Research ArticlePLANTS INTERACTING WITH OTHER ORGANISMS
Open Access

HSPRO Controls Early Nicotiana attenuata Seedling Growth during Interaction with the Fungus Piriformospora indica

Stefan Schuck, Iris Camehl, Paola A. Gilardoni, Ralf Oelmueller, Ian T. Baldwin, Gustavo Bonaventure

Published October 2012. DOI: https://doi.org/10.1104/pp.112.203976

Abstract

In a previous study aimed at identifying regulators of Nicotiana attenuata responses against chewing insects, a 26-nucleotide tag matching the HSPRO (ORTHOLOG OF SUGAR BEET Hs1pro-1) gene was found to be strongly induced after simulated herbivory (Gilardoni et al., 2010). Here we characterized the function of HSPRO during biotic interactions in transgenic N. attenuata plants silenced in its expression (ir-hspro). In wild-type plants, HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv tomato DC3000 and roots with the growth-promoting fungus Piriformospora indica. Reduced HSPRO expression did not affect the regulation of direct defenses against Manduca sexta herbivory or P. syringae pv tomato DC3000 infection rates. However, reduced HSPRO expression positively influenced early seedling growth during interaction with P. indica; fungus-colonized ir-hspro seedlings increased their fresh biomass by 30% compared with the wild type. Grafting experiments demonstrated that reduced HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots. This effect was accompanied by changes in the expression of 417 genes in colonized roots, most of which were metabolic genes. The lack of major differences in the metabolic profiles of ir-hspro and wild-type colonized roots (as analyzed by liquid chromatography time-of-flight mass spectrometry) suggested that accelerated metabolic rates were involved. We conclude that HSPRO participates in a whole-plant change in growth physiology when seedlings interact with P. indica.

Nicotiana attenuata is a wild annual tobacco (Nicotiana spp.) plant native to the deserts of the southwestern United States and it germinates after fires from long-lived seed banks to form monocultures in post-fire nitrogen (N)-rich soils (Baldwin and Morse, 1994). As a result of its life history, N. attenuata grows rapidly after seed germination and the control of seedling growth is critical for plant fitness since young seedlings are more vulnerable to environmental stresses. In addition to water availability and high temperatures and light intensities, N. attenuata plants interact with unpredictable communities of beneficial and nonbeneficial organisms in their natural environment (Baldwin and Preston, 1999; Barazani et al., 2005; Long et al., 2010). With regard to biotic interactions, N. attenuata (and plants in general) readjust their metabolic and growth programs to meet the new requirements of de novo biosynthesis of direct (e.g. accumulation of toxic metabolites) and indirect (e.g. production of volatiles) defense responses as well as to induce tolerance mechanisms (e.g. carbon [C] and N bunkering in roots) or to facilitate symbiotic interactions (Rosenthal and Kotanen, 1994; Bardgett et al., 1998; Schwachtje and Baldwin, 2008). Activation of these responses requires metabolic energy and the redirection of C, N, and additional resources throughout the whole body of the plant (Schwachtje and Baldwin, 2008; Bolton, 2009). With the aim of identifying regulatory components of the pathways mediating defense and tolerance responses against lepidopteran larvae in N. attenuata, a serial analysis of gene expression approach was recently performed by our group to quantify the early transcriptional changes elicited by the insect elicitor N-linolenoyl-Glu (18:3-Glu; Gilardoni et al., 2010). The analysis targeted mRNAs encoding rare transcripts constitutively expressed and showing rapid and transient induction after 18:3-Glu elicitation. Among the approximately 500 differentially expressed transcripts, more than 25% corresponded to putative regulatory components (Gilardoni et al., 2010). One of these components was a homolog of a group of proteins denominated putative nematode resistance protein or ORTHOLOG OF SUGAR BEET Hs1pro-1 (HSPRO) based on their homology to Hs1pro-1 from sugar beet (Beta vulgaris; Cai et al., 1997).

To our knowledge, the study describing the role of Hs1pro-1 was the first to provide functional information for this group of proteins, and Hs1pro-1 was originally identified as a gene conferring resistance to the beet cyst nematode Heterodera schachtii (Cai et al., 1997). Subsequent studies performed in different plant species suggested however that this group of proteins has a more general role in the regulation of plant responses to biotic and abiotic stresses. For example, the Arabidopsis (Arabidopsis thaliana) genome encodes for two homologs of Beta procumbens Hs1pro-1, HSPRO1, and HSPRO2 and these two genes have been categorized as general stress signaling genes (Baena-González and Sheen, 2008). The expression of Arabidopsis HSPRO genes is not induced by nematode feeding (Puthoff et al., 2003) but it can be differentially induced by salicylic acid (SA), Pseudomonas syringae pv tomato (Pst) DC3000, and Xanthomonas campestris pv campestris infection, the bacterial elicitor flagellin22 (flg22), phosphate starvation, salt stress, drought, wounding, and UV-B (Hammond et al., 2003; Zipfel et al., 2004; Cominelli et al., 2005; Gissot et al., 2006; Fujita et al., 2007; Kudla et al., 2007; Murray et al., 2007; Walley et al., 2007). Genetic disruption of HSPRO2 in Arabidopsis increased susceptibility to Pst DC3000 (Murray et al., 2007) and its ectopic expression confers increased resistance against oxidative stress (Luhua et al., 2008). The role of HSPRO genes in these processes is at present unknown. Interestingly, in Arabidopsis, it has been shown that HSPRO1 and HSPRO2 interact with the AKINβγ (adaptor-regulator related to the Suc NONFERMENTING1 [SNF1]/AMPK family) subunit of the SNF1-related protein kinase (SnRK1) complex (Gissot et al., 2006). This complex is a heterotrimeric protein kinase complex related to the Saccharomyces cerevisiae SNF1 kinase (Baena-González and Sheen, 2008). Plant SnRK1s are central regulators of metabolism via the control of gene expression and enzyme activity (Halford et al., 2003; Lovas et al., 2003; Schwachtje et al., 2006; Baena-González and Sheen, 2008).

In this study, we analyzed the role of the HSPRO gene in N. attenuata during diverse biotic interactions, including M. sexta herbivory, Pst DC3000 infection, and association with the growth-promoting fungus Piriformospora indica. P. indica is a root-colonizing basidiomycete of the order Sebacinales (Varma et al., 1999; Weiss et al., 2004) and is closely related to fungal clones isolated from soil samples collected from the rhizosphere of N. attenuata in its natural habitat (Barazani et al., 2005). P. indica has the ability to colonize roots of different plant species including N. attenuata, thereby initiating a mutualistic interaction resulting in plant growth promotion (Sahay and Varma, 1999; Barazani et al., 2005; Achatz et al., 2010; Fakhro et al., 2010). The results demonstrated that HSPRO is not involved in the regulation of traits associated with direct defense responses against M. sexta herbivory or performance of Pst DC3000 during infection, but is a negative regulator of N. attenuata early seedling growth stimulated by P. indica.

RESULTS

Sequence and Localization Analyses of N. attenuata HSPRO

From the serial analysis of gene expression analysis published recently by Gilardoni et al. (2010), a 26-nucleotide tag (UniTag-6205) was identified as a tag whose abundance was 18-fold enriched in leaves of N. attenuata plants within 30 min of elicitation with the fatty acid amino acid conjugate (FAC) 18:3-Glu. The full-length complementary DNA corresponding to UniTag-6205 was obtained by 5′ and 3′ RACE and it was found to encode for an open reading frame of 1,437 bases and for a predicted polypeptide of 478 amino acids (Mr: 54 kD). The protein presented 45% to 70% amino acid sequence identity to the sugar beet Hs1pro-1 protein and to several homologs in other plant species including HSPRO1 and HSPRO2 from Arabidopsis (Fig. 1A; Supplemental Figs. S1 and S2). For consistency with the Arabidopsis nomenclature, we named the N. attenuata homolog HSPRO. The phylogenetic analysis of the 21 closest homologs of HSPRO from different plant species found in GenBank showed that amino acid similarity between paralogs was higher than between orthologs, with sequences from legumes, monocotyledonous, and Arabidopsis species clustering together (Fig. 1B). Interestingly, HSPRO sequences from monocots clustered closer to sequences from the moss Physcomitrella patens than did to sequences from dicots (Fig. 1B), thus not following the phylogeny of the taxa shown. In silico analysis predicted cytosolic localization for HSPRO and this prediction was confirmed by expressing an HSPRO-enhanced GFP (EGFP) C-terminal fusion protein in leaf protoplasts and analysis by fluorescence microscopy (Fig. 1C).

Figure 1.
Figure 1.

Analysis of HSPRO amino acid sequence and cellular localization. A, Schematic protein sequence alignment of N. attenuata HSPRO (JQ354963), Arabidopsis HSPRO1 (At2g4000) and HSPRO2 (At3g55840), and B. procumbes Hs1pro-1 (U79733 plus DQ148271). The cartoon above the sequences shows the percentage of similarity (green bars within the overlapping regions represent identical amino acids in the four sequences). See Supplemental Figure S2 for a detailed amino acid alignment. B, Phylogenetic analysis of HSPRO proteins from different organisms. The tree was constructed using the Geneious Pro software (5.3.4) with the Jukes-Cantor genetic distance model and the neighbor-joining tree building method with bootstrapping (602 random seed, 100 replicates, and 50% support threshold). See Supplemental Figure S1B for a reference to accession numbers. C, Arabidopsis mesophyll protoplasts were isolated and transiently transfected with vectors carrying either EGFP alone (cytosolic localization), EGFP C-terminal fusions with REPRESSOR OF ga1-3 (RGA-EGFP; nuclear localization), and EGFP C-terminal fusions with HSPRO (HSPRO-EGFP) under regulation of the cauliflower mosaic virus 35S promoter. After transfection, protoplasts were incubated for 15 h in the dark at room temperature and images were taken with a Zeiss Axioplan fluorescence microscope with standard settings for EGFP.

HSPRO Expression Is Induced by Different Biotic Stress-Associated Treatments

Analysis of HSPRO mRNA levels in N. attenuata plants showed that this gene was not only differentially induced by 18:3-Glu but also by M. sexta and Spodoptera exigua oral secretions (OS) in leaves (with M. sexta OS being the strongest inducer: approximately 12-fold) when compared with wounding (control treatment; Fig. 2A). FAC elicitation induces a strong jasmonic acid (JA) burst in N. attenuata plants (Kallenbach et al., 2010) and analysis of HSPRO expression in plants deficient in either JA (ir-lox3) and JA-Ile (ir-jar4/6) production or JA signaling (ir-coi1) showed that the induction of HSPRO was negatively affected by JA production (i.e. increased HSPRO mRNA accumulation in ir-lox3 plants compared with the wild type [control]) but not affected in plants deficient in JA-Ile accumulation or CORONATINE-INSENSITIVE1 (COI1) expression (Fig. 2B). Moreover, the induction of HSPRO expression depended on SA-induced protein kinase, a known regulator of JA-mediated responses in N. attenuata (Wu et al., 2007; Supplemental Fig. S3). HSPRO mRNA levels were induced 2.5-fold after 12 h of Pst DC3000 infection and 30-fold after 1 h of exogenous SA treatment (Fig. 2C; Supplemental Fig. S3) whereas they were not induced by Agrobacterium tumefaciens infection compared with control treatment (Fig. 2C). Thus, similar to other plant species, HSPRO responded to multiple biotic-stress-associated stimuli.

Figure 2.
Figure 2.

Analysis of HSPRO expression in wild-type and transgenic N. attenuata plants. The levels of HSPRO mRNA were analyzed by qPCR in leaves of wild-type and transgenic N. attenuata plants after different treatments and in different plant organs and tissues. mRNA levels are expressed relative to the levels of the reference gene Na-EF1A. Quantification was performed by the comparative cycle threshold method (n = 3–6; bars = ±se). A, Elicitation of leaves from wild-type plants with OS from M. sexta and S. exigua larvae, synthetic 18:3-Glu, or wounding. One way-ANOVA with Tukey’s post-hoc test (M. sexta OS versus wounding); ***, P < 0.001. B, Elicitation of leaves from wild-type and transgenic lines with synthetic 18:3-Glu. One way-ANOVA with Tukey’s post-hoc test (ir-lox3 versus the wild type); **, P < 0.01. C, Infection of leaves from wild-type plants with Pst DC3000 and A. tumefaciens (GV3101). One-way ANOVA with Tukey’s post-hoc test (Pst DC3000 versus control); **, P < 0.01; ***, P < 0.001. D, HSPRO mRNA levels in different organs and tissues of wild-type N. attenuata plants. Relative levels of HSPRO mRNA in roots were set arbitrarily to 1. One-way ANOVA with Tukey’s post-hoc test (roots versus other tissues); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Analysis of tissue-specific expression showed that HSPRO was ubiquitously expressed with the highest levels of expression in flower parts, in particular the corolla (Fig. 2D).

Generation of N. attenuata Plants with Stably Reduced Levels of HSPRO Expression

To examine in further detail the function of HSPRO, stably transformed N. attenuata plants with reduced expression of this gene were generated by inverted-repeat-mediated RNA interference (see “Materials and Methods” for a detailed description of the generation of these plants). Two homozygous independently transformed lines, named ir-hspro1 and ir-hspro2, were selected and used for all the experiments described below. These lines harbored a single T-DNA insertion in their genomes (Fig. 3A) and the levels of HSPRO mRNA were reduced on average by 93% (ir-hspro1) and 95% (ir-hspro2) compared with wild-type plants after 18:3-Glu elicitation (a condition that maximizes HSPRO expression; Fig. 3B). A third line, ir-hspro3 harbored two T-DNA insertions in its genome (Figs. 3A) and it was used only for a selected number of experiments. The levels of HSPRO mRNA in this line were reduced by 91% compared with the wild type (Fig. 3B).

Figure 3.
Figure 3.

Characterization of ir-hspro plants. A, Southern-blot analysis of the ir-hspro transgenic lines. Genomic DNA from four independent ir-hspro N. attenuata lines (1–4) and wild-type plants was digested with EcoRV and resolved by agarose gel electrophoresis. A 32P-labeled fragment corresponding to the hygromycin resistance gene nptII was used as a probe. The white arrows point to individual T-DNA insertions (lane 2: ir-hspro1; lane 3: ir-hspro2; lane 4: ir-hspro3). B, Analysis of HSPRO mRNA levels in leaves of ir-hspro lines at 1 h after 18:3-Glu elicitation (n = 6; bars = ±se). Relative mRNA levels were quantified as detailed in caption of Figure 1. One-way ANOVA with Tukey’s post-hoc test (wild type versus ir-hspro); ***, P < 0.001. C, Morphology of wild-type and ir-hspro plants at the late elongation stage. D, Rosette growth curve (measured as rosette diameter) of wild-type and ir-hspro plants (n = 8–20; bars = ±se). [See online article for color version of this figure.]

The growth and morphology of ir-hspro plants grown under standard chamber and glasshouse conditions were indistinguishable from those of the wild type at all stages of development (Fig. 3, C and D; see also below).

HSPRO Does Not Affect Defense Responses against M. sexta Herbivory and Pst DC3000 Infection

Based on the strong expression response of HSPRO to M. sexta OS and FACs (Fig. 2A), we first assessed whether ir-hspro plants were more susceptible to the attack of M. sexta larvae. The results showed that the performance of these larvae (evaluated as the gain of body mass as a function of time) was similar between ir-hspro and wild-type plants (Supplemental Fig. S4A). Consistently, the quantification of the JA-inducible defense metabolites nicotine, chlorogenic acid (3-O-caffeoylquinic acid), and rutin (quercetin-3-O-rutinoside) after elicitation with M. sexta OS showed that their amounts were similar in leaves of ir-hspro and wild-type plants (Supplemental Fig. S4, B–D).

Second, based on the high levels of HSPRO expression in flower parts and in particular the corolla (Fig. 2D), we assessed whether this gene participates in the regulation of traits (defensive and nondefensive) associated with the interaction of insects with flowers. The analyzed traits were: (1) the production of benzyl acetone in corollas, (2) the volume of nectar in flowers, (3) the amount of nicotine in nectar, (4) the amount of sugar in nectar, and (5) the level of trypsin protease inhibitor activity in ovaries and anthers. These traits were quantified in wild-type and ir-hspro plants grown either under control conditions or under the attack of M. sexta caterpillars for 15 consecutive days. All samples were collected at the end of the treatment (15 d). The results showed that the analyzed traits did not differ between ir-hspro and wild-type plants (Supplemental Fig. S5).

Finally, based on the induction of HSPRO mRNA levels by SA and Pst DC3000 infection (Fig. 2C), ir-hspro and wild-type plants were infected with this pathogen by leaf infiltration. In two independent experiments, the number of colony forming units (CFU) retrieved per area of infected leaves was similar between wild-type and ir-hspro plants at 24, 48, and 72 h postinfection (data not shown).

HSPRO Participates in the Mechanisms That Regulate Growth Promotion by the Fungus P. indica

The induction of HSPRO mRNA levels by diverse biotic-related stresses prompted us to investigate the interaction of ir-hspro plants with the growth-promoting fungus P. indica. This fungus has the capacity to establish symbiotic associations with roots of a broad range of plant species including N. attenuata (Varma et al., 1999; Barazani et al., 2005; Waller et al., 2005; Qiang et al., 2011). To study the interaction between ir-hspro seedlings and P. indica, we used a previously described plate system (Camehl et al., 2011; Fig. 4A). In this system, the hyphae reached the seedling’s roots between day 4 and 5 after the start of the experiment (i.e. transfer of seedlings to plates containing P. indica; see “Materials and Methods” for a detailed description of the system used). Unless noted, all the experiments were conducted with seedling tissue harvested at day 14 after the start of the experiment. In wild-type seedlings, HSPRO transcripts were undetected in roots of control treatment but strongly induced upon P. indica colonization (Fig. 4B). In colonized roots of ir-hspro seedlings, the level of this mRNA was also increased but it remained at less than 8% of wild-type levels (Fig. 4B).

Figure 4.
Figure 4.

Induction of differential growth promotion of ir-hspro seedlings by P. indica. A, Plate system used for the experiments. Five days after germination in standard agar media, two seedlings were transferred onto a nylon mesh covering an agar plate at 1 cm distance from a plug transferred from a 2-week-old P. indica culture. B, Analysis of HSPRO mRNA levels in roots of wild-type and ir-hspro seedlings during interaction with P. indica and in control treatment (absence of P. indica). Root samples were harvested at day 14 (n = 3; bars = ±se) and mRNA levels were quantified as detailed in caption of Figure 1. b.d., Below detection limit. C to E, Determination of the fresh biomass of total seedlings, shoots, and roots was performed with a microbalance after 14 d of seedling growth on the plate system (n = 18–20; bars = ±se); one-way ANOVA with Tukey’s post-hoc test (wild type versus ir-hspro during P. indica colonization); **, P < 0.01; ***, P < 0.001. F, Quantification of P. indica root colonization. DNA was extracted from roots of wild-type and ir-hspro seedlings colonized by P. indica at day 14 and fungal colonization was determined by qPCR based on the relative abundance of the π-EF1a gene compared with the Na-EF1a gene (n = 16–18; bars = ±se). b.d., Below detection limit. [See online article for color version of this figure.]

The quantification of root, shoot, and seedling fresh biomasses showed that P. indica-colonized ir-hspro seedlings gained on average 30% more biomass than P. indica-colonized wild-type seedlings (Fig. 4, C–E). This experiment was repeated seven times with consistent results (Supplemental Table S1). The differential growth promotion of ir-hspro seedlings varied between 15% and 73% depending on the experiment and the length of the incubation period (10 or 14 d) with an average growth promotion of 32% (Supplemental Table S1). Thus, in addition to the growth promotion effect of P. indica observed on wild-type seedlings (Fig. 4, C–E; Supplemental Table S1), there was an enhanced differential growth promotion on ir-hspro seedlings. The difference in growth was maintained as the seedlings were transferred to soil and grown in the glasshouse for maturation. In this case, the rosette diameter was determined as a parameter of growth (Supplemental Fig. S6A). At the end of the rosette expansion period (i.e. start of reproductive phase [bolting]), the rosette diameter was similar between the two genotypes (Supplemental Fig. S6A). A higher percentage of ir-hspro plants bolted 1 d earlier than the wild type (25% and 42% of ir-hspro1 and ir-hspro2, respectively) and the rate of stalk elongation and flowering time were similar between the genotypes (Supplemental Fig. S6, B–D). These results showed that the growth of P. indica-colonized ir-hspro seedlings was primarily accelerated during the early stages of seedling growth without consequences for the final plant size at the mature stage.

To analyze if the differential growth promotion of ir-hspro seedlings during interaction with P. indica was the result of a differential assimilation of CO2 produced by the fungus, ir-hspro and wild-type seedlings and P. indica were grown together but physically separated from one another in a three-sector split-plate system (Supplemental Fig. S7A). This setting allowed for the exchange of CO2 between organisms in the absence of physical contact. No differential growth promotion was observed between wild-type and ir-hspro seedlings in this experiment (Supplemental Fig. S7, B–D), indicating that physical interaction between roots and P. indica was required to differentially stimulate the growth of ir-hspro seedlings.

Analysis of P. indica-Root Interactions and P. indica-Induced Changes in Phytohormone Levels

Microscopy analysis of P. indica-colonized roots of wild-type and ir-hspro seedlings at two different times (days 7 and 14 of the plate system) showed a close association between roots and the fungal hyphae (Supplemental Fig. S8). Similar to other plant species (Varma et al., 1999; Stein et al., 2008; Schäfer et al., 2009a; Lee et al., 2011; Zuccaro et al., 2011), the fungus colonized the maturation zone of the root without a strong association with the elongation zone and root tip (Supplemental Fig. S8). Also similar to previous observations performed with N. attenuata seedlings and Sebacina vermifera (a closely related Sebacinales species; Barazani et al., 2005), we could not detect fungal structures in the roots characteristic of endomycorrhiza (e.g. arbuscules and intracellular vesicles). Root growth and hair density after P. indica colonization were not different between wild-type and ir-hspro seedlings (Supplemental Fig. S9) and the number of secondary roots per seedling was also similar between genotypes (wild type: 5.25 ± 0.33; ir-hspro1: 4.50 ± 0.36; ir-hspro2: 4.92 ± 0.23; n = 12). Quantification of P. indica root colonization by quantitative amplification of the P. indica EF1A gene (Deshmukh et al., 2006) showed a lower tendency of root colonization of ir-hspro seedlings compared with wild-type seedlings, however the differences were not statistically significant (Fig. 4F). Hence, the differential growth promotion of ir-hspro seedlings was not associated with increased P. indica root colonization or root growth.

During colonization of Arabidopsis roots by P. indica, the regulation of root cell death by the fungus plays an important role (Jacobs et al., 2011; Qiang et al., 2011). When roots of N. attenuata wild-type and ir-hspro seedlings were analyzed for cell death by trypan blue staining in both the absence and presence of P. indica (at day 14 on the plate system), no differences in the staining pattern were observed between plant genotypes (Supplemental Fig. S10). It has been reported that the interaction of P. indica and closely related Sebacinales species with roots involves changes in phytohormone accumulation and signaling (Barazani et al., 2007; Stein et al., 2008; Vadassery et al., 2008; Schäfer et al., 2009b; Camehl et al., 2010). Quantification of JA, SA, abscisic acid (ABA), and ethylene (ET) levels in P. indica-colonized wild-type and ir-hspro seedlings (at day 14 on the plate system) showed that the levels of SA were reduced approximately 2-fold by root colonization but they did not differ between genotypes (Supplemental Fig. S11A). JA and ABA levels were not affected by root colonization while ET levels were induced. However, the levels of these phytohormones were similar between genotypes (Supplemental Fig. S11B; data not shown). Interestingly, the levels of colneleic acid (CA), a divinyl-ether (DVE) derived from the action of 9-lipoxygenase (9-LOX), were strongly induced (from less than 1 nmol g fresh weight−1 to 40 nmol g fresh weight−1) by P. indica in roots of both ir-hspro and wild-type seedlings (Supplemental Fig. S11C). These results suggested that similar to other plant species (Camehl et al., 2010; Jacobs et al., 2011; Leon-Morcillo et al., 2012), some defense-associated responses were triggered by P. indica in N. attenuata roots.

Gene Expression Profiling of ir-hspro Roots Reveals Significant Changes in Metabolic Processes during P. indica Colonization

To gain further insight into the mechanisms affected in ir-hspro plants, changes in gene expression in roots of ir-hspro and wild-type seedlings were analyzed. RNA was isolated from roots of wild-type and ir-hspro seedlings grown for 14 d on the plate system either in the absence or presence of P. indica, and changes in gene expression were evaluated with an Agilent custom array containing 43,533 N. attenuata probes (Gilardoni et al., 2011). This array represented approximately 70% to 80% of the N. attenuata transcriptome (Gase and Baldwin, 2012). Genes were considered to be differentially regulated when log2 (fold-changes [FCs]) were larger or equal to 1 or smaller or equal to −1 (ir-hspro versus the wild type) and q values were lower than 0.05 (corresponding to a false discovery rate less than 5%). Using these conditions, transcripts corresponding to 11 genes were differentially expressed in control roots of ir-hspro seedlings (Fig. 5A; Supplemental Table S2) while 417 genes were differentially expressed in P. indica-colonized roots of ir-hspro seedlings (Fig. 5B; Supplemental Table S2). In control roots, nine transcripts were up- and two down-regulated while in colonized roots, 293 transcripts were up-regulated and 124 were down-regulated (Fig. 5, A and B). Eight genes (all of unknown function) were differentially up-regulated in both control and P. indica-colonized roots (Fig. 5C; Supplemental Table S2).

Figure 5.
Figure 5.

Microarray and Gene Ontology (GO) analysis of differentially expressed genes in P. indica-colonized roots of ir-hspro and wild-type seedlings. A, Distribution of FC of genes expressed differentially in roots of ir-hspro seedlings (ir-hspro versus the wild type). B, Distribution of FC of genes expressed differentially in roots of ir-hspro seedlings colonized by P. indica (ir-hspro versus the wild type). C, Venn diagram of the number of genes differentially expressed in control and colonized roots of ir-hspro seedlings compared with the wild type. The number in the intersection represents the genes differentially expressed in the two groups. D and E, Annotated genes differentially expressed in P. indica-colonized roots of ir-hspro seedlings were categorized based on biological processes (D) and molecular function (E), using the Blast2Go software. The numbers between brackets represent the percentage (%) of genes in the metabolism category (M) or in the stress response category (RS). [See online article for color version of this figure.]

In P. indica-colonized roots and based on the biological process, 60.6% of the annotated genes were involved in metabolic processes while 18.1% in responses to stimuli (Fig. 5D). Analysis of enzyme codes (Supplemental Tables S2 and S3), revealed that the most prevalent changes in gene expression occurred in enzymes involved in metabolic processes associated with the metabolism of starch and sugars, purines, nicotinate and nicotinamide, and membrane glycerophospholipids (Table I). Moreover, several genes involved in the transport of metabolites or ions were also affected in their expression (Table I). Gene ontology categorization by molecular function showed that genes encoding for enzymes with acyltransferase (14.2%), hydrolase (12.7%), and nucleotide binding (14.2%) activities were the most prevalent genes changing expression levels in ir-hspro roots (Fig. 5E; Table II). The changes in the expression of genes involved in metabolic processes were consistent with the differential growth rate of ir-hspro seedlings, and showed that the growth response was accompanied by significant changes in metabolic gene expression. The changes in the expression of genes involved in responses to stimuli (as the second-most prevalent group of genes; Fig. 5D) most likely reflected the processes affected in ir-hspro seedlings that were more directly connected with the interaction of roots with P. indica. The expression of several genes associated with phytohormone signaling was affected in ir-hspro roots and these included genes associated with JA (jasmonate zim-domain protein [JAZ], COI1), auxin (auxin response factor), and ABA (ABA INSENSITIVITY1b) signaling (Table II).

View this table:
Table I. List of selected genes differentially expressed in P. indica-colonized roots of ir-hspro seedlings and involved in metabolic processes
View this table:
Table II. List of selected genes differentially expressed in P. indica-colonized roots of ir-hspro seedlings and involved in regulatory processes

Interaction of P. indica with ir-hspro Roots Does Not Affect the Accumulation of Polar Metabolites

The changes in the expression of multiple genes involved in metabolic processes prompted us to investigate if the accumulation of primary and secondary metabolites was affected during the association of roots from ir-hspro seedlings with P. indica. For this purpose, we profiled small polar metabolites extracted from P. indica-colonized roots of wild-type and ir-hspro seedlings by liquid chromatography time-of-flight mass spectrometry. Root samples were harvested from seedlings grown for 14 d on the plate system and polar metabolites were extracted (see “Materials and Methods” for details). Ions were selected using the electrospray ionization interface in both positive and negative ion modes and those metabolites eluting from the liquid chromatography column between 125 and 550 s and having mass-to-charge ratio values ranging from 90 to 1,400 were selected for analysis. After data analysis (see “Materials and Methods” for a detailed description), no significant differences in the accumulation of ions in roots of ir-hspro and wild-type seedlings were detected in the negative ion mode (data not shown) and in the positive ion mode, the abundance of only three ions (out of more than 2,500 identified) changed significantly between these genotypes (Supplemental Table S4). The intensities of these ions were however low and the FCs small (between 2- and 2.8-fold down-regulated in ir-hspro roots).

Reduced HSPRO Expression in Roots Is Sufficient to Control Differential Growth Promotion in the Whole Seedling

We reasoned that if HSPRO had a general role associated with the control of growth instead of a more direct role in the control of the association of P. indica with roots, grafting experiments in which root stocks and shoot scions were reduced or not in HSPRO expression, could provide important information about the function of this gene. Hence, shoot scions and root stocks from either wild-type or ir-hspro seedlings were reciprocally grafted (Fig. 6A) and the root, shoot, and seedling biomasses were quantified after 19 d of seedling growth in either the presence or absence of P. indica. A differential growth promotion was observed (compared with the wild type/wild-type seedlings) in all cases in which either the root stock or the shoot scion belonged to ir-hspro seedlings (Fig. 6, B–D). This differential growth promotion was similar to the grafted parental seedlings (ir-hspro-1/ir-hspro-1 and ir-hspro-2/ir-hspro-2; Fig. 6, B–D).

Figure 6.
Figure 6.

Reciprocal grafting of ir-hspro and wild-type seedlings and determination of seedling biomass during root colonization by P. indica. A, Scheme of the grafting combinations used. B to D, Determination of the fresh biomass of total seedlings, shoots, and roots was performed with a microbalance after 19 d of seedling growth on the plate system. One way-ANOVA with Tukey’s post-hoc test (wild type versus ir-hspro during P. indica colonization); *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 18–20; bars = ±se).

DISCUSSION

As mentioned above, the function of Hs1pro-1 in sugar beet was originally associated with resistance to cyst nematodes, however, several subsequent studies performed in different plant species have suggested that homologs of this gene have a more general role in the plant’s response to environmental stresses (see the first section for references). Consistent with the observation that HSPRO homologs are induced by multiple stresses in Arabidopsis, we found that N. attenuata HSPRO mRNA levels were induced by multiple biotic-stress-associated stimuli including simulated lepidopteran herbivory, SA application, Pst DC3000 infection, and P. indica root colonization (Figs. 2 and 4B; Supplemental Figs. S3B and S9).

N. attenuata HSPRO Is a Negative Regulator of Seedling Growth Induced by P. indica

Microarray analysis of P. indica-colonized roots showed that silencing HSPRO expression brought about significant changes in gene expression and that the largest fraction (approximately 60%) of these genes were involved in metabolic processes (Fig. 5; Table I). These changes in gene expression were consistent with the accelerated growth of ir-hspro seedlings; increased growth rates are accompanied by increased metabolic rates to meet growth demands (e.g. cell walls and cellular membranes). Additionally, 18% of the genes affected in their expression in roots of ir-hspro seedlings were categorized as responses to stimuli and stresses (Fig. 5). The genes in this category probably reflected those genes having a more direct association with the interaction of roots with P. indica (Table II). In the absence of P. indica colonization, changes in gene expression in roots of ir-hspro plants were very small, with only 11 genes changing expression compared with wild-type seedlings (Fig. 5A). These results were consistent with a function of HSPRO in the control of metabolism during stress responses.

Similar to the interaction of roots from different plant species with arbuscular mycorrhizal fungi (Strack et al., 2003; Hause and Fester, 2005; Herrera-Medina et al., 2007), the interaction between P. indica and plant roots is controlled by multiple phytohomones including auxin and cytokinins (Vadassery et al., 2008), gibberellins (Schäfer et al., 2009a), ET (Barazani et al., 2007; Camehl et al., 2010), SA, and JA (Jacobs et al., 2011). Changes in the mRNA levels of auxin and ET signaling components and a cytokinin biosynthesis gene were affected in colonized roots of ir-hspro seedlings (Table II). Moreover, changes were also detected in the expression of COI1 and a JAZ homolog (Table II), two components of the JA-signaling pathway (Xie et al., 1998; Turner, 2007; Paschold et al., 2008). Together the results suggested that the phytohormone-signaling network was affected in ir-hspro plants and that these changes are probably part of the mechanisms effecting differential growth promotion of these plants during interaction with P. indica.

Differential expression of genes involved in ET and JA signaling in roots of wild-type and ir-hspro seedlings were not accompanied however by the differential accumulation of these phytohormones. The levels of JA and ABA in roots did not change during P. indica root colonization compared with uncolonized roots (Supplemental Fig. S11B) and although ET and SA levels were affected by P. indica root colonization the levels were similar between wild-type and ir-hspro seedlings (Supplemental Fig. S11A). The reduction in SA levels in P. indica-colonized roots may reflect the suppression of defense and cell death responses in N. attenuata seedlings. Lower SA levels in tobacco and Arabidopsis have been correlated with higher Glomus mosseae (Herrera Medina et al., 2003) and P. indica (Jacobs et al., 2011) root colonization, respectively. However, the accumulation of CA, a DVE strongly induced in leaves of Solanaceae species upon infection by Phytophthora spp. (Weber et al., 1999; Bonaventure et al., 2011), was also strongly induced in roots by P. indica in both ir-hspro and wild-type seedlings, indicating that oxylipin-related defense pathways were activated by this fungi (Supplemental Fig. S11C). CA is a DVE that is strongly induced in leaves of tobacco and potato (Solanum tuberosum) plants in response to pathogens such as Phytophthora parasitica and Phytophthora infestans (Weber et al., 1999; Göbel et al., 2002; Fammartino et al., 2007). It has been shown that DVEs have antimicrobial properties by, for example, inhibiting mycelial growth and spore germination of some Phytophthora species (Prost et al., 2005). Interestingly, during the interaction of G. intraradices with tomato (Solanum lycopersicum) roots, there is a strong induction of genes involved in the formation of oxylipins derived from 9-LOX activity, and it has been suggested that 9-LOX products may control arbuscular mycorrhizal fungal spread in roots (Leon-Morcillo et al., 2012).

Gene expression analysis by microarray hybridization showed only three genes directly associated with defense responses and differentially expressed in P. indica-colonized roots of ir-hspro seedlings (Supplemental Table S2). These observations were consistent with the current understanding of the mechanisms involved in the association of plant-growth-promoting rhizobacteria and fungi with roots; the activation of immune responses by plants and their suppression by microorganisms underlay the establishment of long-term mutualistic interactions (Preston, 2004; Van Wees et al., 2008; Zamioudis and Pieterse, 2012).

Grafting experiments demonstrated that the silencing of HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots of ir-hspro seedlings. Thus, the effect on shoot growth was dependent on the function of HSPRO in the roots. Because small-silencing RNAs generated in shoots of N. attenuata seedlings can be transported to roots (but not vice versa) and reduce gene expression of the targeted gene in the latter tissue (Fragoso et al., 2011), the similar effect on growth promotion observed in wild-type (shoot)/ir-hspro (root) and ir-hspro (shoot)/wild-type (root) grafted seedlings was most likely explained by shoot-induced silencing of HSPRO expression in roots of ir-hspro (shoot)/wild-type (root) grafted seedlings (Fig. 6A). Microscopy analysis of P. indica-colonized and uncolonized roots did not reveal morphological changes in roots of ir-hspro seedlings compared with the wild type, changes in the association pattern of P. indica with the maturation zone of the root, or differential root cell death (Supplemental Figs. S8–S10). The microbe-mediated stimulation of plant growth has been associated with improved plant nutrition via increased uptake of growth-limiting soil nutrients. Since P. indica root colonization and root growth was not different between ir-hspro and wild-type seedlings and seedlings were grown under conditions of high nutrient availability (media or soil), it is unlikely that increased uptake of growth-limiting nutrients was a main factor influencing the P. indica-induced differential growth promotion of ir-hspro seedlings. Barazani et al. (2005) showed that the nutritional status of N. attenuata plants colonized by S. vermifera and P. indica does not depend on N and phosphorous supply, and similar results were reported by Achatz et al. (2010) in P. indica-barley (Hordeum vulgare) symbiosis. Since growth and metabolic rates are mutually dependent, further experiments will be required to disentangle the role of HSPRO in the control of growth and/or metabolism. As mentioned in the introduction, the Arabidopsis HSPRO1 and HSPRO2 proteins interact with the AKINβγ subunit of the SnRK1 complex (Gissot et al., 2006). Thus, although speculative at this point, one possible scenario is that HSPRO affects N. attenuata seedling growth and/or metabolism via its association with SnRK1.

What’s the Role of HSPRO in Responses against Insects and Pathogenic Bacteria?

Herbivore attack elicits metabolically costly defenses that can decrease plant fitness by limiting metabolic resources otherwise invested in growth and reproduction (Schwachtje and Baldwin, 2008; Bolton, 2009). The performance of M. sexta larvae and the M. sexta-OS elicited induction of the defense-associated metabolites nicotine, chlorogenic acid, and rutin were similar between ir-hspro and wild-type plants (Supplemental Fig. S4). Moreover, flower traits associated with the interaction of plants with insects were not affected in M. sexta-attacked or -unattacked ir-hspro plants (Supplemental Fig. S5). Thus, these results suggested that HSPRO was not directly involved in the regulation of induced defenses or plant-insect association traits.

Similarly, the performance of Pst DC3000 on leaves of ir-hspro plants was unaffected compared with wild-type plants. These results contrasted those made with Arabidopsis, where plants disrupted in HSPRO2 expression supported increased Pst DC3000 growth in leaves (Murray et al., 2007). However, similar to HSPRO2 expression in Arabidopsis (Murray et al., 2007), N. attenuata HSPRO was also induced by SA and Pst DC3000 infection (Fig. 2; Supplemental Fig. S3), suggesting these two genes have both overlapping and divergent functions during bacterial infection in these two plant species.

Although speculative at this point, one possible scenario is that HSPRO regulates plant tolerance mechanisms against insect herbivores. In N. attenuata, the mRNA levels of GAL83 (a β-subunit of SnRK1) are rapidly repressed upon insect herbivory and it has been shown that GAL83 is involved in the regulation of tolerance to insect herbivory via the control of resource allocation in roots (Schwachtje et al., 2006). Thus, one possibility is that the HSPRO gene product in N. attenuata participates in the regulation of tolerance mechanisms associated with the regulation of SnRK1 activity.

Plant responses to insect herbivores can also involve changes in the association of roots with belowground beneficial microorganisms such as growth-promoting fungi and rhizobacteria (Bardgett et al., 1998; Pineda et al., 2010). For example, insect herbivory can affect mycorrhizal colonization (Gehring and Whitham, 1991; Gehring and Bennett, 2009) or the composition of the root-associated microbial community (Sthultz et al., 2009). Thus, although speculative at this point, but given the results obtained with P. indica, an alternative (but not excluding) scenario is that HSPRO participates in mechanisms that control the interaction of herbivore-attacked plants with belowground microorganisms (e.g. via changes in nutrient allocation and root exudates). Plants interacting with beneficial microbes can benefit from an increase in tolerance to herbivory, for example, by affecting C and N reallocation used for tissue regrowth after herbivory (Bardgett et al., 1998). Additionally, plants can also benefit via increased resistance to plant pathogens (Pineda et al., 2010). However, association with beneficial microorganisms can also reduce plant fitness by compromising induced defense responses against insect herbivores (Barazani et al., 2005). Thus, a delicate balance of interactions between roots and microorganisms is required to optimize plant fitness in nature and HSPRO may play a role in this process. These hypotheses are the focus of future research.

CONCLUSION

The results presented in this study have unraveled the important role that HSPRO has in the control of early N. attenuata seedling growth stimulated by the growth-promoting fungus P. indica. Since the effect on growth was only observed when ir-hspro seedlings were colonized by this fungus, and HSPRO expression was induced by multiple stress-associated stimuli, the results suggested that HSPRO plays an important role in growth and/or metabolism readjustment during stress responses. Although speculative, the control over metabolism during insect herbivory could involve the regulation of resource partitioning between shoots and roots and its resulting consequences in the interaction of roots with soilborne microbes. The results opened new hypotheses on how this control may be achieved, and the interaction of HSPRO with components of the SnRK1 complex appears as one potential scenario. Future work will focus on the disentangling of the HSPRO-dependent mechanisms underlying the regulation of growth/metabolism during stress responses.

MATERIALS AND METHODS

Please refer to online Supplemental Materials and Methods S1 for additional experimental details.

Plant Growth and Treatments

Seeds of the 31st generation of an inbred genotype of Nicotiana attenuata, originally collected from southwestern Utah in 1988, were used for all experiments. For glasshouse experiments, seeds from wild-type and genetically transformed plants were germinated as previously described (Krügel et al., 2002). Plants were grown in the glasshouse under high-pressure sodium lamps (200–300 μmol s−1 m−2 light) with a day/night cycle of 16 h (26°C–28°C)/8 h (22°C–24°C) and 45% to 55% humidity. For plate experiments with Piriformospora indica see section “P. indica Maintenance and Inoculation of N. attenuata Seedlings” below. N. attenuata ir-lox3 (Allmann et al., 2010), ir-jar4/6 (Kang et al., 2006), ir-coi1 (Paschold et al., 2008), and ir-sipk (Wu et al., 2007) plants have been previously described.

For wounding and elicitation treatments, leaves were wounded by rolling a fabric-pattern wheel three times on each side of the midvein and the wounds were supplemented either with 10 μL of 0.01% (v/v) Tween 20 in water (wounding treatment), 10 μL of 18:3-Glu (0.03 nmol/μL; FAC elicitation), or 10 μL of Manduca sexta or Spodoptera exigua OS (OS elicitation). For analysis of gene expression, Pseudomonas syringae pv tomato (Pst) DC3000 and Agrobacterium tumefaciens (GV3101) were grown on Luria-Bertani liquid medium containing 25 μg mL−1 Rifampicin until optical density at 600 nm (OD600) = 0.5 to 0.6. Bacteria were pelleted and resuspended in 10 mm MgCl2 to a final OD600 = 0.02 (107 CFU mL−1). This suspension was syringe infiltrated into leaves (1 mL leaf−1). As control treatment, leaves were infiltrated with an aqueous solution of 10 mm MgCl2. For infection assays with Pst DC3000, the bacteria pellet was resuspended in 10 mm MgCl2 to a final OD600 = 0.001 (105 CFU mL−1) and the suspension was syringe infiltrated into leaves (1 mL leaf−1). Leaf discs of 8 mm in diameter were harvested at different times (0, 1, 2, and 3 d), ground in 0.3 mL of sterile water, and after centrifugation, one-tenth serial dilutions of the supernatant were plated out on Luria-Bertani agar plates containing 25 μg mL−1 Rifampicin. Plates were incubated for 2 d at 28°C and the CFU were counted. For SA treatment, a solution of 300 μm SA dissolved in 0.2% (v/v) Tween 20/water was used. The solvent alone was used as control treatment. Tissue expression profile of HSPRO was evaluated by collecting different plant tissues from wild-type N. attenuata plants; rosette leaves and roots were collected from 30-d-old (rosette stage) plants whereas stalks, stalk leaves, sepals, pistils, corolla, and stamens were collected from 50-d-old plants.

Generation of Stably Silenced Lines

A PCR fragment generated with primers ir-hspro-fwd and ir-hspro-rev (Supplemental Table S5) and HSPRO complementary DNA as template was subcloned using SacI and XhoI (New England Biolabs) restriction sites into the pSOL8 transformation vector (Bubner et al., 2006) as an inverted-repeat construct. This construct was used to transform N. attenuata wild-type plants using A. tumefaciens-mediated transformation and plant regeneration as previously described (Krügel et al., 2002). T1 transformed plants were analyzed for T-DNA insertion number by DNA gel-blot hybridization (see below). Segregation analysis for hygromycin resistance in T2 seedlings was performed on agar plates supplemented with hygromycin (0.035 mg mL−1). Two lines, ir-hspro1 and ir-hspro2 had a single T-DNA insertion in their genomes, and were used for most of the experiments. A third line, ir-hspro3 had two T-DNA insertions and was used for some experiments. Efficiency of gene silencing (HSPRO mRNA levels) in ir-hspro plants was evaluated by quantitative real-time PCR (qPCR; see below) after 1 h of 18:3-Glu elicitation using the primers listed in Supplemental Table S5. For Southern-blot analysis, genomic DNA from wild-type and ir-hspro plants was isolated by the cetyltrimethylammonium bromide method. DNA samples (5 μg) were digested with EcoRV (New England Biolabs) overnight at 37°C according to commercial instructions and separated on a 0.8% (w/v) agarose gel using standard conditions. DNA was blotted onto gene screen plus hybridization transfer membranes (Perkin Elmer Life and Analytical Sciences) using the capillary transfer method. A gene-specific probe for the hygromycin resistance gene hptII was generated by PCR using the primers HYG1-18 and HYG3-20 (Supplemental Table S5). The probe was labeled with [α-32P]dCTP (Perkin Elmer) using the Rediprime II kit (Amersham Pharmacia) according to commercial instructions.

P. indica Maintenance and Colonization of N. attenuata Seedlings

P. indica was maintained on Kaefer medium (a modified Aspergillus spp. minimal medium; Pham et al., 2004) containing 1% (w/v) agar. For seedling colonization, 9-cm discs of polyamide mesh (pore 70 μm/thickness 80 μm; SEFAR GmbH) were placed on top of 9-cm agar plates containing modified plant nutrient culture medium [5 mm KNO3, 2 mm MgSO4, 2 mm Ca(NO3)2, 0.01 μm FeSO4, 70 μm H3BO3, 14 μm MnCl2, 0.5 μm CuSO4, 1 μm ZnSO4, 0.2 μm Na2MoO4, 0.01 μm CoCl2, 10.5 g L−1 agar, pH 5.6, 0.6% (w/v) agar]. Two 7-d-old N. attenuata seedlings germinated on Gamborg’s B5 medium (pH: 6.8; 0.6% [w/v] agar) were laid on the polyamide discs at a distance of 1 cm from an agar plug placed in the center of the plate and containing a 2-week-old P. indica culture (Fig. 4A). Agar plugs without fungus were used as control. The plates were incubated horizontally for 10 or 14 d at 21°C and light was supplied from the side for 16 h d−1 with a white fluorescent light source (80 μmol m−2 s−1). The fresh biomass of total seedlings, roots, and shoots was determined with a microbalance. Seedling grafting was performed as previously described (Fragoso et al., 2011). After grafting, seedlings were first kept for 5 d on Gamborg’s B5 medium containing 0.8% (w/v) agar for recovery and were then transferred to agar plates covered with polyamide mesh discs and preincubated (7 d before) with P. indica agar plugs. The fresh biomass of total seedlings, roots, and shoots was determined in this case 19 d after the transferring of the seedlings to the P. indica-containing plates (due to the slower growth of grafted seedlings compared with intact seedlings).

qPCR

Total RNA was extracted using the TRIzol reagent (Invitrogen) and 5 μg of total RNA were reverse transcribed using oligo(dT)18 and SuperScript reverse transcriptase II (Invitrogen). qPCR was performed with a Mx3005P Multiplex qPCR system (Stratagene) and the qPCR core kit for SYBR Green I (Eurogentec). Relative quantification of HSPRO mRNA levels was performed by the comparative cycle threshold method using the elongation factor 1A (Na-EF1A) mRNA as an internal standard (Gilardoni et al., 2010). The sequences of the primers used for qPCR are listed in Supplemental Table S5. All the reactions were performed using the following qPCR conditions: initial denaturation step of 95°C for 10 min, followed by 40 cycles each of 95°C for 15 s and 60°C for 1 min, with a final extension step of 95°C for 15 s and 60°C for 30 s. All samples were obtained from at least three independent biological replicates (n = 3) for each time point, plant genotype, and treatment.

For quantification of P. indica colonization rates, DNA was extracted from P. indica-colonized roots and control roots by the cetyltrimethylammonium bromide method. qPCR was performed using SYBR Green and 20 ng of isolated DNA as template. Copy number of the P. indica translation elongation factor 1a (π-EF1A) gene (Deshmukh et al., 2006) relative to the Na-EF1A gene was used to quantify colonization rates of P. indica by the comparative cycle threshold method. The primers used are listed in Supplemental Table S5.

Statistical Analysis

Statistics were calculated using the SPSS software version 17.0. The data were subjected to one-way ANOVA (and means were compared by the Tukey’s post-hoc test). For analysis of differences in bolting and flowering time the Kolmogorov-Smirnov test was used. The number of replicates (n) used in each experiment are detailed in the figures’ captions.

Data from this article can be found under the following accession numbers: Na-HSPRO (JQ354963; GenBank database), Agilent Chip platform (GPL13527; National Center for Biotechnology Information Gene Expression Omnibus database), microarray data (GSE35086; National Center for Biotechnology Information Gene Expression Omnibus database).

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

The authors would like to thank Dr. J. Wu for supplying Gateway cloning material and for his assistance during cloning and protoplast transfection, V. Fragoso for her help with seedling grafting, and A. Wissgott for her help with microarrays.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gustavo Bonaventure (gbonaventure{at}ice.mpg.de).

  • www.plantphysiol.org/cgi/doi/10.1104/pp.112.203976

  • ↵1 This work was supported by the Deutsche Forschungsgesellschaft (grant nos. BO3260/3–1 and 3–2) and the Max Planck Society.

  • ↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.

  • ↵[OA] Open Access articles can be viewed online without a subscription.

  • ↵[W] The online version of this article contains Web-only data.

Glossary

C
carbon
N
nitrogen
SA
salicylic acid
FAC
fatty acid amino acid conjugate
CFU
colony forming units
ET
ethylene
ABA
abscisic acid
CA
colneleic acid
DVE
divinyl-ether
9-LOX
9-lipoxygenase
FCs
fold-changes
EGFP
enhanced GFP
qPCR
quantitative real-time PCR
JA
jasmonic acid
OS
oral secretions
Pst
Pseudomonas syringae pv tomato
OD600
optical density at 600 nm
  • Received July 20, 2012.
  • Accepted August 14, 2012.
  • Published August 14, 2012.

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HSPRO Controls Early Nicotiana attenuata Seedling Growth during Interaction with the Fungus Piriformospora indica
Stefan Schuck, Iris Camehl, Paola A. Gilardoni, Ralf Oelmueller, Ian T. Baldwin, Gustavo Bonaventure
Plant Physiology Oct 2012, 160 (2) 929-943; DOI: 10.1104/pp.112.203976
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