An endophytic microbe from an unusual volcanic swamp corn seeks and inhabits root hair cells to extract rock phosphate

Abstract

In the animal microbiome, localization of microbes to specific cell types is well established, but there are few such examples within the plant microbiome which includes endophytes. Endophytes are non-pathogenic microbes that inhabit plants. Root hairs are single cells, equivalent to the nutrient-absorbing intestinal microvilli of animals, used by plants to increase the root surface area for nutrient extraction from soil including phosphorus (P). There has been significant interest in the microbiome of intestinal microvilli but less is known about the root hair microbiome. Here we describe a bacterial endophyte (3F11) from Zea nicaraguensis, a wild corn discovered in a Nicaraguan swamp above rock-P lava flowing from the San Cristobal volcano. Rock-P is insoluble and a major challenge for plants. Following seed coating and germination on insoluble-P, the endophyte colonized epidermal surfaces, ultimately colonizing root hairs intracellularly. The endophyte promoted root hair growth and secreted acids to solubilize rock-P for uptake by a larger root hair surface. The most interesting observation was that a seed-coated endophyte targeted and colonized a critical cell type, root hair cells, consistent with earlier studies. The endophyte maintained its targeting ability in two evolutionary divergent hosts, suggesting that the host recognition machinery is conserved.

Figure 1

Testing bacterial endophytes from seeds of diverse wild, ancient and modern genotypes of corn (genus Zea) for growth promotion of P-hyperaccumulating annual ryegrass. (A) Geographic origin of Zea seeds used as sources of endophytes in this study. The map image was modified from a photo created by Nicolas Raymond, available at https://www.flickr.com/photos/80497449@N04/10012162166/ and licensed under the Creative Commons Attribution 2.0 Generic (CC BY 2.0, https://creativecommons.org/licenses/by/2.0/) and available at http://freestock.ca/flags_maps_g80-world_map__abstract_acrylic_p2970.html and released under a standard Creative Commons License - Attribution 3.0 Unported, https://creativecommons.org/licenses/by/3.0/deed.en_US). (B) Colonies of bacterial endophytes on R2A agar. (C) Annual ryegrass plants from 3F11 coated seeds grown on rock P containing medium as the sole P source. (D) Root biomass after 4–5 weeks of growth. Shown are 4 independent trials (n = 3 plants for trial 1; n = 7 for trial 2; n = 15 for trials 3 and 4). The black asterisk indicates that the mean is significantly different at p = 0.05 from the control (No endophyte treatment). The green asterisk indicates significance at p = 0.1. (E) San Cristobal volcano (the geographic origin of Zea nicaraguensis, the host from which endophyte 3F11 was isolated). The volcano image was modified from a photo created by Jorge Mejia Peralta, available at https://www.flickr.com/photos/mejiaperalta/9345658009/in/photostream/ and attributed to Creative Commons Attribution 2.0 Generic (CC BY 2.0, https://creativecommons.org/licenses/by/2.0/). (F) Seeds of Zea nicaraguensis. Scale bar is 10 mm. (G,H). Testing for P solubilization by endophyte 3F11 in vitro. (G) Graph showing the concentration of solubilized P. (H) Tubes of NBRIP liquid medium containing insoluble P, either (left) uninoculated or (right) 9 days after inoculation with 3F11 showing clearing. The histograms represent the mean, and the error bars represent the standard error of the mean (SEM).

Figure 2

Testing endophyte 3F11 for its ability to cause ambient acidification using bromocresol purple as a pH indicator. A change in color from purple to yellow indicates a decline in pH. (A) Test for in vitro acid production by a representative colony of 3F11 using NBRIP agar after 24 h compared to a negative control endophyte, 3F7. (B,C) Test for in planta acid production of 3F11 following seed inoculation of annual ryegrass. Shown are three 3F11-inoculated roots (left) or uninoculated roots (right) placed on insoluble-P agar medium (NBRIP) supplemented with bromocresol purple and either (B) no kanamycin or (C) kanamycin. The growth of 3F11 but not annual ryegrass was previously shown to be suppressed by kanamycin. (D) Confirming the colonization of GFP-tagged 3F11 cells on annual ryegrass roots on an LB agar plate following coating onto seeds. (E,F) Testing the co-localization of 3F11 cells with acid production in planta. Shown are three GFP-3F11-inoculated roots (left) or uninoculated roots (right) placed on insoluble-P agar medium (NBRIP) supplemented with bromocresol purple and kanamycin and (E) imaged using UV light to reveal GFP or (F) white light to reveal acid production (yellow). (G,H) Test for the effect of P-bioavailability in the rhizosphere on the in planta acid production of 3F11. Shown are three GFP-3F11-inoculated roots (left) or uninoculated roots (right) placed on (G) soluble-P agar medium supplemented with bromocresol purple and (H) insoluble P containing agar medium (NBRIP) supplemented with bromocresol purple. (I,J) Representative confocal microscopy images showing colonization of GFP-tagged 3F11 cells on annual ryegrass root systems 7 days following seed-inoculation and growth on either (I) rock P, or (J) soluble P, showing the difference in the extent of colonization.

Figure 3

Localization of endophyte 3F11 on the surface and inside root hairs of annual ryegrass along with evidence that 3F11 promotes root hair growth. (A–D) Localization of GFP-tagged endophyte 3F11 on the surface of root hairs of annual ryegrass following inoculation onto seeds at (A) 7 days after planting (DAP), (B) 9 DAP, (C) 15 DAP and (D) 16 DAP. Panels (C) and (D) show 3F11 cells colonizing in between layers of root hairs or in between the root hair to rhizosphere interface. (E) Colonies of GFP-3F11 cells on LB agar plates retrieved following surface sterilization and grinding of annual ryegrass roots from GFP-3F11 coated seeds confirming subsurface plant colonization of 3F11 inside roots (endophytic lifestyle). (F–H) Localization of GFP-tagged endophyte 3F11 cells inside annual ryegrass root hairs following germination of 3F11-inoculated seeds, where (G,H) are the corresponding higher magnification images showing (G) root hairs in cross section and (H) longitudinal section. Confocal microscopy was used in conjunction with propidium iodide (red). For panels (A–H), all seeds were germinated on medium containing insoluble rock P as the sole P source. (I–K) Effect of endophyte 3F11 on root hair length of annual ryegrass growing on media containing rock P as the sole P source. Representative pictures of root hairs of annual ryegrass following germination of seeds treated with (I) buffer (no endophyte control) or (J) endophyte 3F11. The scale bar is 0.2 mm. (K) Average root hair length. Thirty root hairs per plant were measured on the longest crown root (top, middle and bottom segments) from 5 plants per treatment. The error bars represent the standard error of the mean (SEM). The asterisk indicates that the mean is significantly different at p = 0.05 from the control. RH indicates root hairs.

Figure 4

Localization of endophyte 3F11 on the surface and inside corn root hairs. (A,B) Localization of GFP-tagged endophyte 3F11 on the surface of root hairs following inoculation onto seeds at 7 days after planting (DAP). (C,D) Localization of GFP-tagged 3F11 inside root hairs at 5 DAP, where (D) is the corresponding higher magnification showing root hairs in cross section. Confocal microscopy was used in conjunction with propidium iodide (red). RH indicates root hairs.