Medicago truncatula plants overexpressing the early nodulin gene enod40 exhibit accelerated mycorrhizal colonization and enhanced formation of arbuscules

Abstract

The mutualistic symbiosis between flowering plants and arbuscular mycorrhizal fungi is extremely abundant in terrestrial ecosystems. In this symbiosis, obligately biotrophic fungi colonize the root of the host plants, which can benefit from these fungi by enhanced access to mineral nutrients in the soil, especially phosphorus. One of the main goals of research on this symbiosis is to find plant genes that control fungal development in the host plant. In this work, we show that mycorrhizal colonization is regulated by enod40, an early nodulin gene known to be involved in the nodule symbiosis of legumes with nitrogen-fixing bacteria. Medicago truncatula plants overexpressing enod40 exhibited stimulated mycorrhizal colonization in comparison with control plants. Overexpression of enod40 promoted fungal growth in the root cortex and increased the frequency of arbuscule formation. Transgenic lines with suppressed levels of enod40 transcripts, likely via a cosuppression phenomenon induced by the transgene, exhibited reduced mycorrhizal colonization. Hence, enod40 might be a plant regulatory gene involved in the control of the mycorrhizal symbiosis.

Figure 1

Transgenic M. truncatula plants overexpressing Mtenod40 exhibit enhanced colonization by G. mosseae. (a) Degree of mycorrhizal colonization (length of roots containing mycorrhizal structures in percentage of total root length) of M. truncatula wild-type (WT) plants, WT plants regenerated from leaf explants (Control), plants expressing a 35S promoter-GFP fusion (GFP), and line Se40(3) overexpressing the Mtenod40 gene harvested 21 days p.i. (experiment 1; n = 20, χ = 12.58, P = 0.006). (b) Time course of the mycorrhizal colonization of WT and Se40(3) plants (experiment 2). The degree of mycorrhizal colonization was estimated at different time points after inoculation. Differences (indicated by asterisks) were statistically significant (P < 0.05) up to 38 days. (c and d) Mycorrhizal colonization by G. mosseae using low amounts of inoculum. The difference between WT and two different enod40-overexpressing lines was significant (P < 0.05) in both experiments. (c) Degree of mycorrhizal colonization in roots of WT plants and line Se40(3) harvested 35 days p.i. (experiment 3). (d) Degree of mycorrhizal colonization in roots of WT plants and line Se40(10) harvested 49 days p.i. (experiment 4).

Figure 2

Number of arbuscules in wild-type (WT) plants and line Se40(3) overexpressing enod40. Plants were inoculated with G. mosseae and harvested 12 and 18 days later (experiment 2). Asterisks indicate statistically significant differences (P < 0.05) between WT and line Se40(3). (a) Number of arbuscules (per mm root length) on the basis of the total root length (colonized and noncolonized root zones). (b) Number of arbuscules (per mm root length) in root zones colonized by G. mosseae.

Figure 3

Number of vesicles in wild-type (WT) plants and line Se40(3) overexpressing enod40. Plants were harvested 12 and 18 days after inoculation with G. mosseae (experiment 2). (a) Number of vesicles (per mm root length) on the basis of the total root length (colonized and noncolonized root zones). Asterisks indicate statistically significant differences (P < 0.05) between WT and line Se40(3) for root material harvested 12 days p.i. (b) Number of vesicles (per mm root length) in root zones colonized by G. mosseae.

Figure 4

Mycorrhizal colonization by G. intraradices. (a) Degree of mycorrhizal colonization of M. truncatula wild-type (WT) plants, plants expressing a 35S promoter-GFP fusion (GFP) and overexpressing the Mtenod40 gene [lines Se40(3) and Se40(10)] harvested 56 days p.i. (experiment 5; n = 22, χ = 12.39, P = 0.006). (b) Degree of mycorrhizal colonization of M. truncatula wild-type (WT) plants, two lines with a cosuppression phenomenon [lines Se40(2) and Se40(5)] and line Se40(3) overexpressing enod40 harvested 49 days p.i. (experiment 6; n = 62, χ = 27.17, P < 0.001).