Plant growth-promoting rhizobacterium Pseudomonas sp. CM11 specifically induces lateral roots

Abstract

Plant growth-promoting rhizobacteria are involved in altering secondary root (SR) formation, but hitherto there has been no distinction between the different types of SRs upon induction of soil biota, and the genetic pathways involved. By using plate and soil systems, we studied the effects of the Pseudomonas strains CM11 and WCS417 on plant performance with a focus on root development. Through a combination of cellular, molecular and genetic analyses, we investigated the type of SRs induced upon CM11 and WCS417 root inoculation using genetic pathways associated with specific SR types. CM11 was shown to affect the root architecture differently from WCS417. CM11 inoculation leads to primary root arrest, whereas WCS417 reveals a longer primary root. Both CM11 and WCS417 activate the PLETHORA 3,5,7-controlled lateral root pathway, rather than the WUSCHEL-RELATED HOMEOBOX 11,12-controlled adventitious (lateral) root pathway. In addition, CM11 promotes plant growth in model and various crop species. It improves plant fitness traits, such as bigger shoots, faster bolting and higher yield in terms of seeds. Our results indicate that the root system architecture can be promoted by activation of PLETHORA 3,5,7 dependent primed lateral pre-branch sites upon inoculation with CM11, which creates great potential to gain a better understanding of root plasticity.

Keywords: Pseudomonas; PLETHORA; Rhizobacteria; lateral roots; plant growth-promoting; root architecture; secondary roots.

Fig. 1

Inoculation of Pseudomonas spp. strains CM1–CM11 affects plant growth. (a) Representative images of Arabidopsis wild‐type (WT) seedlings growing on ½ Murashige & Skoog (MS) agar plates with CM1–CM11 strains or mock. Closed arrowheads, inoculation sites; open arrowheads, tips of primary roots (PRs). Bar, 1 cm. (b–c) Quantification of PR length (newly grown PR below the inoculation sites, b) and number of emerged secondary roots (SRs) (emerged SRs above the inoculation sites, c) in mock and CM1–CM11 inoculated plants. (d–e) Root (d) and shoot (e) FW production per Arabidopsis seedling with the indicated bacterial strains or mock. In all cases, 7‐d‐old seedlings were inoculated with mock or CM1‐CM11 bacterial suspensions at the root tip and grown 10 d post‐inoculation (dpi). Box plots in (b) and (c) indicate the lower and upper quartiles, bars represent the maximum and minimum values. The line in the box indicates the median. Data in (d) and (e) represent mean ± SD of three biological replicates, each consisting of eight seedlings. Different letters indicate statistically significant differences (LSD and Tukey’s honestly significant difference (HSD) tests; P ≤ 0.05).

Fig. 2

CM11 and WCS417 play additive roles in plant growth promotion. (a) Colonization of CM11 in the rhizosphere of Arabidopsis seedlings grown on plates. 7‐d‐old seedlings were inoculated with CM11‐GFP bacterial suspensions at the root tip and grown 1 d post‐inoculation (dpi). Red, propidium iodide; green, green fluorescent protein (GFP). (b) Representative images of seedlings growing on ½ Murashige & Skoog (MS) agar plates with CM11, WCS417 and CM11+WCS417 (Co‐inoc) or mock. Closed arrowheads, inoculation sites; open arrowheads, root tips of primary root (PR). (c–d) Quantification of PR length (newly grown PR below the inoculation sites, c) and number of emerged secondary roots (SRs) (emerged SRs above the inoculation sites, d) in mock and bacteria inoculated plants. (e–f) Root (e) and shoot (f) FW production per Arabidopsis seedling with the indicated bacterial strains or mock. In (b–f), 7‐d‐old seedlings were inoculated with mock or bacterial suspensions at the root tip and grown 10 dpi. Box plots in (c–d) indicate the lower and upper quartiles, bars represent the maximum and minimum values. The line in the box indicates the median. Data in (e–f) represent mean ± SD of three biological replicates, each consisting of eight seedlings. Different letters indicate statistically significant differences (LSD and Tukey’s honestly significant difference (HSD) tests; P ≤ 0.05). Bars: (a) 100 μm; (b) 1 cm.

Fig. 3

Effects of CM11 on primary root development of Arabidopsis. (a, b) Root meristem length (a) and cell numbers (b) of Arabidopsis seedlings inoculated with CM11, WCS417 and CM11+WCS417 (Co‐inoc) bacteria or mock. Data represent mean ± SD of four biological replicates, each consisting of five seedlings; (c) Cortical cell size in the differentiation zone of mock and CM11 inoculated roots. Data represent means ± SD of 10 individual seedlings. In (a–c), 4‐d‐old seedlings were inoculated at the root tip and grown 5 d post‐inoculation (dpi) in (a) and (b), or 3 dpi in (c). Different letters indicate statistically significant differences (LSD and Tukey’s honestly significant difference (HSD) tests; P ≤ 0.05). (d–e) Representative confocal images showing the expression patterns of the cell cycling reporter pCYCB1;1::GFP in roots under mock (d) and CM11 inoculated conditions (e) (GFP, green fluorescent protein). (f–g) Representative confocal images showing the expression pattern of the endodermis/quiescent center (QC) localized reporter pSCR::SCR::GFP under mock (f) and CM11 inoculated conditions (g). (h–i) Representative confocal images showing the expression patterns of the QC‐localized reporter pWOX5::GFP under mock (h) and CM11‐inoculated conditions (i). (j–k) mPS‐PI stained root tips show the absence of amyloplasts in the columella stem cell layer (orange arrows) in mock (j) and CM11 inoculated conditions (k); (l–o) Effects of CM11 on auxin distribution in the Arabidopsis root tips. Representative confocal images of DR5::vYFP expression in the Arabidopsis root tip of mock (l) and CM11 inoculated conditions at 1 (m), 2 (n) and 3 (o) dpi (YFP, yellow fluorescent protein). In (d–o), 4‐d‐old seedlings were inoculated with mock or CM11 bacterial suspensions at the root tip and confocal images were captured at 3 dpi (d–k) or at the indicated time (l–o). All imaging experiments were repeated twice and showed similar results. Bar: (d–o) 50 μm.

Fig. 4

CM11 induces the PLT3PLT5PLT7‐mediated lateral root pathway. (a) Representative images of wild‐type (WT), plt3plt5plt7, wox11wox12 seedlings growing on ½ Murashige & Skoog (MS) agar plates with CM11 or mock. Closed arrowheads, inoculation sites; open arrowheads, tips of primary roots. (b) Quantification of secondary root (SR) number in WT, plt3plt5plt7, wox11wox12 seedlings under mock and CM11 inoculated conditions. Only the SRs formed above the inoculating sites were counted. (c) Quantification of the numbers of emerged lateral roots (LRE) and initiated lateral root primordia (LRI) using the pPLT7::GUS reporter line under mock and CM11 inoculated conditions. Different letters indicate statistically significant differences (LSD and Tukey’s honestly significant difference (HSD) tests; P ≤ 0.05); (d) Shoot : root ratio of WT, plt3plt5plt7 and wox11wox12 Arabidopsis seedlings under mock and CM11 inoculated conditions. In all cases, 7‐d‐old seedlings were inoculated with mock or bacterial suspensions at the root tip. In (a), (b) and (d), images and data were analyzed 7 d post‐inoculation (dpi), whereas in (c) they were analyzed at 2 dpi. Data represent mean ± SD of three biological replicates, each consisting of eight seedlings in (b) and (d), and 20 seedlings in (c). Asterisks in (b) and (d) indicate significant differences between inoculated and mock plants: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s t‐test). Bar, 1 cm.

Fig. 5

CM11 inoculation improves Arabidopsis plant fitness. On the left of Arabidopsis plant mode downwards are comparisons in plant height, bolting time and root FW of plants grown in soil between CM11 inoculated and mock conditions. On the right are comparisons in total seed number, per seed size, per seed weight and seed germination rate (top), in shoot FW, leaf number and total leaf area (bottom) of plants grown in soil between mock and CM11 inoculated conditions. In all cases, 7‐d‐old Arabidopsis wild‐type (WT) seedlings were transferred to pots with CM11 bacterial suspension or mock in soil. Each parameter was quantified as described in the Materials and Methods section. Data represent mean ± SD of three biological replicates, each consisting of 10–12 seedlings. Asterisks indicate SDs between inoculated and mock plants (ns, no significance): *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s t‐test).

Fig. 6

CM11 alters root system architecture and promotes growth of lettuce and tomato seedlings. (a) Representative images of lettuce seedlings growing on ½ Murashige & Skoog agar plates with CM11 strains or mock at 10 d post‐inoculation (dpi). (b–c) Quantification of primary root (PR) length (newly‐grown PR below the inoculation sites, b) and number of emerged secondary roots (SRs) (emerged SRs above the inoculation sites, c) of lettuce seedlings under mock and CM11 inoculated conditions at 8 dpi. (d) Representative images of tomato seedlings growing on ½MS agar plates with CM11 strains or without (mock) at 10 dpi. (e–f) Quantification of PR length (newly grown primary root below the inoculation sites, e) and number of emerged SRs (emerged SRs above the inoculation sites, f) of tomato seedlings under mock and CM11 inoculated conditions at 3 dpi. Box plots in (b, c, e, f) indicate the lower and upper quartiles, bars represent the maximum and minimum values. The line in the box indicates the median; (g) Representative photographs of lettuce seedlings grown in soil inoculated with CM11 bacterial suspension or without (mock). (h–i) Root (h) and shoot (i) FW production per lettuce seedling with CM11 inoculation or mock. (j) Representative photographs of tomato seedlings grown in soil inoculated with CM11 bacterial suspension or mock. (k–l) Root (k) and shoot (l) FW production per tomato seedling with CM11 inoculation or mock. In (a–c) 3‐d‐old lettuce seedlings and in (d–f) 4‐d‐old tomato seedlings were inoculated with mock or CM11 bacterial suspensions at the root tip. In (g–l) 10‐d‐old lettuce or tomato seedlings were transferred to soil with CM11 bacterial suspension or mock, and measured at 10 dpi. Data in (h, i, k, l) represent mean ± SD of three biological replicates, each consisting of 8–10 seedlings. Asterisks indicate SDs between inoculated and Mock plants: **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s t‐test). Bar, 2 cm.