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The Piriformospora indica effector PIIN_08944 promotes the mutualistic Sebacinalean symbiosis.

Akum FN, Steinbrenner J, Biedenkopf D, Imani J, Kogel KH - Front Plant Sci (2015)

Bottom Line: Current knowledge about fungal effectors has been mainly derived from biotrophic and hemibiotrophic plant pathogenic fungi and oomycetes with restricted host range.We demonstrate that the candidate effector PIIN_08944 plays a crucial role during fungal colonization of Arabidopsis thaliana roots.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Phytopathology, Research Center for BioSystems, Land Use and Nutrition, Justus Liebig University Giessen Giessen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: To confirm previous results and to benchmark our colonization assay, we investigated the increase in fungal biomass during P. indica colonization of Arabidopsis and barley roots by using qPCR. Similar colonization pattern were observed for both plant species over a time period of 21 days. The relative amount of fungal DNA increased in roots from 3 to 21 dpi, with a more than 20-fold increase in barley (Figure 1A) and 300-fold increase in Arabidopsis (Figure 1B) relative to plant DNA. This is in agreement with published results (Pedrotti et al., 2013).


The Piriformospora indica effector PIIN_08944 promotes the mutualistic Sebacinalean symbiosis.

Akum FN, Steinbrenner J, Biedenkopf D, Imani J, Kogel KH - Front Plant Sci (2015)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4620400&req=5

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.
Mentions: To confirm previous results and to benchmark our colonization assay, we investigated the increase in fungal biomass during P. indica colonization of Arabidopsis and barley roots by using qPCR. Similar colonization pattern were observed for both plant species over a time period of 21 days. The relative amount of fungal DNA increased in roots from 3 to 21 dpi, with a more than 20-fold increase in barley (Figure 1A) and 300-fold increase in Arabidopsis (Figure 1B) relative to plant DNA. This is in agreement with published results (Pedrotti et al., 2013).

Bottom Line: Current knowledge about fungal effectors has been mainly derived from biotrophic and hemibiotrophic plant pathogenic fungi and oomycetes with restricted host range.We demonstrate that the candidate effector PIIN_08944 plays a crucial role during fungal colonization of Arabidopsis thaliana roots.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Phytopathology, Research Center for BioSystems, Land Use and Nutrition, Justus Liebig University Giessen Giessen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus