Endophytic Life Strategies Decoded by Genome and Transcriptome Analyses of the Mutualistic Root Symbiont Piriformospora indica

Recent sequencing projects have provided deep insight into fungal lifestyle-associated genomic adaptations. Here we report on the 25 Mb genome of the mutualistic root symbiont Piriformospora indica (Sebacinales, Basidiomycota) and provide a global characterization of fungal transcriptional responses associated with the colonization of living and dead barley roots. Extensive comparative analysis of the P. indica genome with other Basidiomycota and Ascomycota fungi that have diverse lifestyle strategies identified features typically associated with both, biotrophism and saprotrophism. The tightly controlled expression of the lifestyle-associated gene sets during the onset of the symbiosis, revealed by microarray analysis, argues for a biphasic root colonization strategy of P. indica. This is supported by a cytological study that shows an early biotrophic growth followed by a cell death-associated phase. About 10% of the fungal genes induced during the biotrophic colonization encoded putative small secreted proteins (SSP), including several lectin-like proteins and members of a P. indica-specific gene family (DELD) with a conserved novel seven-amino acids motif at the C-terminus. Similar to effectors found in other filamentous organisms, the occurrence of the DELDs correlated with the presence of transposable elements in gene-poor repeat-rich regions of the genome. This is the first in depth genomic study describing a mutualistic symbiont with a biphasic lifestyle. Our findings provide a significant advance in understanding development of biotrophic plant symbionts and suggest a series of incremental shifts along the continuum from saprotrophy towards biotrophy in the evolution of mycorrhizal association from decomposer fungi

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica

Phytohormones in plant root-Piriformospora indica mutualism

Piriformospora indica is a mutualistic root-colonising basidiomycete that tranfers various benefits to colonized host plants including growth promotion, yield increases as well as abiotic and biotic stress tolerance. The fungus is characterized by a broad host spectrum encompassing various monocots and dicots.1,2 Our recent microarray-based studies indicate a general plant defense suppression by P. indica and significant changes in the GA biosynthesis pathway.3 Furthermore, barley plants impaired in GA synthesis and perception showed a significant reduction in mutualistic colonization, which was associated with an elevated expression of defense-related genes. Here, we discuss the importance of plant hormones for compatibility in plant root-P. indica associations. Our data might provide a first explanation for the colonization success of the fungus in a wide range of higher plants

GUS accumulation in roots of <i>ACS1</i>::<i>GUS</i> reporter plants colonized by <i>P. indica</i>.

<p><i>Arabidopsis</i> line <i>ACS1</i>::<i>GUS</i> was harvested at 7 dai and, after GUS and WGA-AF 488 staining, analyzed cytologically. (<b>A</b>, <b>B</b>) <i>P. indica</i> colonization at the base of lateral roots (arrows) or primordia (asterisks) of line <i>ACS1</i>::<i>GUS</i> was associated with enhanced GUS accumulation. <i>P. indica</i> (arrowsheads in A) was visualized by staining with WGA-AF 488. (<b>C</b>) In mock-treated <i>ACS1</i>::<i>GUS</i>, GUS staining was weakly detectable e.g. at the lateral root base. Bars = 60 µm.</p

Colonization of ethylene synthesis and signaling mutants by <i>P. indica</i>.

<p>(<b>A</b>) Three-week-old plants were inoculated with <i>P. indica</i> and fungal biomass was determined in <i>ein2-1</i>, <i>etr1-3, eto1-1, ctr1-1</i>, and <i>35S</i>::<i>ERF1</i> by qRT-PCR at 3 and 14 dai. (<b>B</b>) Three-week-old <i>35S::ERF1</i> plants were injured with foreceps and inoculated with <i>P. indica</i> at 1 day after wounding. Fungal biomass was determined by qRT-PCR at 3 and 7 dai. All values were related to Col-0 (set to one). The data are based on at least three independent experiments. Students <i>t</i>-test indicated significant difference in <i>P. indica</i>-colonization (* P<0.05, ** P<0.001).</p

Colonization of barley and <i>Arabidopsis</i> by <i>P. indica</i> in response to ACC and MCP.

<p>(<b>A</b>) Two-day-old barley seedlings or (<b>B</b>) two-week-old <i>Arabidopsis</i> seedlings were inoculated with <i>P. indica</i> and subsequently treated with 500 ppt 1-methylcyclopropene (MCP) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035502#s4" target="_blank">Materials and Methods</a>. Barley was also treated with 100 µM 1-aminocyclopropane-1-carboxylic acid (ACC). MCP inhibited <i>P. indica</i> colonization at 3 or 7 dai in <i>Arabidopsis</i> or barley, respectively. The values are normalized to colonization in mock-treated roots (set to one). The data are based on three independent biological experiments. Student's <i>t</i>-test indicates a significant difference in <i>P. indica</i>-colonization of MCP-treated roots (* P<0.05).</p

List of barley genes differentially regulated by <i>P. indica</i> and involved in ethylene synthesis or signaling.

1<p>Gene expression data was published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035502#pone.0035502-Schfer2" target="_blank">[27]</a>.</p

Suppression of chitin-induced oxidative burst by <i>P. indica</i>.

<p>Chitin (1 µM <i>N</i>-acetylchitooctaose) was applied to barley root segments of seedlings harvested at 3 days after <i>P. indica</i>- or <i>Rhizoctonia solani</i> inoculation or mock-treatment, respectively. Values are given as relative light units (RLU) over time as means with standard errors of two biological experiments with three independent measurements per treatment and experiment. GP, barley cv. Golden Promise.</p

ACC content in barley roots during <i>P. indica</i> colonization.

<p>Free (<b>A</b>) and malonylated (<b>B</b>) 1-aminocyclopropane-1-carboxylic acid (ACC) contents were determined in <i>P. indica</i> and mock-treated roots at 1, 3, and 7 days after treatments. At 1 dai, the complete roots were harvested and forwarded to ACC measurements. At 3 and 7 dai, the upper two centimeters (basal part) and the remaining part of the roots (apical part) were analyzed separately. Absolute values are given in nmol • g FW⁻¹ for mock-treated and <i>P. indica</i>-colonized roots. (<b>A</b>) Free ACC levels were significantly enhanced at 3 and 7 dai in the apical zone and 7 dai in the basal part as indicated by Students <i>t</i>-test (* P<0.05, ** P<0.01, *** P<0.001). (<b>B</b>) Malonylated ACC was not significantly altered during <i>P. indica</i> colonization at any timepoint or in any tissue. Data show the mean content of four biological experiments (with at least two technical repetitions per experiment) and bars indicate standard errors.</p

GUS accumulation in roots of <i>ACS1</i>::<i>GUS</i> and <i>ACS8</i>::<i>GUS</i> reporter plants colonized by <i>P. indica</i>.

<p><i>Arabidopsis</i> lines <i>ACS1</i>::<i>GUS</i> and <i>ACS8</i>::<i>GUS</i> were harvested at 7 dai and, after GUS and WGA-AF 488 staining, analyzed cytologically. GUS staining was more pronounced in root tip regions of colonized roots as compared to mock-treated roots (upper images). At 7 dai, <i>P. indica</i>-colonized roots of both lines showed a significant increase of dark blue tips and a significant reduction in pale blue tips as compared to mock-treated roots. GUS staining did not colocalize with colonization sites of <i>P. indica</i> or extracellular fungal growth. The data base on at least two biological experiments. Asterisks indicate significant differences between control and <i>P. indica</i>-colonized roots according to Students <i>t</i>-test (* P<0.05, ** P<0.001).</p