University of Groningen Bacillus Mycoides

University of Groningen

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

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Download date: 07-10-2021 1 Introduction PLANT-MICROBE INTERACTION 1 Plants naturally harbor diverse species of microorganisms, because they offer a wide range of habitats, supporting microbial growth. These microorganisms form a complex biological community interacting with the plant host, e.g. being pathogenic, or employing mutualism (symbi- Introduction 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 environmental 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 interaction. 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 competition, antimicrobial production, and induced systematic resistance.

9 inorganic acids, oxygen and water. These compounds may directly affect has an important role in root colonization by Pseudomonas fluorescens Pf0-1 1 the biogeochemistry of the soil (Jones et al., 2009). However, the major- (Oku et al., 2012). In Azospirillum brasilense, the chemoreceptor-like pro- 1 ity of the root exudates are formed by organic materials, which can be di- tein Tlp1 serves as an energy taxis transducer and affects the coloniza- vided into low-molecular-weight compounds and high-molecular-weight tion on roots (Greer-Phillips et al., 2004). Chemotaxis towards A. thaliana compounds. The low-molecular-weight compounds include amino acids, root exudates was mediated by two well-characterized chemoreceptors, organic acids (citric, malic, succinic, oxalic and pyruvic), sugars (glucose, McpB and McpC, as well as by the orphan receptor TlpC in Bacillus subtilis Introduction xylose, fructose, maltose, sucrose, ribose), phenolics, fatty acids and an (Allard-Massicotte et al., 2016). Moreover, plant roots also secrete com- array of secondary metabolites. The high-molecular weight compounds pounds that mimic quorum-sensing (QS) signals of bacteria to stimulate

Plant-microbe interaction Plant-microbe consist of mucilage and proteins (Badri and Vivanco, 2009; Rohrbacher QS-regulated responses of associated bacteria (Huang et al., 2014). The and St-Arnaud, 2016), and rhizosphere microorganisms can use some of most common QS system occurs via N-acyl homoserine lactones (AHLs) these compounds as an energy source for growth and development or as binding to members of LuxR-family transcriptional regulators. Thus, they signaling molecules (Carvalhais et al., 2015). The composition of root ex- regulate the expression of downstream genes with the lux box in the pro- udates varies by plant species, developmental stage and plant growth sub- moter region (Hartmann et al., 2014). However, the LuxR-family regu- strate (Doornbos et al., 2011). For example, the organic acids and sugars lator (Lux-R solo) found in plant-associated bacteria lacks the LuxI pro- in root exudates of tomato and cucumber increase during plant growth tein when compared with other LuxR families. These regulators do not on stone wool (Kamilova et al., 2006). A systematic proteomic analysis of bind AHLs but to plant-produced compounds (González et al., 2013; Ven- root exudates showed that the defense-related proteins such as chitinases, turi and Fuqua, 2013). For instance, a LuxR solo regulator of P. fluorescens, glucanases, and myrosinases showed more secretion during the flower- PsoR, was identified to have a function in responding to plant compounds ing stage of Arabidopsis thaliana (De-la-Peña et al., 2010), while the release and regulating genes involved in the biosynthesis of various antimicro- of amino acids and phenolics increased at later stages of life of Arabidopsis bial compounds including 2,4-diacetylphloroglucinol, which has antago- (Chaparro et al., 2013). nistic activity against the damping-off disease pathogenPythium ultimum It is believed that the dynamic nature of root exudates is one of the driv- (Subramoni et al., 2011). ing forces for the plant to select associated microbial communities that are Following recognition and recruitment, bacteria physically interact favorable for the plant. The best-known plant-microbe symbiosis is the with different parts of the roots from the tip to the elongation zone to form nodulation of legumes by rhizobia. This interaction is very specific, allow- complex multicellular and often multispecies assemblies, including bio- ing certain rhizobial strains to nodulate with specific host legumes (Bais films and smaller aggregates (Danhorn and Fuqua, 2007). The rhizobac- et al., 2006). In soil, a Rhizobium spp. finds its host legume plant from a teria form biofilms in a heterogeneous way along the root surface, where distance by chemotaxis, being attracted to the root exudates due to the the root tips usually have the least bacterial biofilm formed (Rudrappa presence of flavonoids (Sugiyama and Yazaki, 2012). Flavonoids also reg- et al., 2008). In general, the zone immediately behind the root tip is con- ulate the expression of nod genes that play important roles in nodulation sidered to be a major site of colonization (Badri and Vivanco, 2009). This establishment (Abdel-Lateif et al., 2012). Another widely-occurring plant- is partially due to the pH difference from the root tip to basal regions associated microbiome part is formed by arbuscular mycorrhizal fungi (Peters, 2004), which might affect the bacterial growth and biofilm devel- (AMF), which form a symbiotic relationship with more than 80% of terres- opment. The nutrient availability is also quite different since particular trial plants, from bryophytes to tracheophytes (Lee et al., 2013). Although cell types in the root system might be more important than others in the AMF have low host specificity in their symbiosis with plants, they may also secretion of particular compounds. The biofilm formation of rhizobacte- recognize their host by signals released by plant roots, similar to the rec- ria on root surfaces is a mutualistic interaction. In B. subtilis-A. thaliana ognition by rhizobia. It has been shown that mycorrhizal fungi are me- interaction, plant polysaccharides act as an environmental cue that trig- diated by root-secreted compounds, such as strigolactone 5-deoxystrigol gers biofilm formation by the bacterium (Beauregard et al., 2013). In turn, (Yoneyama et al., 2008), sugars, and carbohydrates (Kiers et al., 2011). plants benefit from the biofilm of rhizobacteria. The inhibitory effect of Some specific root-released chemical signals have been identified, that Lysobacter sp. strain SB-K88 against pathogenic Aphanomyces cochlioides play a role in recruiting specific bacteria to build mutualistic interactions is due to a combination of antibiosis and biofilm formation at the rhizo- with the plant. For example, the chemotaxis to amino acids of root exudates plane of the host plant (Islam et al., 2005).

10 11 Although extensive studies have been performed to investigate plant- 1 associated bacteria, we are far from understanding the plant-microbe in- 1 teraction mechanisms, especially when indigenous multispecies bacterial communities are considered. So far, the studies on plant-growth promoting rhizobacteria (PGPR) and their beneficial effects on host plants have drawn considerable attention, but more research is needed to increase Introduction 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- Figure 2. Schematic diagram showing that plant growth-promoting bacteria affect

Plant growth promoting rhizobacteria (PGPR) rhizobacteria promoting Plant growth mote plant growth due to certain traits of the rhizobacteria. A diverse ar- plant growth directly and indirectly. ISR: induced systemic resistance. ray of bacterial species including Azospirillum, Azotobacter, Bacillus, Burk- holderia, Enterobacter, Klebsiella, Pseudomonas, Serratia, and Xanthomonas growth. PGPR produce various phytohomones including auxin, gibber- were identified as PGPR of which Bacillus and Pseudomonas spp. are pre- ellins, and cytokinins to enhance plant growth (Ruzzi and Aroca, 2015). dominant (Podile and Kishore, 2007). PGPR promote plant growth di- Some PGPR possessing 1-aminocyclopropane-1-carboxylate (ACC) deam- rectly by either facilitating resource acquisition (nitrogen, phosphorus, inase activity are able to reduce the ethylene level to relieve plants from and essential minerals) or modulating plant hormone levels, or indirectly, several forms of stress (Bal et al., 2013). Rhizobacteria, such as B. subtilis, B. by decreasing the inhibitory effects of various pathogens on plant growth megaterium, and P. chlororaphis, promote plant growth by producing vol- and development in the form of biocontrol agents (Ahemad and Kibret, atiles like 2,3-butanediol, dimethylhexadecylamine, and 2-pentylfuran 2014) (Figure 2). (Chung et al., 2010; Zou et al., 2010; Velázquez-Becerra et al., 2011). Of all the essential nutrients, nitrogen (N) is required by plants in the The indirect plant growth stimulation by PGPR basically involves the largest quantity and is most frequently the limiting factor in crop pro- ability of PGPR to reduce the deleterious effects of plant pathogens on ductivity. However, 78% of N is in the atmosphere and thus is unavaila- crop yield. Competition for the rhizosphere nutrients and niches is a ble for growing plants. Biological N2 fixation by nitrogen-fixing microor- fundamental mechanism by which PGPR protect plants from pathogens ganisms is widely distributed in nature. Rhizobia such as Rhizobium and (Compant et al., 2005). Prior to infection by a plant pathogen, PGPR col- Bradyrhizobium associated with legume plants are well known for their onization could trigger plant defense mechanisms to prevent or reduce N2 fixing activity and are defined as symbiotic N2-fixing bacteria (Zah- the severity of the disease. This phenomenon is known as the induction ran, 2001). Free-living PGPR with N2-fixing capacity are defined as diaz- of systemic resistance (ISR), which is activated by PGPR and functions otrophs (Kennedy et al., 2004), which can form a non-obligate interaction throughout the plant. On the other hand, PGPR produce a wide variety of with host plants. The second most important essential nutrient, phos- compounds with antimicrobial properties to defend itself against other mi- phate (P), is ubiquitously present in soil but has low solubility or can eas- croorganisms including phytopathogens. Siderophores, bacteriocins and ily be converted to insoluble forms (Hanif et al., 2015). Some PGPR pro- antibiotics are three of the most effective and well-known mechanisms duce organic acids or enzymes including phosphatases and phytases to that PGPR employ to minimize or prevent phytopathogenic proliferation release soluble phosphorus from organic compounds in soil (Lugten- (Beneduzi et al., 2012). Under iron-limiting conditions, PGPR produce low- berg and Kamilova, 2009). Also, balanced and sufficient potassium and/ molecular-weight chelating compounds called siderophores. The produc- or iron availability is essential to maintain a healthy active root system. tion of siderophores plays an important role in reducing phytopathogen PGPR that contribute to the uptake of these nutrients have been reported proliferation by iron deprivation and enhancing plant development by in- and offer a novel solution for sustainable agriculture (Sheng, 2005; Gupta creased uptake of iron (Masalha et al., 2000; Hibbing et al., 2010). A vari- et al., 2015). Hormones, also known as plant growth regulators, can reg- ety of microorganisms also exhibit antagonistic activity against pathogens ulate plant growth and development by stimulating or inhibiting plant by excreting cell wall hydrolytic enzymes including chitinases, proteases,

12 13 and β-1,3-glucanase to destroy the cell wall integrity of pathogens (Com- the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pum- 1 pant et al., 2005). It has long been known that bacteriocins and antibiotics ilus, and B. mycoides elicit a significant reduction in the incidence or se- 1 are involved in shielding host against pathogens and mediating local pop- verity of various diseases on a diversity of plant hosts via ISR mechanisms ulation dynamics in the rhizosphere (Thomashow et al., 2008; Subrama- (Kloepper et al., 2004; Ryu et al., 2004). nian and Smith, 2015). B. clausii GM17 isolated from the rhizosphere of On- Several studies have shown that Bacillus species are highly diverse, not onis angustissima have been found to produce bacteriocin Bac-GM17 with only among different species but also between strains of the same spe- Introduction bactericidal effect on Agrobacterium tumefaciens and fungistatic effect on cies. Analyses of whole bacterial communities showed a significant dis- Candida tropicalis (Mouloud et al., 2013). The bacteriocin thuricin 17, iso- tinction between soil and rhizosphere communities (Smalla et al., 2001). A lated from B. thuringiensis NEB17, shows plant growth stimulation effects significant difference was observed in the total and relative abundance of on plants (Lee et al., 2009). Another mechanism involved in disease sup- Bacillus-like sequences amplified from bulk soil and crop roots (Choudhary pression of PGPR is the production of antibiotics. Pseudomonads produce and Johri, 2009). Mavingui et al. (1992) studied the genetic and phenotypic lipopeptides (LPs) such as 2,4-diacetyl phloroglucinol, hydrogen cyanide, diversity of 130 strains of B. polymyxa isolated from non-rhizosphere soil

Rhizosphere Bacillus function and phylogeny Rhizosphere pyrrolnitrin, tensin, pederin, tropolone, phenazine, that have been exten- (32 strains), rhizosphere soil (38 strains), and the rhizoplane (60 strains). sively studied (Gross and Loper, 2009). Bacillus is well-known to produce Their results showed that diversity within populations of B. polymyxa iso- fengycin and surfactin family LPs. In addition, the production of zwitter- lated from non-rhizosphere and rhizosphere soil is higher than that of micin A and bacillomycin, has also been reported (Arguelles-Arias et al., B. polymyxa isolated from the rhizoplane. This phenomenon can be ex- 2009; Kinsella et al., 2009; Pérez-García et al., 2011). plained by the selection effects of plant roots on particular strains of a species. On the other hand, the strains living in association with plants are well adapted to the specific niche and did evolve some beneficial features RHIZOSPHERE BACILLUS FUNCTION AND PHYLOGENY for the host. Comparative genomics revealed a core set of genes that might be found in beneficial B. amyloliquefaciens strain. The phylogenetic tree Bacillus spp. are well-known rhizosphere residents of many plants and based on concatenated sequences of 11 conserved genes of various other may directly or indirectly contribute to crop productivity. By standard Bacillus strain genomes, results in a plant-associated clade (Figure 3A) isolation on nutrition-rich medium, isolates phylogenetically related to (Magno-Perez-Bryan et al., 2015). Phylogenomic analysis of B. subtilis and B. subtilis and B. cereus have been frequently recovered (Bai et al., 2002; B. amyloliquefaciens indicated that strains isolated from plant-associated Cavaglieri et al., 2005; Cazorla et al., 2007; Egamberdieva et al., 2008). (PA) habitats could be clearly distinguished from those from non-plant- Pandey and Palni (1997) reported that Bacillus species are the dominant associated (nPA) niches in both species (Figure 3B). Furthermore, the core bacteria of the rhizosphere of established tea bushes. B. subtilis and B. my- genomes of PA strains are more abundant in genes relevant to interme- coides comprise a major part of the bacterial population, even during un- diate metabolism and secondary metabolite biosynthesis, as compared to favorable periods, also due to their ability to sporulate. Moreover, culture- those of nPA strains. Moreover, they possess specific additional genes in- independent rhizosphere microbiome studies have indicated the presence volved in the utilization of plant-derived substrates and the synthesis of of a large uncultured diversity in Bacillus (Felske et al., 2003; Choudhary antibiotics (Zhang et al., 2016). and Johri, 2009; Pereira et al., 2011). In a disease-suppressive soil, Bacillus was identified as the dominant bacterial species. The strain B. amyloliquefaciens NJN-6 isolated from this suppressive soil decreased Panama dis- ENDOPHYTIC BACILLUS SPP. 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- As soil-borne microorganism, the species of Bacillus is not only found in spectively, devoted to antibiotic synthesis (Stein, 2005; Chen et al., 2007). the rhizosphere but also in the endosphere of plants. Endophytic bacte- Among all the synthesized antimicrobial compounds by Bacillus, cyclic li- ria are defined as bacteria colonizing the internal tissues of plants with- popeptides (LPs) of the surfactin, iturin and fengycin families have been out causing symptoms of infection or negative effects on their host (Col- well-characterized, with potential uses in biotechnology and agriculture lins et al., 2004). According to their life strategy and the dependency on (Ongena and Jacques, 2008). It has been shown that specific strains of the host, endophytic bacteria can be classified as obligate endophytes,

14 15 facultative endophytes or opportunistic endophytes. Obligate endophytes 1 depend entirely on their host for all of their needs and spend all of their 1 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 Introduction turn, the endophytic bacteria bring beneficial attributes to their hosts, as they enhance plant growth and health in similar ways as PGPR. For exam- Endophytic Bacillus spp. Endophytic ple, direct plant growth promotion can be achieved by producing plant growth regulators including auxins, cytokinins, and gibberellings, reducing ethylene stress by 1-aminocyclopropane-1-carboxylate (ACC) deaminase; 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 promoting 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 microbial 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 Figure 3. Phylogenetic analysis of several Bacillus strains. (A) Phylogenetic tree of iso- the main source of endophytic colonizers. Among them, the genus Ba- lates closely related to species of B. amyloliquefaciens, B. subtilis, B. atrophaeus, and B. li- cillus stands out as one of most reported endophytic bacteria from var- cheniformis. 11 concatenated genes (nusA, rpoA, dnaA, rpoB, gyrA, gyrB, rpoC, spoVG, sigW, ious plant species. The spore-forming property with additional features sigH, and sigB) were handled to build a neighbor-joining tree, using MEGA 5 bootstrap of being natural soil dwellers makes Bacillus suitable for further devel- values (10,000 repetitions), which are shown on the branches. (B) The maximum-likeli- opment into commercial biocontrol agents. A recent metagenomics hood phylogeny derived from the alignment of 1835 concatenation core genes of B. subtilis study revealed the dominance of Bacillus as a major endophytic genus and B. amyloliquefaciens. Leaf clip art indicates an association with plants. Colors indicate in rice roots, probably playing a key role in nitrogen fixation (Sengupta the biogeographic origin of the strains (PA: plant-associated; nPA: non-plant-associated). et al., 2017). Bodhankar et al. (2017) isolated 80 seed endophytic bacte- (Images modified from Magno-Perez-Bryan et al. (2015); Zhang et al. (2016)) ria from 30 maize genotypes. Their results showed that Bacillus was the

16 17 most dominant encountered genus affiliated with the Phylum Firmicutes. 1 To date, the species that have been demonstrated to be endophytes are 1 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- Introduction 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- Figure 4. The growth of B. mycoides in (A) LB liquid medium & (B) LB agar plate; and its ance (Gagne-Bourque et al., 2016). cell morphology observed by a phase contrast microscope (C).

A general introduction on B. mycoides mycoides on B. introduction A general of the causing pathogen Glomerella cingulata var. orbiculare, when used A GENERAL INTRODUCTION ON B. MYCOIDES to induce SAR in cucumber (Neher et al., 2009). Bacillus mycoides SU-23 could completely suppress the damping-off symptoms caused by Pythium Bacillus mycoides is a Gram positive, rod-shaped, and spore-forming bac- mamillatum on cucumber and has growth promoting activity on seedlings terium. The growth of B. mycoides on agar plates has a particular rhizoid (Paul et al., 1995). Application of a surfactin producing B. mycoides culture shape, resulting from cells linked end to end, and grouped in filaments completely suppressed the formation of water-soaked lesions on cucum- bundles (Figure 4). It belongs to the B. cereus species-group, which com- ber leaves and reduced Pythium damping-off by 35% in the greenhouse prises several closely related species including B. anthracis, B. cereus, B. (Peng et al., 2017). Nitrogen fixation activity of B. mycoides was reported thuringiensis, B. mycoides, B. weihenstephanensis, B. pseudomycoides and (Ambrosini et al.), which makes this a strain with biofertilizer potential. some recently identified species (Fiedoruk et al., 2017). However, B. mycoides received far less attention compared to other members, since it is neither pathogenic, as B. cereus and B. anthracis, nor pesticidal as B. thur- METHODS USED IN STUDYING PLANT-MICROBE ingiensis. To date, there are more than twenty B. mycoides strains that have INTERACTIONS been sequenced, with genome sizes ranging from 5.17 megabases (mb) to 6.55 mb. All the sequenced strains contain several plasmids of various Genome sequencing and comparative genomics have a major impact sizes. The type strain ATCC6462 has one chromosome of 5.25 mb, one big on our understanding of the genetic potential, ecology, and evolution of plasmid, pBMX_1, of 360 kilobases (kb), and two other plasmids with sizes microorganisms. Thanks to new methods developed over the past two of about 10 kb (Figure 5). It is psychrotolerant with optimal growth be- decades, genome sequencing is now much faster and less expensive than tween 25 °C and 30 °C (Fiedoruk et al., 2017). it was before. There is an increasing number of rhizobacteria and endo- The economic and biological significance ofB. mycoides is being gradu- phytic bacteria genome sequences being published, and it is now pos- ally recognized. For example, B. mycoides TKU038 isolated from soil is able sible to get a detailed insight into the evolution and genetic adaptation to produce a novel chitosanase converting chitinous into chito-oligomers of rhizobacteria by comparative genomics approaches. The genomes of with antioxidant and anti-inflammatory potential (Liang et al., 2016). A B. plant-associated bacteria are relatively large and versatile, often compris- mycoides strain isolated from an oil field produced a high level of biosur- ing more than one chromosome and/or multiple plasmids. Endophytes factant (Najafi et al., 2010). Most of the studies on B. mycoides are focusing and rhizobacteria can display a range of different life-styles associated on its biocontrol and PGPR activity. B. mycoides Bac J (BmJ) isolated from with plants, differing in their time spent free-living in the soil (Hardoim sugar beet leaves reduces Cercospora leaf-spot disease of sugar beet up to et al., 2008). Such differences in life-style should be reflected in their ge- 91% (Bargabus et al., 2002). The induced systemic-acquired resistance via nomes. The presence of genes involved in motility and chemotaxis in the the induction of pathogenesis-related proteins and oxidative burst was genome is implicating the strategy that rhizobacteria use to move towards the main mechanism (Bargabus et al., 2003). This strain delayed the onset the site of colonization. Many of the PGPR that have been sequenced are of anthracnose disease and reduced the total and live spore production of interest because of their role in plant growth promotion. The genes

18 19 the activation or suppression of specific gene expression. The expression 1 of several genes from P. aeruginosa involved in metabolism, chemotaxis 1 and type II secretion was regulated by sugar-beet root exudates (Mark et al., 2005). It has been suggested that the availability of particular metabolites in root exudates, especially amino acids and aromatic compounds, support P. putida to colonize the rhizosphere, and the expression of several Introduction genes implicated in plant-bacteria interactions was upregulated upon contact 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- Methods used in studying plant-microbe interactions plant-microbe Methods used in studying pression, protein localization and cell motility. In the merging fields of microbial ecology and plant physiology, visualization of FP-labeled rhizobacteria 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 visualization 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 Figure 5. Circular representations of the B. mycoides ATCC6462 genome displaying rel- far-red at 630 nm for multicolor imaging. When mixed populations of dif- evant genome features. From the inner to the outer concentric circle: circle 1, genomic ferent FP-labeled bacteria are used, inter-bacteria interactions can be ob- position; circle 2, GC skew. the origin of replication was clearly detectable by a bias of served. Four FPs including enhanced cyan (ECFP), enhanced green (EGFP), G toward the leading strand; circles 3, GC content; circles 4 and 5, predicted protein- enhanced yellow (EYFP), and the red fluorescent protein (DsRed) can be coding sequences (CDS) on the forward and the reverse strand. used to simultaneously visualize different populations of pseudomonads in the rhizosphere (Bloemberg et al., 2000). The dual-labeling of wild-type for synthesis of indole-3-acetic acid (IAA), the major naturally occurring versus mutant strains of bacteria will provide important insights whether auxin, are found in various species (Kaneko et al., 2010; Wu et al., 2011). a particular mutant is compromised for rhizosphere competence. Other important PGPR-related genes are encoding ACC deaminase, vola- Genetic manipulation is a crucial prerequisite for detailed analysis of tile compounds, siderophores, as well as antimicrobials. the genetic basis of the physiological and ecological properties of rhizo- Transcriptome analysis is an efficient approach to study gene expres- bacteria. The development of efficient genetic manipulation tools has al- sion during plant-microbe interactions at a genome-wide scale. Plant lowed to identify bacterial mechanisms and genes involved in the PGPR roots produce and secrete numerous small molecular weight compounds effects. Such tools offer possibilities to improve the expression of useful into the rhizosphere. These plant-derived extracellular metabolites and traits into a certain bacterium or to remove one or more genes of undesir- signals can influence the behavior of rhizosphere-associated bacteria via able traits. In laboratory conditions, many plant-associated bacteria have

20 21 been identified with PGPR or biocontrol properties. However, the effec- optimized GFP and RFP for B. mycoides with highly improved brightness. In 1 tiveness varied greatly from test to test when used as inoculants in field chapter 6, we developed a CRISPR/Cas9 genome editing system for rhizos- 1 conditions, due to the difficulty in maintaining them in the rhizosphere. phere isolated B. mycoides and B. subtilis. Gene deletion and insertion were Thus, improving bacterial root colonization through genetic modifica- performed to explore their plant-microbe interactions. These tools will fa- tion should be considered. The introduction of a DNA fragment contain- cilitate future mechanistic studies on bacteria-plant interactions. ing the sss (site-specific recombinase) gene into strain P. fluorescens F113 Introduction and WCS307 greatly increased its root tip colonization ability (Dekkers Scope of this thesis et al., 2000). 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