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Bacillus mycoides: novel tools for studying the mechanisms of its interaction with plantsYi, Yanglei

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1Introduction

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PLANT-MICROBE INTERACTION

Plants naturally harbor diverse species of microorganisms, because they offer a wide range of habitats, supporting microbial growth. These mi-croorganisms form a complex biological community interacting with the plant host, e.g. being pathogenic, or employing mutualism (symbi-onts), and commensalism (Pini et al., 2012). Bacteria that associate with plants are diverse in their ability to affect plant health, their genotypes, and their phenotypic characteristics. Although the majority of research on plant-associated microorganisms has focused on phytopathogens and diazotrophic (nitrogen-fixing) phytosymbionts, it is clear that many plant-associated microbes, even those that comprise only a small pro-portion of a community, have functions that are of agricultural or envi-ronmental importance (Beattie, 2006). Bacteria could reside in/on seeds, roots, leaves, and fruits of plants. The rhizosphere, the narrow zone of soil influenced by the root, is much richer in bacteria than the surround-ing bulk soil and even more densely populated than other parts of a plant (Lugtenberg and Kamilova, 2009) (Figure 1).

The rhizosphere contains an enormous range of compounds excreted by roots. The roots exudate a range of inorganic compounds like ions,

Figure 1. Plant-associated microorganisms, plant-microbe and microbe-microbe inter-action. Soil inhibiting bacteria are attracted by root secreted signals to become plant- associated bacteria. They may live in the rhizosphere, rhizoplane or endosphere. Some of them are aiding plant health by fighting against pathogens through niche competi-tion, antimicrobial production, and induced systematic resistance.

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inorganic acids, oxygen and water. These compounds may directly affect the biogeochemistry of the soil (Jones et al., 2009). However, the major-ity of the root exudates are formed by organic materials, which can be di-vided into low-molecular-weight compounds and high-molecular-weight compounds. The low-molecular-weight compounds include amino acids, organic acids (citric, malic, succinic, oxalic and pyruvic), sugars (glucose, xylose, fructose, maltose, sucrose, ribose), phenolics, fatty acids and an array of secondary metabolites. The high-molecular weight compounds consist of mucilage and proteins (Badri and Vivanco, 2009; Rohrbacher and St-Arnaud, 2016), and rhizosphere microorganisms can use some of these compounds as an energy source for growth and development or as signaling molecules (Carvalhais et al., 2015). The composition of root ex-udates varies by plant species, developmental stage and plant growth sub-strate (Doornbos et al., 2011). For example, the organic acids and sugars in root exudates of tomato and cucumber increase during plant growth on stone wool (Kamilova et al., 2006). A systematic proteomic analysis of root exudates showed that the defense-related proteins such as chitinases, glucanases, and myrosinases showed more secretion during the flower-ing stage of Arabidopsis thaliana (De-la-Peña et al., 2010), while the release of amino acids and phenolics increased at later stages of life of Arabidopsis (Chaparro et al., 2013).

It is believed that the dynamic nature of root exudates is one of the driv-ing forces for the plant to select associated microbial communities that are favorable for the plant. The best-known plant-microbe symbiosis is the nodulation of legumes by rhizobia. This interaction is very specific, allow-ing certain rhizobial strains to nodulate with specific host legumes (Bais et al., 2006). In soil, a Rhizobium spp. finds its host legume plant from a distance by chemotaxis, being attracted to the root exudates due to the presence of flavonoids (Sugiyama and Yazaki, 2012). Flavonoids also reg-ulate the expression of nod genes that play important roles in nodulation establishment (Abdel-Lateif et al., 2012). Another widely-occurring plant- associated microbiome part is formed by arbuscular mycorrhizal fungi (AMF), which form a symbiotic relationship with more than 80% of terres-trial plants, from bryophytes to tracheophytes (Lee et al., 2013). Although AMF have low host specificity in their symbiosis with plants, they may also recognize their host by signals released by plant roots, similar to the rec-ognition by rhizobia. It has been shown that mycorrhizal fungi are me-diated by root-secreted compounds, such as strigolactone 5-deoxystrigol ( Yoneyama et al., 2008), sugars, and carbohydrates (Kiers et al., 2011).

Some specific root-released chemical signals have been identified, that play a role in recruiting specific bacteria to build mutualistic interactions with the plant. For example, the chemotaxis to amino acids of root exudates

has an important role in root colonization by Pseudomonas fluorescens Pf0-1 (Oku et  al., 2012). In Azospirillum brasilense, the chemoreceptor- like pro-tein Tlp1 serves as an energy taxis transducer and affects the coloniza-tion on roots (Greer-Phillips et al., 2004). Chemotaxis towards A. thaliana root exudates was mediated by two well-characterized chemoreceptors, McpB and McpC, as well as by the orphan receptor TlpC in Bacillus subtilis (Allard-Massicotte et  al., 2016). Moreover, plant roots also secrete com-pounds that mimic quorum-sensing (QS) signals of bacteria to stimulate QS-regulated responses of associated bacteria (Huang et  al., 2014). The most common QS system occurs via N-acyl homoserine lactones (AHLs) binding to members of LuxR-family transcriptional regulators. Thus, they regulate the expression of downstream genes with the lux box in the pro-moter region (Hartmann et  al., 2014). However, the LuxR-family regu-lator (Lux-R solo) found in plant-associated bacteria lacks the LuxI pro-tein when compared with other LuxR families. These regulators do not bind AHLs but to plant-produced compounds (González et al., 2013; Ven-turi and Fuqua, 2013). For instance, a LuxR solo regulator of P. fluorescens, PsoR, was identified to have a function in responding to plant compounds and regulating genes involved in the biosynthesis of various antimicro-bial compounds including 2,4-diacetylphloroglucinol, which has antago-nistic activity against the damping-off disease pathogen Pythium ultimum (Subramoni et al., 2011).

Following recognition and recruitment, bacteria physically interact with different parts of the roots from the tip to the elongation zone to form complex multicellular and often multispecies assemblies, including bio-films and smaller aggregates (Danhorn and Fuqua, 2007). The rhizobac-teria form biofilms in a heterogeneous way along the root surface, where the root tips usually have the least bacterial biofilm formed (Rudrappa et al., 2008). In general, the zone immediately behind the root tip is con-sidered to be a major site of colonization (Badri and Vivanco, 2009). This is partially due to the pH difference from the root tip to basal regions ( Peters, 2004), which might affect the bacterial growth and biofilm devel-opment. The nutrient availability is also quite different since particular cell types in the root system might be more important than others in the secretion of particular compounds. The biofilm formation of rhizobacte-ria on root surfaces is a mutualistic interaction. In B. subtilis-A. thaliana interaction, plant polysaccharides act as an environmental cue that trig-gers biofilm formation by the bacterium (Beauregard et al., 2013). In turn, plants benefit from the biofilm of rhizobacteria. The inhibitory effect of Lysobacter sp. strain SB-K88 against pathogenic Aphanomyces cochlioides is due to a combination of antibiosis and biofilm formation at the rhizo-plane of the host plant (Islam et al., 2005).

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Although extensive studies have been performed to investigate plant- associated bacteria, we are far from understanding the plant- microbe in-teraction mechanisms, especially when indigenous multispecies bacterial communities are considered. So far, the studies on plant-growth promot-ing rhizobacteria (PGPR) and their beneficial effects on host plants have drawn considerable attention, but more research is needed to increase our understanding of the underlying processes.

PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR)

PGPR form a group of bacteria that colonize the host plant roots and pro-mote plant growth due to certain traits of the rhizobacteria. A diverse ar-ray of bacterial species including Azospirillum, Azotobacter, Bacillus, Burk-holderia, Enterobacter, Klebsiella, Pseudomonas, Serratia, and Xanthomonas were identified as PGPR of which Bacillus and Pseudomonas spp. are pre-dominant (Podile and Kishore, 2007). PGPR promote plant growth di-rectly by either facilitating resource acquisition (nitrogen, phosphorus, and essential minerals) or modulating plant hormone levels, or indirectly, by decreasing the inhibitory effects of various pathogens on plant growth and development in the form of biocontrol agents (Ahemad and Kibret, 2014) (Figure 2).

Of all the essential nutrients, nitrogen (N) is required by plants in the largest quantity and is most frequently the limiting factor in crop pro-ductivity. However, 78% of N is in the atmosphere and thus is unavaila-ble for growing plants. Biological N2 fixation by nitrogen-fixing microor-ganisms is widely distributed in nature. Rhizobia such as Rhizobium and Bradyrhizobium associated with legume plants are well known for their N2 fixing activity and are defined as symbiotic N2-fixing bacteria (Zah-ran, 2001). Free-living PGPR with N2-fixing capacity are defined as diaz-otrophs (Kennedy et al., 2004), which can form a non-obligate interaction with host plants. The second most important essential nutrient, phos-phate (P), is ubiquitously present in soil but has low solubility or can eas-ily be converted to insoluble forms (Hanif et  al., 2015). Some PGPR pro-duce organic acids or enzymes including phosphatases and phytases to release soluble phosphorus from organic compounds in soil (Lugten-berg and Kamilova, 2009). Also, balanced and sufficient potassium and/or iron availability is essential to maintain a healthy active root system. PGPR that contribute to the uptake of these nutrients have been reported and offer a novel solution for sustainable agriculture (Sheng, 2005; Gupta et al., 2015). Hormones, also known as plant growth regulators, can reg-ulate plant growth and development by stimulating or inhibiting plant

growth. PGPR produce various phytohomones including auxin, gibber-ellins, and cytokinins to enhance plant growth (Ruzzi and Aroca, 2015). Some PGPR possessing 1-aminocyclopropane-1-carboxylate (ACC) deam-inase activity are able to reduce the ethylene level to relieve plants from several forms of stress (Bal et al., 2013). Rhizobacteria, such as B. subtilis, B. megaterium, and P. chlororaphis, promote plant growth by producing vol-atiles like 2,3-butanediol, dimethylhexadecylamine, and 2-pentylfuran (Chung et al., 2010; Zou et al., 2010; Velázquez-Becerra et al., 2011).

The indirect plant growth stimulation by PGPR basically involves the ability of PGPR to reduce the deleterious effects of plant pathogens on crop yield. Competition for the rhizosphere nutrients and niches is a fundamental mechanism by which PGPR protect plants from pathogens ( Compant et  al., 2005). Prior to infection by a plant pathogen, PGPR col-onization could trigger plant defense mechanisms to prevent or reduce the severity of the disease. This phenomenon is known as the induction of systemic resistance (ISR), which is activated by PGPR and functions throughout the plant. On the other hand, PGPR produce a wide variety of compounds with antimicrobial properties to defend itself against other mi-croorganisms including phytopathogens. Siderophores, bacteriocins and antibiotics are three of the most effective and well-known mechanisms that PGPR employ to minimize or prevent phytopathogenic proliferation (Beneduzi et al., 2012). Under iron-limiting conditions, PGPR produce low-molecular- weight chelating compounds called siderophores. The produc-tion of siderophores plays an important role in reducing phytopathogen proliferation by iron deprivation and enhancing plant development by in-creased uptake of iron (Masalha et al., 2000; Hibbing et al., 2010). A vari-ety of microorganisms also exhibit antagonistic activity against pathogens by excreting cell wall hydrolytic enzymes including chitinases, proteases,

Figure 2. Schematic diagram showing that plant growth-promoting bacteria affect plant growth directly and indirectly. ISR: induced systemic resistance.

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and β-1,3-glucanase to destroy the cell wall integrity of pathogens (Com-pant et al., 2005). It has long been known that bacteriocins and antibiotics are involved in shielding host against pathogens and mediating local pop-ulation dynamics in the rhizosphere (Thomashow et al., 2008; Subrama-nian and Smith, 2015). B. clausii GM17 isolated from the rhizosphere of On-onis angustissima have been found to produce bacteriocin Bac-GM17 with bactericidal effect on Agrobacterium tumefaciens and fungistatic effect on Candida tropicalis (Mouloud et  al., 2013). The bacteriocin thuricin 17, iso-lated from B. thuringiensis NEB17, shows plant growth stimulation effects on plants (Lee et al., 2009). Another mechanism involved in disease sup-pression of PGPR is the production of antibiotics. Pseudomonads produce lipopeptides (LPs) such as 2,4-diacetyl phloroglucinol, hydrogen cyanide, pyrrolnitrin, tensin, pederin, tropolone, phenazine, that have been exten-sively studied (Gross and Loper, 2009). Bacillus is well-known to produce fengycin and surfactin family LPs. In addition, the production of zwitter-micin A and bacillomycin, has also been reported (Arguelles-Arias et al., 2009; Kinsella et al., 2009; Pérez-García et al., 2011).

RHIZOSPHERE BACILLUS FUNCTION AND PHYLOGENY

Bacillus spp. are well-known rhizosphere residents of many plants and may directly or indirectly contribute to crop productivity. By standard isolation on nutrition-rich medium, isolates phylogenetically related to B. subtilis and B. cereus have been frequently recovered (Bai et al., 2002; Cavaglieri et  al., 2005; Cazorla et  al., 2007; Egamberdieva et  al., 2008). Pandey and Palni (1997) reported that Bacillus species are the dominant bacteria of the rhizosphere of established tea bushes. B. subtilis and B. my-coides comprise a major part of the bacterial population, even during un-favorable periods, also due to their ability to sporulate. Moreover, culture- independent rhizosphere microbiome studies have indicated the presence of a large uncultured diversity in Bacillus (Felske et al., 2003; Choudhary and Johri, 2009; Pereira et al., 2011). In a disease- suppressive soil, Bacillus was identified as the dominant bacterial species. The strain B. amylolique-faciens NJN-6 isolated from this suppressive soil decreased Panama dis-ease of banana by 68.5% (Xue et  al., 2015). The rhizobacteria B. subtilis and B. amyloliquefaciens FZB42 have 4–5% and 8.5% of their genome, re-spectively, devoted to antibiotic synthesis (Stein, 2005; Chen et al., 2007). Among all the synthesized antimicrobial compounds by Bacillus, cyclic li-popeptides (LPs) of the surfactin, iturin and fengycin families have been well- characterized, with potential uses in biotechnology and agriculture (Ongena and Jacques, 2008). It has been shown that specific strains of

the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pum-ilus, and B. mycoides elicit a significant reduction in the incidence or se-verity of various diseases on a diversity of plant hosts via ISR mechanisms ( Kloepper et al., 2004; Ryu et al., 2004).

Several studies have shown that Bacillus species are highly diverse, not only among different species but also between strains of the same spe-cies. Analyses of whole bacterial communities showed a significant dis-tinction between soil and rhizosphere communities (Smalla et al., 2001). A significant difference was observed in the total and relative abundance of Bacillus- like sequences amplified from bulk soil and crop roots (Choudhary and Johri, 2009). Mavingui et al. (1992) studied the genetic and phenotypic diversity of 130 strains of B. polymyxa isolated from non- rhizosphere soil (32 strains), rhizosphere soil (38 strains), and the rhizoplane (60 strains). Their results showed that diversity within populations of B. polymyxa iso-lated from non-rhizosphere and rhizosphere soil is higher than that of B. polymyxa isolated from the rhizoplane. This phenomenon can be ex-plained by the selection effects of plant roots on particular strains of a spe-cies. On the other hand, the strains living in association with plants are well adapted to the specific niche and did evolve some beneficial features for the host. Comparative genomics revealed a core set of genes that might be found in beneficial B. amyloliquefaciens strain. The phylogenetic tree based on concatenated sequences of 11 conserved genes of various other Bacillus strain genomes, results in a plant- associated clade (Figure  3A) (Magno-Perez-Bryan et al., 2015). Phylogenomic analysis of B. subtilis and B. amyloliquefaciens indicated that strains isolated from plant-associated (PA) habitats could be clearly distinguished from those from non-plant- associated (nPA) niches in both species (Figure 3B). Furthermore, the core genomes of PA strains are more abundant in genes relevant to interme-diate metabolism and secondary metabolite biosynthesis, as compared to those of nPA strains. Moreover, they possess specific additional genes in-volved in the utilization of plant-derived substrates and the synthesis of antibiotics (Zhang et al., 2016).

ENDOPHYTIC BACILLUS SPP.

As soil-borne microorganism, the species of Bacillus is not only found in the rhizosphere but also in the endosphere of plants. Endophytic bacte-ria are defined as bacteria colonizing the internal tissues of plants with-out causing symptoms of infection or negative effects on their host (Col-lins et al., 2004). According to their life strategy and the dependency on the host, endophytic bacteria can be classified as obligate endophytes,

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facultative endophytes or opportunistic endophytes. Obligate endophytes depend entirely on their host for all of their needs and spend all of their life within the host. In contrast, facultative and opportunistic endophytes have a free-living stage outside the plant host (Hardoim et  al., 2008). Once the mutualistic symbiosis has been established, the plants provide a uniform, nutrient-rich, and non-competitive niche for endophytes. In turn, the endophytic bacteria bring beneficial attributes to their hosts, as they enhance plant growth and health in similar ways as PGPR. For exam-ple, direct plant growth promotion can be achieved by producing plant growth regulators including auxins, cytokinins, and gibberellings, reduc-ing ethylene stress by 1-aminocyclopropane-1-carboxylate (ACC) deami-nase; or fixing nitrogen or solubilizing phosphorus to aid plant nutrient acquisition(Rosenblueth and Martínez-Romero, 2006; Reinhold-Hurek and Hurek, 2011). Endophytic bacteria also indirectly benefit plant growth by the production of antimicrobial compounds and the induction of plant defense mechanisms (Weyens et al., 2009). Moreover, endophytic bacteria can help the host to cope with extreme biotic (pathogen, pest) and abiotic (temperature, drought, and heavy metals contaminations) stresses, which can severely reduce crop production. When applied as plant growth pro-moting or biocontrol agents, endophytic bacteria have advantages over rhizosphere bacteria due to their close interaction with the plant. For the seed-transmitted endophytes, there is an extra advantage for commer-cialization. Seed treatment is an efficient delivery technique to place mi-crobial inocula into the soil, where they will be well positioned to colonize seedling roots and protect against soil-borne diseases (Card et al., 2016).

The dominant endophytic bacteria belong to three major phyla i) Prote-obacteria, including Azorhizobium, Rhizobium, Enterobacter, Pseudomonas, and Burkholderia (Rosenblueth and Martínez-Romero, 2006; Taghavi et al., 2010; Dudeja et al., 2012; Ali et al., 2014); ii) Actinobacteria e.g., Microbac-terium, Streptomyces, Curtobacterium, and Arthrobacter (Conn and Franco, 2004; Chung et al., 2010; Verma et al., 2011); and iii) Firmicutes, including Bacillus and Staphylococcus (Luo et al., 2012; Phukon et al., 2013). Species of these genera are ubiquitous in the soil/rhizosphere, which represents the main source of endophytic colonizers. Among them, the genus Ba-cillus stands out as one of most reported endophytic bacteria from var-ious plant species. The spore-forming property with additional features of being natural soil dwellers makes Bacillus suitable for further devel-opment into commercial biocontrol agents. A recent metagenomics study revealed the dominance of Bacillus as a major endophytic genus in rice roots, probably playing a key role in nitrogen fixation (Sengupta et  al., 2017). Bodhankar et  al. (2017) isolated 80 seed endophytic bacte-ria from 30 maize genotypes. Their results showed that Bacillus was the

Figure 3. Phylogenetic analysis of several Bacillus strains. (A) Phylogenetic tree of iso-lates closely related to species of B. amyloliquefaciens, B. subtilis, B. atrophaeus, and B. li-cheniformis. 11 concatenated genes (nusA, rpoA, dnaA, rpoB, gyrA, gyrB, rpoC, spoVG, sigW, sigH, and sigB) were handled to build a neighbor-joining tree, using MEGA 5 bootstrap values (10,000 repetitions), which are shown on the branches. (B) The maximum-likeli-hood phylogeny derived from the alignment of 1835 concatenation core genes of B. subtilis and B. amyloliquefaciens. Leaf clip art indicates an association with plants. Colors indicate the biogeographic origin of the strains (PA: plant-associated; nPA: non-plant-associated). (Images modified from Magno-Perez-Bryan et al. (2015); Zhang et al. (2016))

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most dominant encountered genus affiliated with the Phylum Firmicutes. To date, the species that have been demonstrated to be endophytes are mainly distributed among B. subtilis, B. amyloliquefaciens, B. mojavensis, B. cereus, B. thuringiensis, B. mycoides, B. velezensis, B. pumilus, B. megaterium, and Paenibacillus, with a few reports on B. altitudinis, B. capparidis, and B. endoradicis. Most of the endophytes exhibit at least one beneficial func-tion on plants, including growth promotion (Falcao et al., 2014), ISR (Yi et al., 2013), antifungal activity (Gond et al., 2015), antibacterial activity (Jasim et  al., 2016), phytomediation (Bisht et  al., 2014), and stress toler-ance (Gagne-Bourque et al., 2016).

A GENERAL INTRODUCTION ON B. MYCOIDES

Bacillus mycoides is a Gram positive, rod-shaped, and spore-forming bac-terium. The growth of B. mycoides on agar plates has a particular rhizoid shape, resulting from cells linked end to end, and grouped in filaments bundles (Figure 4). It belongs to the B. cereus species-group, which com-prises several closely related species including B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. weihenstephanensis, B. pseudomycoides and some recently identified species (Fiedoruk et  al., 2017). However, B. my-coides received far less attention compared to other members, since it is neither pathogenic, as B. cereus and B. anthracis, nor pesticidal as B. thur-ingiensis. To date, there are more than twenty B. mycoides strains that have been sequenced, with genome sizes ranging from 5.17 megabases (mb) to 6.55 mb. All the sequenced strains contain several plasmids of various sizes. The type strain ATCC6462 has one chromosome of 5.25 mb, one big plasmid, pBMX_1, of 360 kilobases (kb), and two other plasmids with sizes of about 10 kb (Figure 5). It is psychrotolerant with optimal growth be-tween 25 °C and 30 °C (Fiedoruk et al., 2017).

The economic and biological significance of B. mycoides is being gradu-ally recognized. For example, B. mycoides TKU038 isolated from soil is able to produce a novel chitosanase converting chitinous into chito-oligomers with antioxidant and anti-inflammatory potential (Liang et al., 2016). A B. mycoides strain isolated from an oil field produced a high level of biosur-factant (Najafi et al., 2010). Most of the studies on B. mycoides are focusing on its biocontrol and PGPR activity. B. mycoides Bac J (BmJ) isolated from sugar beet leaves reduces Cercospora leaf-spot disease of sugar beet up to 91% (Bargabus et al., 2002). The induced systemic-acquired resistance via the induction of pathogenesis-related proteins and oxidative burst was the main mechanism (Bargabus et al., 2003). This strain delayed the onset of anthracnose disease and reduced the total and live spore production

of the causing pathogen Glomerella cingulata var. orbiculare, when used to induce SAR in cucumber (Neher et al., 2009). Bacillus mycoides SU-23 could completely suppress the damping-off symptoms caused by Pythium mamillatum on cucumber and has growth promoting activity on seedlings (Paul et al., 1995). Application of a surfactin producing B. mycoides culture completely suppressed the formation of water-soaked lesions on cucum-ber leaves and reduced Pythium damping-off by 35% in the greenhouse (Peng et al., 2017). Nitrogen fixation activity of B. mycoides was reported (Ambrosini et al.), which makes this a strain with biofertilizer potential.

METHODS USED IN STUDYING PLANT-MICROBE INTERACTIONS

Genome sequencing and comparative genomics have a major impact on our understanding of the genetic potential, ecology, and evolution of microorganisms. Thanks to new methods developed over the past two decades, genome sequencing is now much faster and less expensive than it was before. There is an increasing number of rhizobacteria and endo-phytic bacteria genome sequences being published, and it is now pos-sible to get a detailed insight into the evolution and genetic adaptation of rhizobacteria by comparative genomics approaches. The genomes of plant- associated bacteria are relatively large and versatile, often compris-ing more than one chromosome and/or multiple plasmids. Endophytes and rhizobacteria can display a range of different life-styles associated with plants, differing in their time spent free-living in the soil (Hardoim et al., 2008). Such differences in life-style should be reflected in their ge-nomes. The presence of genes involved in motility and chemotaxis in the genome is implicating the strategy that rhizobacteria use to move towards the site of colonization. Many of the PGPR that have been sequenced are of interest because of their role in plant growth promotion. The genes

Figure 4. The growth of B. mycoides in (A) LB liquid medium & (B) LB agar plate; and its cell morphology observed by a phase contrast microscope (C).

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for synthesis of indole-3-acetic acid (IAA), the major naturally occurring auxin, are found in various species (Kaneko et al., 2010; Wu et al., 2011). Other important PGPR-related genes are encoding ACC deaminase, vola-tile compounds, siderophores, as well as antimicrobials.

Transcriptome analysis is an efficient approach to study gene expres-sion during plant-microbe interactions at a genome-wide scale. Plant roots produce and secrete numerous small molecular weight compounds into the rhizosphere. These plant-derived extracellular metabolites and signals can influence the behavior of rhizosphere-associated bacteria via

the activation or suppression of specific gene expression. The expression of several genes from P. aeruginosa involved in metabolism, chemotaxis and type II secretion was regulated by sugar-beet root exudates (Mark et al., 2005). It has been suggested that the availability of particular metab-olites in root exudates, especially amino acids and aromatic compounds, support P. putida to colonize the rhizosphere, and the expression of several genes implicated in plant-bacteria interactions was upregulated upon con-tact with those compounds (Matilla et al., 2007). Fan et al. (2012) studied the B. amyloliquefaciens FZB42 transcriptomic profiles in response to maize root exudates. Their results showed that several groups of genes that were strongly induced by root exudates are involved in metabolic pathways relating to nutrient utilization, bacterial chemotaxis and motility, and non-ribosomal synthesis of antimicrobial peptides and polyketides.

Fluorescent proteins (FPs) are powerful tools for studying gene ex-pression, protein localization and cell motility. In the merging fields of microbial ecology and plant physiology, visualization of FP-labeled rhizo-bacteria is a key prerequisite to gain detailed insights into colonization behavior and plant-bacteria interaction mechanisms. One advantage of using FP marker systems for in situ studies is that they allow direct visuali-zation of the tagged bacteria at the single cell level, without the addition of exogenous substrates (unlike luciferase-based systems), in a nondestruc-tive way (Larrainzar et  al., 2005). By using FP-labeled cells and confocal laser scanning microscopy, it is possible to visualize bacteria colonizing and growing along with a host plant. Such technology has been applied to monitor GFP-Rhizobium in association with the root at early stages of nodulation (Gage et al., 1996). Until now, the engineering and screening of FPs has resulted in more color variants, expanded from blue at 448 nm to far-red at 630 nm for multicolor imaging. When mixed populations of dif-ferent FP-labeled bacteria are used, inter-bacteria interactions can be ob-served. Four FPs including enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the red fluorescent protein (DsRed) can be used to simultaneously visualize different populations of pseudomonads in the rhizosphere (Bloemberg et al., 2000). The dual-labeling of wild-type versus mutant strains of bacteria will provide important insights whether a particular mutant is compromised for rhizosphere competence.

Genetic manipulation is a crucial prerequisite for detailed analysis of the genetic basis of the physiological and ecological properties of rhizo-bacteria. The development of efficient genetic manipulation tools has al-lowed to identify bacterial mechanisms and genes involved in the PGPR effects. Such tools offer possibilities to improve the expression of useful traits into a certain bacterium or to remove one or more genes of undesir-able traits. In laboratory conditions, many plant-associated bacteria have

Figure 5. Circular representations of the B. mycoides ATCC6462 genome displaying rel-evant genome features. From the inner to the outer concentric circle: circle 1, genomic position; circle 2, GC skew. the origin of replication was clearly detectable by a bias of G toward the leading strand; circles 3, GC content; circles 4 and 5, predicted protein- coding sequences (CDS) on the forward and the reverse strand.

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been identified with PGPR or biocontrol properties. However, the effec-tiveness varied greatly from test to test when used as inoculants in field conditions, due to the difficulty in maintaining them in the rhizosphere. Thus, improving bacterial root colonization through genetic modifica-tion should be considered. The introduction of a DNA fragment contain-ing the sss (site-specific recombinase) gene into strain P. fluorescens F113 and WCS307 greatly increased its root tip colonization ability (Dekkers et al., 2000). It is well-known that many lepidopteran insects are suscep-tible to endotoxins produced by B. thuringiensis. However, this bacterium has a short field-life. When the Bt toxin is produced in P. fluorescens, it can be encapsulated and retain its effectiveness for two- to three times longer than other Bt formulations (Peng et al., 2003). Molecular genetic studies also enable researchers to analyze the relevance of antibiotic production by plant-associated bacteria in the biological control of deleterious bac-teria and fungi (Lindow et al., 1989). Antimicrobial deficient mutants of several bacteria showed a reduced biocontrol activity (Girard et al., 2006; Diego Romero et al., 2007).

SCOPE OF THIS THESIS

In this thesis, we use various approaches to investigate the role of B. my-coides in the rhizosphere and the molecular basis of its plant-associated life style. Preliminary studies by our collaborators have shown that there is an inter-strain variation among B. mycoides species. A large number of strains were isolated from either the endosphere of healthy potato roots or from bulk soil. In chapter 2, we sequenced the genomes of seven B. my-coides strains with four isolated from endosphere and three isolated from bulk soil. Their plant-associated or non-plant-associated phenotype was confirmed by plant inoculation assays. Comparative genomics showed that there is a putative “endophytic clade” in the species of B. mycoides. In chapter 3, we subsequently selected one representative of the endophytic strains and one of the soil strains and compared their plant colonization ability and transcriptome profile in response to potato root exudates. The results showed that the endophytic strain has a more profound transcrip-tional response to root exudates than the soil strain, which might explain its good rhizosphere fitness.

For environmental isolates, the lack of genetic manipulation tools is the bottleneck for deep molecular genetics studies. In chapter 4, we devel-oped an efficient electroporation method for B. mycoides to facilitate fur-ther molecular genetics studies on this species. Strong fluorescent proteins are needed for in-planta visualization of rhizobacteria. In chapter 5, we

optimized GFP and RFP for B. mycoides with highly improved brightness. In chapter 6, we developed a CRISPR/Cas9 genome editing system for rhizos-phere isolated B. mycoides and B. subtilis. Gene deletion and insertion were performed to explore their plant-microbe interactions. These tools will fa-cilitate future mechanistic studies on bacteria-plant interactions.

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