The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses

Abstract

Background: Verticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years.

Results: Here we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host.

Conclusions: We conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.

Figure 1

Pi inhibits growth of Vd on agar plates. (A) Typical plates from 3 independent experiments are shown. (B) Quantification of the colony. The diameter of the Pi and Vd mycelia on the agar plate is given in cm. Bars represent SDs. Asterisks indicate significant differences, as determined by ANOVA (*** P ≤ 0.001).

Figure 2

Pi protects Arabidopsis seedlings from Vd infection. (A) Fresh weights of seedlings after 10, 14 and 21 days of co-cultivation or mock-treatments on Petri dishes. The seedlings were exposed to either Pi or Vd alone or in combination as described in the Methods and Additional file 1: Figure S1. C: seedlings treated without fungi; Vd: seedlings treated with Vd; Pi: seedlings treated with Pi; 1P2V: seedlings first treated with Pi for 4 days followed by Vd; 1V2P: vice-versa as 1P2V. n.d: no detectable (seedlings were dead, no fresh weight could be determined). The data are based on 3 independent experiments with 16 seedlings each. Bars represent SDs. Asterisks indicate significant differences, as determined by ANOVA (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). (B) The phenotype of typical seedlings on 21^st day. (C) PDI for seedlings exposed to Vd. For treatments, cf. Methods and Additional file 1: Figure S1. The data are based on 3 independent experiments with 16 seedlings each. Bars represent SDs.

Figure 3

Total chlorophyll content (mg/g fresh weight) in shoots. The data were obtained 4, 10, 14 and 21 days after the fungal treatments (cf. Methods, Additional file 1: Figure S1 and legend to Figure 2A). The data are based on 3 independent experiments with 16 seedlings each. Bars represent SDs. Asterisks indicate significant differences to the untreated control, as determined by Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Figure 4

Stomata closure rate in leaves after 3 (A) and 7 (B) days. The data are based on 3 independent experiments with 16 seedlings each. Bars represent SDs. Asterisks indicate significant differences to the untreated control, as determined by Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Figure 5

Induction of defense genes in the shoots of Arabidopsis seedlings 1 and 14 days after the fungal treatments, relative to the untreated control. The data represents fold induction (mRNA level _{+fungal treatments}/mRNA level _{-fungal treatments}; fold of control is set as 1.0). For experimental details, cf. Methods, Additional file 1: Figure S1 and legend to Figure 2A. The data are based on 5 independent experiments with 16 seedlings each. Bars represent SDs. Asterisks indicate significant differences, as determined by Student’s t-test (* P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001).

Figure 6

Phytohormone levels in the shoots 21 days after the different fungal treatments. For experimental details, cf. Methods, Additional file 1: Figure S1, and legend to Figure 2A. The data are based on 3 independent experiments with 12 seedlings each. Bars represent SDs. Asterisks indicate significant differences, as determined by Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Figure 7

The amount of fungal DNA in the roots and shoots of Arabidopsis seedlings exposed to the 5 treatments (cf. legend to Figure 2 A). For experimental details, cf. Methods and Additional file 1: Figure S1. The measurements were performed for the 14^th (A, B, C) and 21st (D, E, F) day. The data are based on 3 independent experiments with 12 seedlings each. Bars represent SDs. Asterisks indicate significant differences compared to Vd (A, B, D, E) or to Pi (C and F), as determined by Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Figure 8

Pi inhibits the formation of Vd micosclerotia in roots, irrespective of whether the roots were first exposed to Pi ( 1P2V ) or first to Vd ( 1V2P ). The analysis was performed 21 days after infection. Left: microscopy of root sections with microslerotia (black spots). Right: Quantification of the number of microsclerotia. The data are based on 3 independent experiments with 12 seedlings each. Bars represent SDs. Asterisks indicate significant differences to Vd, as determined by Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Figure 9

Confirmation of the results for adult plants, grown in sterile vermiculite. After exposure of the seedlings to the 5 treatments in Petri dishes for 10 days (cf. legend to Figure 2A), they were transferred to Magenta boxes with sterile vermiculite for 14 days. (A) Number of survived plants. (B) Fresh weight of plants. (C) Fungal DNA content in roots and shoots. The data are based on 3 independent experiments with 16 seedlings each. Bars represent SDs. Asterisks indicate significant differences to Vd, as determined by Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Figure 10

EIN3 is required for full susceptibility of Arabidopsis to Vd . (A) The representative picture (3 independent experiments with 32 plants each) was taken after 21 days inoculation with Vd. (B) Number of survived seedlings. (C) Ethylene levels in WT and ein3 seedlings after exposure to Vd. Bars represent SDs. Asterisks indicate significant differences, as determined by Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Figure 11

Vd exudate preparation induces [Ca ²⁺ ] _cyt elevation in A. thaliana seedlings expressing cytosolic aequorin. (A) Roots of 21-day old pMAQ2 in Col-0 seedlings were dissected and incubated overnight in 7.5 μM coelenterezine. The roots were challenged with 50 μl of the Vd preparations. [Ca²⁺]_cyt level was calculated from the relative light unit (RLU) at 5 s integration time for 10 min. The arrow indicates the time (60 s) of addition of the stimuli/PBS buffer. For all experiments, 10 mM phosphate buffer (PBS, pH 7.0) was used as control and gave background readings. All curves and values represent average of five independent experiments with eight replications in each experiment. (B) Vd exudate preparation does not induce [Ca²⁺]_cyt elevation in the cycam1 mutant, but induces [Ca²⁺]_cyt elevation in pMAQ2 lines in the glr2.4, glr2.5 and glr3.3 background.