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Review
. 2014 Mar 7:5:94.
doi: 10.3389/fmicb.2014.00094. eCollection 2014.

Scanning a microhabitat: plant-microbe interactions revealed by confocal laser microscopy

Massimiliano Cardinale  1
Affiliations
Review

Scanning a microhabitat: plant-microbe interactions revealed by confocal laser microscopy

Massimiliano Cardinale. Front Microbiol. .

Abstract

No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.

Keywords: DsRed; GFP; confocal laser scanning microscopy (CLSM); cryptogams; endophyte; fluorescence in situ hybridization (FISH); pathogen; plant-microbe interactions.

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Figures

Figure 1
Figure 1
Combination of FISH with autofluorescence. Confocal images showing root colonization by the PGPR Burkholderia terricola ZR2-12. (A) In the maximum projection it is not possible to assess the colonization pattern of Burkholderia terricola ZR2-12 (red) on this 3 weeks-old sugar beet root (blue); it is impossible as well to discriminate endophytism from ectophytism. (B) The volume rendering of the same confocal stack shows the cells colonizing the internal root tissues but only in the three-dimensional models (C,D) it appears clear that the same bacterium shows a double colonization style: ectophytic at the sides of the root (C) and endophytic, following the apoplastic spaces (D); furthermore, the data from the three-dimensional models (number of spots, volume, etc.) can be easily retrieved and treated with statistics. This confocal stack has a thickness of 70.16 μm and was acquired with a Leica TCS SPE (Leica Microsystems GmbH, Mannheim, Germany) using the oil immersion objective Leica ACS APO 40.0x1.15. Z-step was 0.8 μm. Three-dimensional models were created with the software Imaris 7.3 (Bitplane, Zurich, Switzerland). Figure was prepared with Adobe Creative Suite version 3 (Adobe Systems Inc., San Jose, CA, USA).
Figure 2
Figure 2
Combination of FISH with histochemical staining. Volume rendering of a confocal stack showing the bacterial colonization of salad root (Lactuca sativa) by the native bacterial community, stained by FISH. Gammaproteobacterial (A), betaproteobacterial (B), and other bacterial cells (C) (green, blue, and red, respectively) stained with the FISH probes Gam42a (Cy5-labeled), Bet42a (ATTO488-labeled) and EUBMIX (Cy3-labeled), respectively. (D) Compounds and tissues stained with calcofluor white (0.15% in H2O, 15 min incubation) appear gray. (E) Overlap of images (A–D); yellow, Gammaproteobacteria; pink, Betaproteobacteria; red: other bacteria; gray: compounds surrounding some Betaproteobacteria stained by calcofluor white. Scale bars: 20 μm. Confocal stack has a thickness of 16.99 μm, and was acquired with a Leica TCS SPE (Leica Microsystems GmbH, Mannheim, Germany) using the oil immersion objective Leica ACS APO 40.0x1.15. The Z-step was 0.38 μm. Volume rendering was created with the software Imaris 7.3 (Bitplane, Switzerland). Figure was prepared with Adobe Creative Suite version 3 (Adobe Systems Inc., CA, USA).
Figure 3
Figure 3
Microbial interactions in the rhizosphere. Maximum projections of a confocal stack showing the colonization pattern of salad root (Lactuca sativa) by the native bacterial community stained by FISH. (A,D) Gammaproteobacterial and betaproteobacterial cells (green and blue, respectively) stained with the FISH probes Gam42a (Cy5-labeled) and Bet42a (ATTO488-labeled), respectively. (B,E) All bacterial cells (red) stained with the FISH probe EUB338-MIX (Cy3-labeled). (C,F) Overlap of images (A,B,D,E), respectively; yellow, Gammaproteobacteria; pink, Betaproteobacteria; red: other bacteria. Different taxa do not share the habitats, but instead colonize microniches of the rhizoplane dominantly, excluding each other (see text for more explanations). Scale bars: 20 μm. Confocal stacks (A,C,D,E) have a thickness of 30.72 and 37.26 μm, respectively, and were acquired with a Leica TCS SPE (Leica Microsystems GmbH, Mannheim, Germany) using the oil immersion objective Leica ACS APO 40.0x1.15. The Z-step was 0.5 μm. Maximum projections were created with the software Imaris 7.3 (Bitplane, Zurich, Switzerland). Figure was prepared with Adobe Creative Suite version 3 (Adobe Systems Inc., San Jose, CA, USA).
Figure 4
Figure 4
Integration of CLSM with other techniques. The workflow shows the combination of CLSM with other methods for plant-microbes interactions studies. A case study of PGPR is used in this example.
See this image and copyright information in PMC

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