Volatile signalling by sesquiterpenes from ectomycorrhizal fungi reprogrammes root architecture

Abstract

The mutualistic association of roots with ectomycorrhizal fungi promotes plant health and is a hallmark of boreal and temperate forests worldwide. In the pre-colonization phase, before direct contact, lateral root (LR) production is massively stimulated, yet little is known about the signals exchanged during this step. Here, we identify sesquiterpenes (SQTs) as biologically active agents emitted by Laccaria bicolor while interacting with Populus or Arabidopsis. We show that inhibition of fungal SQT production by lovastatin strongly reduces LR proliferation and that (-)-thujopsene, a low-abundance SQT, is sufficient to stimulate LR formation in the absence of the fungus. Further, we show that the ectomycorrhizal ascomycote, Cenococcum geophilum, which cannot synthesize SQTs, does not promote LRs. We propose that the LR-promoting SQT signal creates a win-win situation by enhancing the root surface area for plant nutrient uptake and by improving fungal access to plant-derived carbon via root exudates.

Figure 1. Laccaria volatile organic compounds (VOCs) stimulate lateral root development in poplar and Arabidopsis.

(a) P. tremula × P. alba (Pc) plantlets grown in the presence of L. bicolor (Lb) in a bi-compartmented Petri dish avoiding direct contact and solute exchange between the plant and the fungus. Red dots indicate lateral roots. Scale bar, 10 mm. (b) Time course of lateral root development. The poplar roots were exposed to fungal VOCs. (c) Arabidopsis seedlings (At) grown in the presence of L bicolor in a bi-compartmented Petri dish. Scale bar, 10 mm. (d) Time courses of lateral root development of A. thaliana grown in the presence of L. bicolor (Lb) or C. geophilum (Cg) VOCs. Asterisks (*) in b and d indicate significant differences compared with controls at P<0.05 (t-test); when error bars are not visible they were smaller than the symbols. Data are means (n=8–10, ±s.e.).

Figure 2. L. bicolor VOCs stimulate root hair growth through a ROS-dependent mechanism.

(a) Root hairs of Arabidopsis plants (At) grown in the presence or absence of L. bicolor VOCs (Lb). Scale bar, 1,000 μm. (b) Diphenyleneiodium (100 nM, DPI) suppresses the L. bicolor VOC-mediated stimulation of root hairs. Scale bar=500 μm. (c) Quantification of root hair size of plants shown in a. Different letters indicate significant differences of the mean values at P<0.05 (HSD test, n=100–121). (d) Root hairs of Arabidopsis WT plants (At) and rhd2 mutants grown in plates (pH 5.0) in the presence or absence of L. bicolor VOCs (Lb). Scale bar=500 μm. (e) Quantification of root hair size of plants shown in d. Different letters indicate significant differences of the mean values at P<0.05 (honest significant difference (HSD) test). (f) Quantification of LR density of plants shown in d. Different letters indicate significant differences of the mean values at P<0.05 (HSD test). (g) Superoxide anion radical staining reveals ROS accumulation in the root of an Arabidopsis plant grown in the presence of L. bicolor VOCs. Scale bar, 100 μm.

Figure 3. Sesquiterpenes (SQTs) are the main regulators of LR development.

(a) In the presence of Lovastin (Lov), an inhibitor of SQT synthesis, the L. bicolor (Lb)-induced lateral root formation of Arabidopsis (At) is abolished. Arabidopsis seedlings were grown in bi-compartmented Petri dishes without physical contact to the second compartment, which was supplemented with L. bicolor (Lb) in the presence or absence of Lov (5 μM). The SQT (−)-thujopsene (Th, 100 p.p.b.) rescues lateral root formation when L. bicolor SQT production was inhibited by Lov (Lb+Lov+Th). Plants were analysed after 10 days of exposure. Controls in the absence of Lov are shown in b. The asterisks indicate significant differences (Tukey’s test, P<0.05; n=10, mean±s.e.). (b) The SQT (−)-thujopsene (Th) induces LR formation of Arabidopsis seedlings. In bi-compartmented Petri dishes, the plants were grown on one side and a filter paper with the VOC (100 p.p.b.) was placed on the other side. The asterisks (*) indicate significant differences to mock-treated Arabidopsis controls (Student’s t-test, P<0.05; n=10, mean±s.e.). (c) In the presence of Lov, the L. bicolor (Lb)-induced lateral root formation of poplar plantlets (Pc) is abolished. Poplar plantlets were grown in bi-compartmented Petri dishes without physical contact to the second compartment, which was supplemented L. bicolor (Lb) in the presence or absence of Lov (5 μM). The SQT (−)-thujopsene (Th, 100 p.p.b.) rescues lateral root formation when L. bicolor SQT production was inhibited by Lov (Lb+Lov+Th). Plants were analysed after 10 days of exposure. Controls in the absence of Lov are shown in d. The asterisks (***) indicate significant differences (Tukey’s test, P<0.001; n=8–10, mean±s.e.). (d) (−)-Thujopsene (Th) induces LR formation in poplar plantlets (Pc), which were grown in bi-compartmented Petri dishes with (−)-thujopsene (100 p.p.b.) on filter paper in the adjacent compartment. The asterisks (*) indicate significant differences to mock-treated control poplars (Student’s t-test, P<0.05; n=9; mean±s.e.).