The isolation and characterization of resident yeasts from the phylloplane of Arabidopsis thaliana

Abstract

The genetic model plant Arabidopsis thaliana (arabidopsis) has been instrumental to recent advances in our understanding of the molecular function of the plant immune system. However, this work has not yet included plant associated and phytopathogenic yeasts largely due to a lack of yeast species known to interact with arabidopsis. The plant phylloplane is a significant habitat for neutral-residents, plant-growth and health-promoting species, and latent-pathogenic species. However, yeast phylloplane residents of arabidopsis remain underexplored. To address this, resident yeasts from the phyllosphere of wild arabidopsis collected in field conditions have been isolated and characterized. A total of 95 yeast strains representing 23 species in 9 genera were discovered, including potentially psychrophilic and pathogenic strains. Physiological characterization revealed thermotolerance profiles, sensitivity to the arabidopsis phytoalexin camalexin, the production of indolic compounds, and the ability to activate auxin responses in planta. These results indicate a rich diversity of yeasts present in the arabidopsis phylloplane and have created culture resources and information useful in the development of model systems for arabidopsis-yeast interactions.

Figure 1

(a), The colonies of leaf resident filamentous fungi (5–15 mm in diameter), yeasts and bacteria (0.1–2 mm in diameter) visualized by leaf printing onto a 0.2 × PDA medium plate from a wild Arabidopsis thaliana. (b), Typical yeast colonies on 0.2 × PDA medium, seven days after plating 20 μL of leaf-wash solution from sample A (Table 1). Similar plates with lower colony density from plating 1:10 or 1:100 dilutions of leaf-wash were used for colony picking. Scale bar: 1 cm.

Figure 2

Composition of arabidopsis surface yeasts isolated from the view of genus (a) and the species or strain composition of the dominant genus Cryptococcus (b).

Figure 3. Production of indolic compounds by yeasts.

Selected isolates were cultivated in nitrogen base with 1% glucose (N + gluc), glucose-yeast-peptone (GYP) media with and without 0.1% L-tryptophan (Trp). Standard curve was calibrated by using indole acetic acid (IAA) and results are presented as equivalents of IAA (IAA eq.) concentration. ^#Not detected. Three independent biological replicates were conducted with similar results.

Figure 4. Effect of indolic compounds produced by yeasts on gene expression of auxin regulated genes, monitored as expression of the artificial auxin responsive DR5 promoter.

Two-week-old in vitro grown DR5::GUS arabidopsis seedlings were treated with the filtered supernatants of five day old yeast cultures for 15 hours. As an example of a positive response, strong GUS expression were detected in seedling roots treated with Taphrina sp. OTU 3 supernatant and in the positive control 5 μM IAA treatment. As representative negative results, only light root tip staining was seen with Cryptococcus sp. OTU 4 supernatant and the negative control GYP medium treatments. Similar results were observed in three independent biological replicate experiments. Scale bar = 2 mm. See also Supplementary Fig. S1 for results of all treatments.

Figure 5. Effect of arabidopsis phytoalexin camalexin on yeast growth.

Yeasts were cultured in liquid GYP medium containing 0, 5, 15, 25 μg/ml camalexin, from starting cell density OD = 0.1. Similar results were observed from three independent biological replicate experiments.

Figure 6. Effect of temperature on yeast growth.

Yeasts were first grown on liquid GYP medium for three days and then diluted to OD = 1. Serial of dilutions (OD = 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷) were plated on GYP agar medium for grown at different temperature conditions (8 °C, 21 °C, 30 °C, 37 °C). Colony forming units (CFUs) were counted to quantify growth after seven days. No isolate grew at 37 °C. Similar results were observed from three independent biological replicate experiments.