Table 1.
Strains and plasmids used in this study.
Table 2.
qRT-PCR primer sets used in this study.
Figure 1.
Congo-red binding and flocculation of A. brasilense strains.
A. Wild type strain Sp7 (1) and flcA complemented strain Sp7-flcAΔ[pAB2053] (4) form dark red, rough surfaced colonies on NFM agar containing Congo-Red, whereas, Sp7-flcAΔ (2) and Sp72001 (3) form light-red, mucoid, smooth-surfaced colonies on the same medium. B. Flocculation of Sp7 (5) and Sp7-flcAΔ[pAB2053] (8) in flocculation liquid medium; Sp7-flcAΔ (6) and Sp72001 (7) do not flocculate under the same conditions.
Figure 2.
Plant-root binding abilities of A. brasilense strain.
Colonization of wheat root by Sp7 (1 and 4), Sp7-flcAΔ (2 and 5) and Sp72001 (3 and 6). A. brasilense strains harbor the reporter plasmid pLA-lacZ, containing a constitutively expressed lacZ gene and were stained with X-gal. Sp7 (1, 4) has strong binding ability to wheat roots and can be found all over the root surface; Sp7-flcAΔ (2, 5) and Sp72001 (3, 6) lost the ability to bind to wheat roots and could only be found in lateral root emergence areas. Scale bars indicate 50 µm (group A) and 5 µm (group B) (Magnification ×100 in group A and ×1000 in group B).
Figure 3.
2-DE gels of A. brasilense Sp7 and Sp7-flcAΔ under flocculation conditions.
Blue silver stained 2-DE gel of proteins extracted from Azospirillum Sp7 (A) and Sp7-flcAΔ mutant (B) under flocculation conditions. Circled spots were found to be differentially expressed and were able to be excised and analyzed by LC-MS/MS. Green circles indicate increased expression and red circles indicate decreased expression in either Sp7 or Sp7-flcAΔ.
Table 3.
Proteins regulated by FlcA.
Figure 4.
Differential protein expression in Sp7 and Sp7-flcAΔ analyzed by 2-DE.
The left section shows the normalized expression volume of the spot in wild type (Sp7) and flcA knock out strain (Sp7-flcAΔ) under flocculation conditions; the relative fold change is shown above each column (Nx indicates that relative fold change could not be calculated as the protein was only detected in either Sp7 or Sp7-flcAΔ). The right section is a 3D representation of the area of interest as provided by PDquest software. Arrows indicate spots with relatively higher expression.
Figure 5.
Differential gene expression in Sp7 and Sp7-flcAΔ under both nutrient and flocculation conditions, analyzed by qRT-PCR.
Transcription levels of atpD, clpP, glnA, livK, nark, narL1, nos and phaA were significantly higher in Sp7-flcAΔ than in Sp7 under flocculation conditions. Transcription levels of cheW, galU and gltA were significantly lower in Sp7-flcAΔ than in Sp7 under flocculation conditions. Transcription levels of ahpC, gloA, lpxC and sodA did not differ significantly in Sp7 and Sp7-flcAΔ under either culture condition. * indicates a significant difference between group means (P<0.05). Data is presented as mean ±SEM, relative to expression of two reference genes, n = 4 biological replicates.