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Pseudomonas putida and Pseudomonas fluorescens Influence Arabidopsis Root System Architecture Through an Auxin Response Mediated by Bioactive Cyclodipeptides

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Abstract

Plant growth-promoting rhizobacteria modulate root development through different mechanisms. This work was conducted to evaluate the effects of root colonization by Pseudomonas putida and Pseudomonas fluorescens in biomass production, lateral root formation, and activation of auxin signaling in Arabidopsis thaliana. Selected strains of P. putida and P. fluorescens were tested for modification of DR5::uidA, BA3::uidA and HS::AXR3NT-GUS auxin-related gene expression, and to promote root hair and lateral root formation in WT and tir1-1, tir1-1afb2-1afb3-1, arf7-1, arf19-1, arf7-1arf19-1, and rhd6 mutants. Production of cyclodipeptides with possible roles in auxin signaling was also determined in P. putida and P. fluorescens culture supernatants by gas chromatography–mass spectrometry. P. putida and P. fluorescens stimulated lateral root and root hair formation and increased plant biomass, which correlated with an induction of the auxin response. Genetic analyses suggested that growth promotion involves auxin signaling as tir1-1, tir1-1afb2-1afb3-1, arf7-1, arf19-1, and arf7-1arf19-1 mutants showed decreased lateral root response to inoculation and because P. putida and P. fluorescens restored root hair development in the rhd6 mutant. It was also found that these bacteria produce the cyclodipeptides cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Phe), and cyclo(L-Pro-L-Tyr), which modulates auxin-responsive gene expression in roots. Our results suggest a role of cyclodipeptides for bacterial phytostimulation.

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Abbreviations

AHL:

N-Acyl-l-homoserine lactones

ARFs:

Auxin response transcription factors

CDPs:

Cyclodipeptides

DAPG:

2, 4-Diacetylphloroglucinol

IAA:

Indole-3-acetic acid

LRD:

Lateral root density

LRN:

Lateral root number

LRP:

Lateral root primordia

PGPR:

Plant growth-promoting rhizobacteria

QS:

Quorum-sensing

TIR1/AFB:

Transport inhibitor response/auxin receptor F-box proteins

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Acknowledgements

We thank Drs. Peter Doerner, Christian Luschnig, Athanasios Theologis, Tom Guilfoyle, and Mark A. Estelle for kindly providing seeds of transgenic and mutant lines. This work was supported by grants from the Consejo Nacional de Ciencia y Tecnología (CONACYT, México, Grant No. 80916, 177775, and PDCPN 2015-882), Consejo de la Investigación Científica (UMSNH, México, Grant No. CIC 2.26), and the Marcos Moshinsky Foundation.

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  1. Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B3, Ciudad Universitaria, C.P. 58030, Morelia, Michoacán, Mexico

    Randy Ortiz-Castro, Jesús Campos-García & José López-Bucio

  2. Catedrático CONACYT-Instituto de Ecología A.C., Red de Estudios Moleculares Avanzados, Edificio B, Campus III, Carretera Antigua a Coatepec 351, El Haya, C.P. 91070, Xalapa, Veracruz, Mexico

    Randy Ortiz-Castro

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  1. Randy Ortiz-Castro
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Correspondence to Randy Ortiz-Castro.

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344_2019_9979_MOESM1_ESM.eps

Supplementary material 1 Fig. S1. Effect of co-inoculation of P. putida and P. fluorescens on root hair development in Arabidopsis (WT) seedlings. (A–C) Arabidopsis WT (Col-0) seedlings uninoculated or co-cultivated with P. putida or P. fluorescens. (D) Root hair number, (E) Root hair length. Data represent the mean ± SD (n=100). Different letters indicate means statistically different at P<0.05. (Scale bar = 500 µm). (EPS 143303 kb)

344_2019_9979_MOESM2_ESM.eps

Supplementary material 2 Fig. S2. Effect of co-cultivation with P. putida and P. fluorescens on root hair development in WT (Col-0) seedlings and auxin-related mutants. Arabidopsis WT (Col-0), tir1-1, arf7-1arf19-1 and tir1-1afb2-1afb3-1 mutants seedlings were co-cultivated with P. putida or P. fluorescens at a distance of 5 cm from the primary root tip and grown for additional time. Representative photographs of axenically grown Arabidopsis seedlings or seedlings co-cultivated with P. putida or P. fluorescens (Scale bar = 500 µm). (EPS 105728 kb)

344_2019_9979_MOESM3_ESM.eps

Supplementary material 3 Fig. S3. Effect of co-cultivation with P. putida and P. fluorescens on root hair development of rhd6 Arabidopsis mutant. Arabidopsis rhd6 mutant seedlings were co-cultivated with P. putida or P. fluorescens at a distance of 5 cm from the primary root tip and grown for additional time. Representative photographs of axenic Arabidopsis seedlings or seedlings co-cultivated with P. putida or P. fluorescens (Scale bar = 500 µm) (EPS 76917 kb)

344_2019_9979_MOESM4_ESM.eps

Supplementary material 4 Fig. S4. Effects of cell-free extracts produced by P. putida and P. fluorescens on root system architecture of Arabidopsis WT (Col-0) seedlings and auxin-related mutants. Arabidopsis WT (Col-0) and tir1-1, arf7-1, arf19-1, arf7-1arf19-1 and tir1-1afb2-1afb3-1 mutant seedlings were germinated and grown for 6 days on 0.2x MS medium and then transferred into 24-well cell culture plates (15 seedlings per well) containing 2 mL 0.2x MS liquid medium supplied with the indicated concentrations of compounds and incubated for 12 h. Data represent mean ± SD (n=15). Different letters indicate means statistically different at P<0.05. (EPS 166479 kb)

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Ortiz-Castro, R., Campos-García, J. & López-Bucio, J. Pseudomonas putida and Pseudomonas fluorescens Influence Arabidopsis Root System Architecture Through an Auxin Response Mediated by Bioactive Cyclodipeptides. J Plant Growth Regul 39, 254–265 (2020). https://doi.org/10.1007/s00344-019-09979-w

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