was a red pigmented protein. No bands were observed with potassium ferricyanide staining (Fig 6C) which indicates that the isolated red protein was not nonheme iron protein. Major band at 38.5kDa was observed in filtrate and unfiltered water extracted red pigment by coomassie brilliant blue staining (Fig 6D). Further experiments are required for characterizing the RPP.

Effect of RPP on root architecture:

Crude extract of RPP was used to observe if this protein is involved in modifying root architecture. Wild type Col-0 roots was treated with 10µl of filtered RPP extract as a treatment, 10µl of water as a negative control and 10µl of UCMB5113 culture as a positive control. There was a slight significant difference observed from total root length and primary root lengths of negative control and RPP treated plants (Fig 7A and 7C p<0.05 mean values of RPP treated versus Negative control). There was a significant difference between the RPP and positive control with total root length, number of lateral roots and primary root length (Fig 7A, 7B and 7C p<0.001 mean values of RPP treated versus Positive control). From these results it indicates that RPP has no significant role in modulating plant root architecture.

Discussion:

Characterizing PGPB is essential to understand its plant interaction mechanisms to develop a better formulation for boosting plant growth promotion and protection against biotic stress. UCMB5113 treated plants induced a number of lateral roots and decrease in primary root length was observed as Pseudomonas strains colonizing A. thaliana were found to modify the root architecture either in  vivo or in  vitro (Persello-Cartieaux et al., 2001) . In this study, we observed that the beneficial effect of UCMB5113 on plant growth (Fig 1A, 1B and 1C) and protection against pathogen (Fig 2) was a dose dependent. Boosting growth promotion of sugar beet at a dosage of 105 CFU/seed or 107 CFU/g dry inoculums was reported by (Suslow, 1982) and approximately 105 CFU/g roots of WCS358 and WCS374 strains are required to suppress Fusarium wilt in radish (Raaijmakers et al., 1995). We observed that dosage of a 10µl volume of 107 _{CFU/ml UCMB5113 culture was required for boosting plant growth and}

protecting against pathogens.

Inoculating plants with PGPB induces and boost faster defensive response (Thoudal, 2012) against biotic and abiotic stress (Conrath et al., 2006). During symbiotic approach, the bacteria need to detect and tolerate plant defensive mechanism for colonization on plant roots (Yssel et al., 2011). Brassicaceae plants produce glucosinolates as biologically active secondary metabolites against pest and pathogens. Based on our experimental observation UCMB5113 has a capability to modulate glucosinolate over expressed transgenic Arabidopsis plants (Fig 3A, 3B and 3C).

Normal root hair elongation is induced by RHD2, RHD3 and RHD4 proteins (Schiefelbein and Somerville, 1990). Root hair formation was a common phenomenon observed among Gram positive PGPB’s (Fan et al., 2012). UCMB5113 has shown no significant difference to increase total root length, number of lateral roots and modulation in primary root under absence of rhd2, rhd3 and rhd4 genes (Fig 4A, 4B and 4C)compare with control plant Col-0. UCMB5113 can be effectively promote root growth and increase number of lateral roots when these genes are available which is common in most plants.

Ethylene is a phytohormone which required by plants for their normal growth and during stress conditions (Deikman, 1997). Ethylene regulates lateral root formation, but at higher concentrations ethylene shows a negative impact on lateral root formation (Negi et al., 2008). Over expressed ethylene production has shown less susceptibility to club root pathogens (Knaust et al., 2013). Most of the PGPB modulates ethylene levels by ACC deaminase (Glick,

2005). UCMB5113 has significantly inhibited primary root length and induced more lateral roots compare with untreated plants (Fig 5C and 5F). Further research is required to understand this mechanism.

UCMB5113 a red pigmented, rod shaped Gram positive bacteria (Reva et al., 2004) (Fig 6A). Water soluble red pigment protein has a maximum absorbance at 422 nm (Fig 6B) whereas pure carotenoid absorbance was estimated to be 455 nm (Wilson et al., 2008). This protein is not a nonheme iron protein as it has shown negative results with potassium ferricyanide staining (Fig 6C). From the coomassie brilliant blue staining it has been observed that filtered RPP extract has shown major protein band at 38.5 kDa (Fig 6D). Molecular weight of pure Orange Carotenoid Protein (OCP) of Synechocystis PCC 6803 is 35 kDa (Wilson et al., 2008), and OCP acts as a photoreceptor and mediate to reduce energy transfer from phycobilisomes to photosystem (Wilson et al., 2006) The red color of the UCMB5113 may be carotene, but the decline of absorbance may be due to encapsulation of carotene in protein. Red pigmented protein has no effect to modulate plant root system. Further experiments are needed to characterize this red pigmented protein.

To conclude, the results suggest that UCMB5113 is highly effective for plant growth promotion and protection at a concentration of 10µl of 107 _{CFU/ml and UCMB5113 can}

tolerate high pressure of plant defensive system. UCMB5113 is involved in regulation of ethylene signaling pathway of plants. To show a positive effect on plant root hair elongation it requires RHD proteins. RPP has shown no effect in modulating plant root system. Further biochemical and molecular studies are required to confirm the above observations for development of better formulation to increase agricultural production.

Acknowledgements:

I heartily thank Prof. Johan Meijer for providing me an opportunity to work with his group and providing help & support to finish my project successfully. I thank Asst. Prof. Sarosh Bejai for his extensive help and scientific support during my project work and Shashidar, Mohammad, Martin and Adnan for their support.

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Figure legends

Figure 1:

UCMB5113 dose response of A. thaliana. Wild type Col-0 plants were exposed to different volumes of UCMB5113. Total root length (cm) (A), no. of lateral roots (B) and primary root length (cm) (C) of Col-0 were measured and analyzed. Data represents mean ± s.e.m. n=12. *p<0.05, ***p<0.001 versus control (unpaired student’s t-test). These observations indicate that 10µl volume of UCMB5113 was an optimum concentration for UCMB5113 Arabidopsis interactions.

Figure 2:

Alternaria brassicicola damaging score of A. thaliana. Plants treated with different volumes

of UCMB5113 were exposed to 5µl of A. brassicicola. Damaging score of leaves was given from 1-5 (1-10 to 20% severity, 2- 30 to 40% severity, 3- 50 to 60% severity, 4- 70 to 80% severity and 5- 90 to 100% severity) based on the severity. These observations indicate that 10µl volume of UCMB5113 was an optimum concentration for inducing resistance and recovering against pathogens. Data represents mean ± s.e.m. n=12. ***p<0.001 versus control (unpaired student’s t-test).

Figure 3:

Effect of UCMB5113 on over expressed glucosinolate transgenics. CYP79A1.6.3.1.1 represents p-hydroxybenzyl over expressed glucosinolate, CYP79A2.30.6 represents Benzyl over expressed glucosinolate, CYP79D2.28 represents Isopropyl over expressed glucosinolate and Col-0 represents wild type. Total root length (cm) (A), no. of lateral roots (B) and primary root length (cm) (C) of the UCMB5113 treated and untreated plants were measured and analyzed. Data represents mean ± s.e.m. n=12. ***p<0.001 versus untreated (unpaired student’s t-test). These observations indicate that UCMB5113 can withstand high pressure of plant defensive system to modulate plant root architecture.

Figure 4:

Effect of UCMB5113 on root hair defective (rhd) mutants. N2259 represents rhd2 mutant, N2260 represents rhd3 mutant, N2261 represents rhd4 mutant and Col-0 represents wild type. Total root length (cm) (A), no. of lateral roots (B) and primary root length (cm) (C) of the UCMB5113 treated and untreated plants were measured and analyzed. Data represents mean

± s.e.m. n=12. ***p<0.001 versus untreated (unpaired student’s t-test). These observations indicate that UCMB5113 requires rhd genes for root hair elongation.

Figure 5:

Effect of UCMB5113 on ethylene over expressing (eto2) mutant. eto2 represents ethylene over expressing mutant and Col-0 represents wild type. Total root length (cm) (A and D), no. of lateral roots (B and E) and primary root length (cm) (C and F) at 5 days after inoculation and 14 days after inoculation of the UCMB5113 treated and untreated plants were measured and analyzed. Data represents mean ± s.e.m. n=12. ***p<0.001 versus untreated (unpaired student’s t-test). These observations indicate that the UCMB5113 might be acting on plant ethylene signaling pathway in modulating root architecture.

Figure 6:

UCMB5113 Red Pigment Protein characterizing.UCMB5113 is a red pigmented bacterium (A). The Red pigment extract was scanned under wavelength ranging from 330-600nm has shown maximum absorbance at 422nm (B). There was no band detected when this red pigment extract was electrophoresis and stained with potassium ferricyanide for nonheme iron protein (C), which indicated that the red pigment extract was not nonheme iron protein. A predominant protein band at 38.5KDa was detected by coomassie brilliant blue staining (D). These observations indicate that the UCMB5113 red pigmentation may be due to the presence of OCP as in Synechocystis PCC 6803 which need to be confirmed by further experiments. Figure 7:

UCMB5113 RPP treatment of A. thaliana roots. Wild type Col-0 roots was treated with 10µl of filtered RPP extract, 10µl of water treated plants as a negative control and 10µl of UCMB5113 culture treated plants as a positive control. Total root length (cm) (A), no. of lateral roots (B) and primary root length (cm) (C) of Col-0 were measured and analyzed. Data represents mean ± s.e.m. n=12. *p<0.05, ***p<0.001 versus positive and negative controls (unpaired student’s t-test). These observations indicate that the RPP of UCMB5113 has no role in modulating plant root architecture.

Figure 1: 0 50 100 150 200 250 300 350 400 Control 5 µl 10 µl 15 µl 20 µl 25 µl T otal R oot Le n gth (c m) UCMB5113 volumes ***   0 5 10 15 20 25 30 35 40 45 Control 5 µl 10 µl 15 µl 20 µl 25 µl N o. of Late ral R oots UCMB5113 volumes ***   0 5 10 15 20 25 30 35 Control 5 µl 10 µl 15 µl 20 µl 25 µl P ri mar y R oot Le n gth (c m) UCMB5113 volumes ***   ***   ***        *   A B C

Figure 2: 0 1 2 3 4 5 6 Control 5 µl 10 µl 15 µl 20 µl 25 µl A.  b ra ssi ci co la  Damage  Sco re   UCMB5113 volumes ***

0 1 2 3 4 5 6 7 8 9 10 P ri mar y R oot Le n gth (c m)

Glucosinolate over expressed transgenics

Control Treated *** *** *** 0   2   4   6   8   10   12   14   16   18   N o. of Late ral R oots

Glucosinolate over expressed transgenics

Control Treated *** *** *** *** 0 5 10 15 20 25 T otal R oot Le n gth (c m)

Glucosinolate over expressed transgenics

Control Treated *** *** * A B C

Figure 4: 0 5 10 15 20 25 Col-0 N2259 N2260 N2261 N o. of Late ral R oots

Root hair defective mutants

Control Treated *** 0 10 20 30 40 50 60 Col-0 N2259 N2260 N2261 T otal R oot Le n gth (c m)

Root hair defective mutants

Control Treated *** 0 2 4 6 8 10 12 14 Col-0 N2259 N2260 N2261 P ri mar y R oot Le n gth (c m)

Root hair defective mutants

Control Treated *** A B C

Figure 5: 0 5 10 15 20 25 30 35 40 Col-0 eto2 T otal R oot Le n gth (c m)

5 days after inoculation

Control Treated 0 1 2 3 4 5 6 7 8 9 Col-0 eto2 N o. of Late ral R oots

5 days after inoculation

Control Treated 0 1 2 3 4 5 6 7 8 9 10 Col-0 eto2 P ri mar y R oot Le n gth (c m)

5 days after inoculation

Control Treated 0 20 40 60 80 100 120 140 160 180 200 Col-0 eto2 T otal R oot Le n gth (c m)

14 days after inoculation

Control Treated 0 5 10 15 20 25 30 35 40 45 50 Col-0 eto2 N o. of Late ral R oots

14 days after inoculation

Control Treated 0 2 4 6 8 10 12 14 16 Col-0 eto2 P ri mar y R oot Le n gth (c m)

14 days after inoculation

Control Treated *** *** *** *** * *** *** * *** A B C D E F

Figure 6: 0,000   0,200   0,400   0,600   0,800   1,000   330n m   358n m   386n m   414n m   442n m   470n m   498n m   526n m   554n m   582n m   OD

Wave  Length  in  nm  

RPP Maximum Absorbance

15 % SDS-PAGE for Nonheme Iron Protein Marker

(kDa) _{RPP extract} Unfiltered Filtered

RPP extract

100

70

55

40

35

25

15

10

Figure 7:

70

55

40

35

25

15

10

15 % SDS-PAGE for Red Pigmented

Protein

65.5

63.6

62.4

58.5

55.6

40.0

48.5

46.6

49.8

39.7

38.5

37.3

28.7

25.0

20.6

18.4

15.0

10.0

10.0

15.0

25.0

18.4

20.6

39.7

38.5

37.3

28.7

32.7

35.0

0   5   10   15   20   25   30   35   Control   RPP   Culture   N o.  o f  L at er al  Ro ot s   Treatments   Col-­‐0   *** *** 0   2   4   6   8   10   12   14   16   Control   RPP   Culture   Pr im ar y   Ro ot  L en gt h   (c m )   Treatments   Col-­‐0   * *** 0   10   20   30   40   50   60   70   80   90   100   Control   RPP   Culture   To ta l  Ro ot  L en gt h   (c m )   Treatments   Col-­‐0   *** * A B C