The Piriformospora indica effector PIIN_08944 promotes the mutualistic Sebacinalean symbiosis

Abstract

Pathogenic and mutualistic microbes actively suppress plant defense by secreting effector proteins to manipulate the host responses for their own benefit. Current knowledge about fungal effectors has been mainly derived from biotrophic and hemibiotrophic plant pathogenic fungi and oomycetes with restricted host range. We studied colonization strategies of the root endophytic basidiomycete Piriformospora indica that colonizes a wide range of plant species thereby establishing long-term mutualistic relationships. The release of P. indica's genome helped to identify hundreds of genes coding for candidate effectors and provides an opportunity to investigate the role of those proteins in a mutualistic symbiosis. We demonstrate that the candidate effector PIIN_08944 plays a crucial role during fungal colonization of Arabidopsis thaliana roots. PIIN_08944 expression was detected during chlamydospore germination, and fungal deletion mutants (PiΔ08944) showed delayed root colonization. Constitutive over-expression of PIIN_08944 in Arabidopsis rescued the delayed colonization phenotype of the deletion mutant. PIIN_08944-expressing Arabidopsis showed a reduced expression of flg22-induced marker genes of pattern-triggered immunity (PTI) and the salicylic acid (SA) defense pathway, and expression of PIIN_08944 in barley reduced the burst of reactive oxygen species (ROS) triggered by flg22 and chitin. These data suggest that PIIN_08944 contributes to root colonization by P. indica by interfering with SA-mediated basal immune responses of the host plant. Consistent with this, PIIN_08944-expressing Arabidopsis also supported the growth of the biotrophic oomycete Hyaloperonospora arabidopsidis while growth of the necrotrophic fungi Botrytis cinerea on Arabidopsis and Fusarium graminearum on barley was not affected.

Keywords: Piriformospora indica; endophyte; fungal effectors; mutualist; root; small secreted proteins; symbiosis.

FIGURE 1

Colonization of plant roots by Piriformospora indica increases over time. (A) Roots of 3-day-old barley grown on 1/10 PNM agar in sterile glass jars, were inoculated with chlamydospores of P. indica. Colonization levels were determined at 3, 7, 14, and 21 dpi as the relative amount of fungal DNA by qPCR using barley (HvUBQ-60-Deg) and fungal (ITS) specific primers. Values represent the mean ± SE of two independent experiments. (B) Seven-day-old Arabidopsis seedlings were inoculated with chlamydospores of P. indica. Colonization levels were determined at 3, 7, 14, and 21 dpi as the relative amount of fungal DNA by qPCR using Arabidopsis (AtUBQ4) and fungal (ITS) specific primers. Data represents the Ct thresholds of ITS relative to the Ct thresholds of AtUBQ-4 (±SE obtain from three technical replicates of one biological experiment). Experiments were repeated twice with similar results. Asterisks indicate significance between time points at ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 analyzed by student’s t-test. (C) Analysis of PIIN_08944 expression by semi-quantitative RT-PCR. Transcripts of PIIN_08944 were detected in in vitro germinated P. indica chlamydospores (CS) grown in CM liquid medium for 7 days and in planta during colonization of Arabidopsis roots by P. indica by RT-PCR. Transcript abundance increased over time from 3 to 21 dpi. The P. indica ubiquitin (UBQ) gene served as reference.

FIGURE 2

Characterization of P. indica Knockout Strains Pi_Δ08944 (PiT1-PiT5). (A) Roots of 7-day-old Arabidopsis seedlings were inoculated with chlamydospores of Pi_Δ08944 strains PiT1 or PiT2, respectively. The roots were harvested at 3, 7, 14, and 21 dpi and PIIN_08944 transcript levels were determined from extracted RNA by RT-PCR using PIIN_08944 specific primer. PIIN_08944 transcripts from Pi_Δ08944 strains were not detected in planta. The P. indica ubiquitin UBQ gene served as reference. (B) Deletion of PIIN_08944 delays colonization of Arabidopsis roots. Roots of 7-day-old Arabidopsis seedlings were inoculated with Pi_Δ08944 (PiT1) and wt, respectively. (C) Colonization of PIIN_08944-expressing Arabidopsis with Pi_Δ08944 (PiT1) mutant. Roots of 7-day-old AtPIIN_08944OE seedlings or wt were inoculated with Pi_Δ08944. Fungal biomass was determined at 3, 7, 14, and 21 dpi as relative amount of fungal DNA by qPCR using fungal (ITS) and plant (AtUBQ4) specific primers. Data displays the Ct thresholds of ITS relative to the Ct thresholds of AtUBQ- 4 (±SE obtain from three technical replicates of one biological experiment). Experiments were repeated twice with similar results. Asterisks indicate significance at ^∗P < 0.05, ^∗∗P < 0.01 analyzed by Student’s t-test.

FIGURE 3

Assessment of the immune status of AtPIIN_08944OE plants. (A) Two-week-old AtPIIN_08944OE and the respective wt plants were treated with 100 nM flg22 in six well plates. RNA was extracted at 0 (untreated control), 2, 6, and 12 hpt and qPCR was performed. Suppression of flg22-induced transcription was observed for AtWRKY22 and CBP60g. Arabidopsis UBQ-4 was used for normalization. Values are means ± SE of two independent experiments. (B) Detached leaves of four to 6-week-old AtPIIN_08944OE (lines #L6, #L10) and the respective wt plants were inoculated with conidia of Botrytis cinerea. Photographs were taken 5 dpi. (C) Average lesion size of AtPIIN_08944OE (lines #L6 and #L10) and the wt plants after inoculation with B. cinerea. Averages were calculated from 10 to 15 leaves per line. Values are means ± SE of three independent experiments. (D) Two-week-old AtPIIN_08944OE seedlings (lines #4 #L6 #L10) and wt plants were sprayed with spores of Hyaloperonospora arabidopsidis and sporangiophores development on true leaves determined 4 dpi. For each line at least 100 plants were analyzed. Values are means ± SE of two independent experiments. Asterisks indicate significant differences at ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 as analyzed by student’s t-test.

FIGURE 4

Assessment of flg22 and chitin-induced reactive oxygen species (ROS) accumulation in barley and Arabidopsis tissue. Leaf disks of 3-week-old barley HvPIIN_08944-OE or EV control plants were treated with (A) 100 nM flg22, and (B) 200 mg/ml crab shell chitin. (C) Leaf disks of 4-week-old AtPIIN_08944-OE or wt Arabidopsis plants were treated with 100 nM flg22. ROS was determined by measuring the relative light unit over time with a luminol-chemiluminescence assay using a Tecan reader. Error bars represent the mean ± SE of three independent experiments. (RLU, relative light units; EV, empty vector).

FIGURE 5

Characterization of barley HvPIIN_08944OE plants inoculated with macroconidia of Fusarium graminearum. Root length (A) and root fresh weight (B) of six plants per line inoculated with F. graminearum. Values represent the mean with ± SE of three independent experiments. (C) Detached leaves of 3-week-old HvPIIN_08944OE plants (#4 #8 #14) and EV control were inoculated with F. graminearum. Photographs were taken 6 dpi. The experiment was repeated three times with similar outcome (D) Relative fungal biomass on detached HvPIIN_08944OE (#4 #8 #14) and EV control leaves by qPCR. Values represent the mean with ±SE of three independent experiments.