University of Groningen
Bacillus mycoides: novel tools for studying the mechanisms of its interaction with plants Yi, Yanglei
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Yi, Y. (2018). Bacillus mycoides: novel tools for studying the mechanisms of its interaction with plants.
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7 General discussion
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General discussion
The world population is expected to reach 8.5 billion by 2030, 9.7 bil- lion in 2050 and 11.2 billion in 2100, according to a report by the United Nations Department of Economic and Social Affairs (www.un.org/en/
development/desa/news/population/2015-report.html). To produce suf- ficient food remains a major task for modern societies. This means there will be an increasing demand for agricultural productivity of food crops, as plants form the basis of every food chain (Morrissey et al., 2004). Over the last decades, the agriculture and horticulture productivity has largely depended on the use of chemical fertilizers, pesticides, and herbicides.
These chemicals can help in boosting the growth of plants, but they also have devastating side effects in the long run for the environment. How to increase crop yields without causing environmental damage is a great challenge globally. Great efforts have been made by scientists to increase the productivity, disease resistance and stress tolerance of crops through breeding or genetic modification methods. However, these efforts have mainly focused on plant phenotypes. The effects of the beneficial plant- associated microbial community, also referred to as the second genome of the plants, on the host health and development have been a topic of in- creasing attention by the scientific community in the last years.
The root-associated microbiome plays a primary role in controlling
phytopathogens. One well-known example is the occurrence of disease-
suppressive soils, which are exceptional ecosystems in which little or
no disease occurs under conditions that are favorable for disease devel-
opment (Bulgarelli et al., 2013). The suppressive soil can be induced af-
ter a severe disease outbreak of a monoculture of the same crop species
for several years. This is because plants can exploit microbial consortia
from soil for protection against infections when attacked by pathogens
(Mendes et al., 2011). Meanwhile, the legume–rhizobium symbiosis is a
classic model of mutualistic plant-microbe interaction in which the bac-
teria bring fixed nitrogen to the plant, receiving, in turn, the sanctuary
and sugars that the plant cell can provide (Long, 2001). The root-nodule
bacteria can be manipulated ecologically, agronomically, edaphically and
genetically to improve legume productivity and, as a consequence, soil
fertility (Brockwell et al., 1995). Apart from disease prevention and nitro-
gen fixation, rhizosphere dwelling microbes have various plant promot-
ing effects including plant hormone production (Idris et al., 2007), host
stress tolerance enhancement (Barka et al., 2006), and systematic resist-
ance induction (Schuhegger et al., 2006). An understanding of the funda-
mental mechanisms of how plants and soil microbes co-exist and benefit
each other can therefore provide new strategies to improve plant produc-
tivity and reduce the use of chemicals, which destruct the biological com-
munity and destabilize the agro-ecosystem. In this study, we showed that
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General discussion
General discussion
Bacillus mycoides is abundantly present in healthy potato roots, and lives inside root tissue as an endophytic bacterium. Moreover, there is a large diversity among the rhizosphere- and endosphere-isolated strains. Other researchers also reported the biocontrol and nitrogen fixation activity of B. mycoides isolated from various plant species. This thesis work aimed to understand the molecular mechanism of B. mycoides-plant interactions.
To this end, a multidisciplinary approach was adopted and novel molecu- lar genetic tools were developed (Figure 1).
During the adaptation to the rhizosphere niche, the rhizosphere-associ- ated strains evolved specific properties different from the non-rhizosphere strains. Comparative genomics of plant-associated Pseudomonas spp. re- vealed that most of the biological control genes are in the variable regions of the genome (Loper et al., 2012), indicating that horizontal gene transfer played a key role in the adaptation process. The strains isolated from dif- ferent niches also have different metabolic preferences. Timm et al. (2015) compared the genome of rhizosphere and endosphere isolated Pseudomonas fluorescens strains. Their results showed that multiple pathways relevant for
UP_EC18 UP_SB8
61 53 52
148 54 294
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DOWN_EC18 DOWN_SB8
Chapter 3 Chapter 4
AB marker HR fragment
cas 9 gRNA CAS9-gRNA complex
?
Chapter 6 Chapter 5
Figure 1. A multidisciplinary approach was adopted to study the plant-microbe inter- actions of B. mycoides. Novel molecular genetics tools and methods were developed that can also be useful in other rhizosphere dwelling Bacilli.
plant-bacterial interactions are enriched in endosphere-isolated genomes.
In the case of B. mycoides, only some strains have the ability to penetrate plant tissue and start an endophytic life stage. Since the plant beneficial ef- fects of endophytes are greater than those of many rhizosphere-colonizing bacteria (Hardoim et al., 2008), we were interested in exploring the genetic difference of the endophytic and soil strains of B. mycoides. In chapter 2, we selected 7 strains based on their origin of isolation and plant colonization ability: four strains isolated from the endosphere that could be endophytic again (plant-associated), and three strains isolated from soil which could not be endophytic (non-plant-associated). The whole genome phylogenetic tree showed that the endophytic strains are clustered together, indicating that the endophytic strains have some common features.
It has been hypothesized that rhizosphere bacteria start root coloniza- tion when encountering root exudates. Understanding the bacterial re- sponse to root exudates is a key step in deciphering plant-microbe interac- tion mechanisms. In chapter 3, we compared the transcriptome profiles of an endophytic strain and a soil strain of B. mycoides in response to po- tato root exudates by RNA-seq. We first used confocal laser scanning mi- croscope (CLSM) to confirm that the endophytic strain has better root col- onization ability than the soil strain. Our transcriptome results revealed that the endophytic strain showed a more active response than the soil strain to potato root exudates (endophyte strain has more genes altered by root exudates). The upregulation of genes related to amino acid metab- olism, several proteolytic enzymes, and O-glycosyl hydrolases points to- ward a specific adaptation to the ecological niche and a good rhizosphere fitness of the endophytic strain. In comparison, the upregulation of sugar transport and metabolite genes of the soil isolate indicates a narrow nu- trition source, which might hamper its colonization ability and prolifera- tion rate in the rhizosphere.
For environmental isolated bacteria, the lack of efficient transforma- tion methods is a bottleneck to establish the genetic manipulation sys- tems, e.g., enabling gene deletion and mutation within their genome. It has been found that many environmental isolated bacteria are quite resist- ant to natural transformation due to their low genetic competence levels (Duitman et al., 2007). So far, different methodologies have been proposed for transforming exogenous DNA into bacterial cells. Phage-mediated transduction has high efficiency in some bacteria (Winstel et al., 2015).
However, bacteriophages have a limited host range and homologous re-
combination is required to perform such method. Although protoplast
transformation (Li et al., 2016) and protoplast electroporation (Romero
et al., 2006) have also been described, a possible disadvantage with the
protoplast is that some bacteria are vulnerable to cell wall degrading
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General discussion
enzymes and very precise manipulation is needed. In chapter 4, we de- veloped a method for electroporation of environmental Bacillus mycoides strains by optimizing several conditions that affect the electroporation efficiency. We found that the transformation efficiency positively corre- lated to the media richness (LB<2XTY<BHIS). The special chaining prop- erty of B. mycoides might play a role in determining the transformation ef- ficiency, because the cells were less aggregated in rich medium. We found that cells collected from the early exponential phase (OD600 = 0.9~1) re- sult in a higher electroporation efficiency. This is different with B. subti- lis WB800, which has high electroporation efficiency at late growth-stage (OD600 = 2.2–2.3) (Lu et al., 2012). The addition of 2% glycine to the culture for 1 hour significantly improved the electroporation efficiency because glycine can reduce the peptidoglycan bonds and loosen up the cell wall by replacing the L- and D-alanine bridges. Moreover, most B. mycoides strains show a higher transformation efficiency when using non- methylated plasmids isolated from the methylation-defect strain E. coli JM110, which indicates that restriction/modification is the main barrier for electropo- ration of this bacterium.
Visualization of fluorescent protein (FP)-labeled rhizobacteria in the rhizosphere and endosphere is a key prerequisite to gain detailed insights into endophytic behavior and plant-bacterial interaction mechanisms.
However, the performance of fluorescent proteins has to be optimized for the bacterial host. A widely used approach to obtain improved FP variants is the adaptation of FP genes to the typical codon usage of the host organ- ism. Such codon optimized FPs have been developed for the cyan fluores- cent protein and a yellow fluorescent protein in Bacillus anthracis (Sastalla et al., 2009), for GFP and RFP in Botrytis cinerea (Leroch et al., 2011) and for GFP in Zymoseptoria tritici (Kilaru et al., 2015). In chapter 5, we used a different in vivo cell sorting approach to select the best performing FP variants in B. mycoides from mutational libraries of the superfolder green fluorescent protein sfGFP and the red fluorescent protein mKate2. After selection, the sfGFP variant SPS6 and the mKate2 variant KPS12, with sig- nificantly increased brightness, were isolated. Both optimized variants were highly suitable for in situ localization studies. The strain EC18 rap- idly attached to plant roots and formed a multicellular matrix on the sur- face. During the process of colonization, the root hairs and their branch- ing regions were hot spots for plant-microbe interactions and probably constitute entrance sites for B. mycoides to establish an endophytic life- style, although both the endophytic strain B. mycoides EC18 and the soil strain B. mycoides SB8 were detected in the rhizosphere. When a 1:1 mix- ture of the EC18 and SB8 cells was inoculated to plants, the roots were pre- dominantly colonized by EC18 (Chapter 2).
The CRISPR-Cas9 system is a powerful and revolutionary genome- editing tool for eukaryotic genomes, but its use in undomesticated envi- ronmental isolated Bacillus strains is still underdeveloped. In Chapter 6, we implemented the CRISPR-Cas9 system in rhizosphere-isolated B. sub- tilis HS3 and B. mycoides EC18 to study their plant-microbe interactions.
All required genetic elements were incorporated into one plasmid, which was transformed into the bacterial cells with the electroporation method developed in chapter 3. It was found that the B. mycoides produced bacil- libactin (siderophore) aid the growth of iron-starved plants. The advan- tage of the CRISPR-Cas9 system is that the plasmid can be cured after the genome has been edited. The marker-free mutant can be subjected to the next round of mutagenesis with the same procedure. Based on this prin- ciple, we further inserted a gfp gene into the genome of the mutants and the wild type strain. The GFP-tagged strains were inoculated with cab- bage plants and the roots were observed by confocal laser scanning micro- scope. The results showed that the siderophore-deficient mutants have a reduced plant colonization ability compared with wild type.
In summary, different strategies were applied to investigate the plant-microbe interactions of B. mycodies. Some of the tools described here are also useful in other Bacillus species, e.g., the FP variants and the CRISPR-Cas9 system. However, there are still many questions that are open and more research is needed to fully understand the relationship of B. mycoides and its host plant.
REFERENCES
Barka, E.A., Nowak, J., and Clément, C. (2006) Enhancement of chilling resistance of in- oculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burk- holderia phytofirmans strain PsJN. Appl. En- viron. Microbiol. 72: 7246–7252.
Brockwell, J., Bottomley, P.J., and Thies, J.E.
(1995) Manipulation of rhizobia micro- flora for improving legume productivity and soil fertility: A critical assessment.
Plant and Soil 174: 143–180.
Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E., and Schulze-Lefert, P. (2013) Structure and functions of the
bacterial microbiota of plants. Annu Rev Plant Biol. 64: 807–838.
Duitman, E.H., Wyczawski, D., Boven, L.G., Venema, G., Kuipers, O.P., and Hamoen, L.W. (2007) Novel methods for genetic transformation of natural Bacillus subti- lis isolates used to study the regulation of the mycosubtilin and surfactin syn- thetases. Appl. Environ. Microbiol. 73:
3490–3496.
Hardoim, P.R., van Overbeek, L.S., and Elsas, J.D. (2008) Properties of bacterial endo- phytes and their proposed role in plant growth. Trends Microbiol. 16: 463–471.
7
General discussion
Idris, E.E., Iglesias, D.J., Talon, M., and Borriss, R. (2007) Tryptophan-dependent produc- tion of indole-3-acetic acid (IAA) affects the level of plant growth promotion by Ba- cillus amyloliquefaciens FZB42. Mol. Plant Microbe Interact. 20: 619–626.
Kilaru, S., Schuster, M., Studholme, D., Soanes, D., Lin, C., Talbot, N.J., and Steinberg, G.
(2015) A codon-optimized green fluores- cent protein for live cell imaging in Zy- moseptoria tritici. Fungal Genet. Biol. 79:
125–131.
Leroch, M., Mernke, D., Koppenhoefer, D., Schneider, P., Mosbach, A., Doehlemann, G., and Hahn, M. (2011) Living colors in the gray mold pathogen Botrytis cinerea:
codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence. Appl. Environ.
Microbiol. 77: 2887–2897.
Li, H., Zhang, L., Guo, W., and Xu, D. (2016) Development of a genetically engineered Escherichia coli strain for plasmid transfor- mation in Corynebacterium glutamicum. J.
Microbiol. Methods 131: 156–160.
Long, S.R. (2001) Genes and signals in the Rhi- zobium-legume symbiosis. Plant Physiol.
125: 69–72.
Loper, J.E., Hassan, K.A., Mavrodi, D.V., Da- vis, E.W., 2nd, Lim, C.K., Shaffer, B.T. et al.
(2012) Comparative genomics of plant-as- sociated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions. PLoS Genet.
8: e1002784.
Lu, Y.P., Zhang, C., Lv, F.X., Bie, X.M., and Lu, Z.X. (2012) Study on the electro-transfor- mation conditions of improving transfor- mation efficiency for Bacillus subtilis. Lett.
Appl. Microbiol. 55: 9–14.
Mendes, R., Kruijt, M., de Bruijn, I., Dekkers, E., van der Voort, M., Schneider, J.H. et al.
(2011) Deciphering the rhizosphere mi- crobiome for disease-suppressive bacteria.
Science 332: 1097–1100.
Morrissey, J.P., Dow, J.M., Mark, G.L., and O’Gara, F. (2004) Are microbes at the root of a solution to world food production?
EMBO Reports 5: 922–926.
Romero, D., Perez-Garcia, A., Veening, J.W., de Vicente, A., and Kuipers, O.P. (2006) Trans- formation of undomesticated strains of Bacillus subtilis by protoplast electropora- tion. J. Microbiol. Methods 66: 556–559.
Sastalla, I., Chim, K., Cheung, G.Y., Pomer- antsev, A.P., and Leppla, S.H. (2009) Co- don-optimized fluorescent proteins designed for expression in low-GC gram-positive bacteria. Appl. Environ. Mi- crobiol. 75: 2099–2110.
Schuhegger, R., Ihring, A., Gantner, S., Bahnweg, G., Knappe, C., Vogg, G. et al. (2006) Induc- tion of systemic resistance in tomato by N-acyl-L-homoserine lactone- producing rhi zosphere bacteria. Plant, Cell and Envi- ronment 29: 909–918.
Timm, C.M., Campbell, A.G., Utturkar, S.M., Jun, S.R., Parales, R.E., Tan, W.A. et al.
(2015) Metabolic functions of Pseudo- monas fluorescens strains from Populus deltoides depend on rhizosphere or endo- sphere isolation compartment. Front. Mi- crobiol. 6: 1118.
Winstel, V., Kühner, P., Krismer, B., Peschel, A., and Rohde, H. (2015) Transfer of plasmid DNA to clinical coagulase-negative staph- ylococcal pathogens by using a unique bacteriophage. Appl. Environmental Micro- biol. 81: 2481–2488.
