HSPRO controls early Nicotiana attenuata seedling growth during interaction with the

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 Hs1 pro-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 (WT), HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv. tomato (Pst 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 Pst 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 to WT. 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 WT 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.


Introduction
Nicotiana attenuata is a wild annual tobacco plant native to the deserts of Southwestern US and it germinates after fires from long-lived seed banks to form monocultures in post-fire nitrogen-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 non-beneficial 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., 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 carbon (C), nitrogen (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 SuperSAGE (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-glutamic acid (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 (PNRP) or HSPRO based on their homology to Hs1 pro-1 from Beta procumbens (sugar beet) (Cai et al., 1997).
stresses. For example, the Arabidopsis thaliana genome encodes for two homologs of B.
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.

Sequence and localization analyses of N. attenuata HSPRO
From the SuperSAGE analysis published recently by Gilardoni et al (2010), a 26-nt 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 cDNA corresponding to UniTag-6205 was obtained by 5' and 3' rapid amplification of cDNA ends (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 (molecular weight: 54 kDa). The protein presented 45% to 70% amino acid sequence identity to the sugar beet Hs1 pro-1 protein and to several homologs in other plant species including HSPRO1 and HSPRO2 from Arabidopsis ( Fig. 1a and Suppl. 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 patents 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-EGFP (Enhanced Green Fluorescent Protein) C-terminal fusion protein in leaf protoplasts and analysis by fluorescence microscopy (Fig. 1c).

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 S. exigua OS in leaves (with M. sexta OS being the strongest inducer: ~12-fold) when compared to wounding (control treatment; Fig. 2a). FAC elicitation induces a strong 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 to WT (control)) but not affected in plants deficient in JA-Ile accumulation or COI1 expression (Fig. 2b). Moreover, the induction of HSPRO expression depended on SIPK (Salicylic acid Induced Protein Kinase), a known regulator of JA-mediated responses in N. attenuata (Wu et al., 2007)(Suppl. Fig. S3). HSPRO mRNA levels were induced 2.5-fold after 10 h of Pst DC3000 infection and 30-fold after 1 h of exogenous SA treatment ( Fig. 2c and Suppl. Fig. S3) whereas they were not induced by Agrobacterium tumefaciens infection compared to control treatment (Fig. 2c). Thus, similar to other plant species, HSPRO responded to multiple biotic stress-associated stimuli.
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 (ir-RNAi; 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 to WT 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 to WT (Fig. 3b).
The growth and morphology of ir-hspro plants grown under standard chamber and glasshouse conditions were indistinguishable from those of WT at all stages of development (Figs. 3c and 3d; 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 WT plants (Suppl. 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 WT plants (Suppl.

Piriformospora 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 Piriformospora 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 WT 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 WT levels (Fig. 4b).
The quantification of root, shoot and seedling fresh biomasses showed that P. indicacolonized ir-hspro seedlings gained on average 30% more biomass than P. indica-colonized WT seedlings (Fig. 4c-e). This experiment was repeated seven times with consistent results (Suppl. Table SI). The differential growth promotion of ir-hspro seedlings varied between 15 to 73% depending on the experiment and the length of the incubation period (10 or 14 days) with an average growth promotion of 32% (Suppl . Table SI). Thus, in addition to the growth promotion effect of P. indica observed on WT seedlings (Fig. 4c-e and Suppl. Table SI), 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 (Suppl. 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 (Suppl. Fig. S6a). A higher percentage of ir-hspro plants bolted one day earlier than WT (25% and 42% of ir-hspro1 and ir-hspro2, respectively) and the rate of stalk elongation and flowering time were similar between the genotypes (Suppl. Fig. S6b-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 CO 2 produced by the fungus, ir-hspro and WT seedlings and P. indica were grown together but physically separated from one another in a three-sector split-plate system (Suppl. Fig. S7a). This setting allowed for the exchange of CO 2 between organisms in the absence of physical contact. No differential growth promotion was observed between WT and ir-hspro seedlings in this experiment (Suppl. Fig. S7b-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 WT 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 (Suppl. Fig. S8). Similar to other plant species (Varma et al., 1999;Stein et al., 2008;Schafer et al., 2009;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 (Suppl. 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 WT and ir-hspro seedlings (Suppl. Fig. S9) and the number of secondary roots per seedling was also similar between genotypes (WT: 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 to WT 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 WT 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 (Suppl. 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;Schafer et al., 2009;Camehl et al., 2010). Quantification of JA, SA, ABA and ethylene (ET) levels in P. indica-colonized WT and ir-hspro seedlings (at day 14 of the plate system) showed that the levels of SA were reduced ~2-fold by root colonization but they did not differ between genotypes (Suppl. 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 (Suppl. Fig. S11b and data not shown).

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 WT seedlings were analyzed. RNA was isolated from roots of WT and ir-hspro seedlings grown for 14 days 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 log 2 (fold-changes; FCs) were larger or equal to 1 or smaller or equal to -1 (ir-hspro vs. WT) and q-values were lower than 0.05 (corresponding to a false discovery rate (FDR) less than 5%). Using these conditions, transcripts corresponding to 11 genes were differentially expressed in control roots of ir-hspro seedlings ( Fig. 5a and Suppl. Table SII) while 417 genes were differentially expressed in P. indica-colonized roots of ir-hspro seedlings ( Fig. 5b and Suppl. Table SII). In control roots, nine transcripts were up-and two down-regulated while in colonized roots, 293 transcripts were up-124 were down-regulated ( Fig. 5a and b). Eight genes (all of unknown function) were differentially up-regulated in both control and P. indica-colonized roots ( Fig.   5c and Suppl. Table SII).
In P. indica-colonized roots and based on the biological process (BP), 60.6% of the annotated genes were involved in metabolic processes while 18.1% in responses to stimuli (Fig. 5d). Analysis of enzyme codes (EC; Suppl. Tables SII and SIII), 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). GO categorization by molecular function (MF) 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 and 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], coronatineinsensitive 1 [COI1]), auxin (auxin response factor [ARF]) and ABA (abscisic acid insensitivity 1b [ABI1b]) signaling (Table II).

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 WT and ir-hspro seedlings by LC-ToF-MS (liquid chromatography-time-of-flight-mass spectrometry). Root samples were harvested from seedlings grown for 14 days on the plate system and polar metabolites were extracted (see Materials and Methods for details). Ions were selected using the ESI (electrospray ionization) interface in both positive and negative ion modes and those metabolites eluting from the LC column between 125 and 550 sec and having m/z values ranging from 90 to 1400 were selected for analysis. After data analysis (see Material and Methods for a detailed description), no significant differences in the accumulation of ions in roots of ir-hspro and WT 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 (Suppl. Table   SIV). The intensities of these ions were however low and the fold changes 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 WT or ir-hspro seedlings were reciprocally grafted (Fig. 6a) and the root, shoot and seedling biomasses were quantified after 19 days of seedling growth in either the presence or absence of P. indica. A differential growth promotion was observed (compared to the WT/WT seedlings) in all cases in which either the root stock or the shoot scion belonged to ir-hspro seedlings ( Fig. 6b to 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.   6b to d).

Discussion
As mentioned in the introduction, the function of Hs1 pro-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 Introduction 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 stressassociated stimuli including simulated lepidopteran herbivory, SA application, Pst DC3000 infection, and P. indica root colonization (Figs. 2 and 4b and Suppl. Fig. 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 (~60%) of these genes were involved in metabolic processes ( Fig. 5 and 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 to WT seedlings (Fig. 5a). These results were consistent with a function of HSPRO in the control of metabolism during stress responses.
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 WT 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 to uncolonized roots (Suppl. Fig. S11b) and although ET and SA levels were affected by P. indica root colonization the levels were similar between WT and irhspro seedlings (Suppl. 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 divinyl-ether strongly induced in leaves of Solanaceae species upon infection by Phytophthora species (Weber et al., 1999;Bonaventure et al., 2011), was also strongly induced in roots by P. indica in both ir-hspro and WT seedlings, indicating that oxylipin-related defense pathways were activated by this fungi (Suppl. Fig. S11c). CA is a DVE which is strongly induced in leaves of tobacco (N. tabacum) and potato (Solanum tuberosum) plants in response to pathogens such as Phytophthora parasitica and P. infestans (Weber et al., 1999;Gobel 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 AM fungal 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 WT(shoot)/ir-hspro(root) and ir-hspro(shoot)/WT(root) grafted seedlings was most likely explained by shoot-induced silencing of HSPRO expression in roots of irhspro(shoot)/WT(root) grafted seedlings (Fig. 6a). Microscopy analysis of P. indicacolonized and uncolonized roots did not reveal morphological changes in roots of ir-hspro seedlings compared to WT, changes in the association pattern of P. indica with the maturation zone of the root, or differential root cell death (Suppl. Figs. S8, S9 and S10). The microbemediated stimulation of plant growth has been associated with improved plant nutrition via increase uptake of growth-limiting soil nutrients. Since P. indica root colonization and root growth was not different between ir-hspro and WT 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)

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 WT plants (Suppl. 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 (Suppl. 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 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.

Conclusions
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 Suppl. Experimental Procedures S1 for additional experimental details.

Plant growth and treatments
Seeds of the 31 st generation of an inbred genotype of Nicotiana attenuata, originally

Generation of stably silenced lines
A PCR fragment generated with primers ir-hspro-fwd and ir-hspro-rev (Suppl. insertion number by DNA gel blot hybridization (see below). Segregation analysis for hygromycin resistance in T 2 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 qPCR (see below) after 1 h of 18:3-Glu elicitation using the primers listed in Suppl. Table SV 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 (Suppl. Table SV).

P. indica maintenance and colonization of N. attenuata seedlings
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 days at 21°C and light was supplied from the side for 16 h day -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 days 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 pre-incubated (7 days before) with P.
indica agar plugs. The fresh biomass of total seedlings, roots and shoots was determined in this case 19 days after the transferring of the seedlings to the P. indica-containing plates (due to the slower growth of grafted seedlings compared to intact seedlings).

Quantitative real-time PCR
Total RNA was extracted using the TRIzol ® reagent (Invitrogen, Karlsruhe, Germany) and 5 µg of total RNA were reverse transcribed using oligo(dT) 18

Accession numbers
Data from this article can be found under the following accession numbers: Na-HSPRO (JQ354963; GenBank database), Agilent Chip platform (GPL13527; NCBI GEO database), microarray data (GSE35086; NCBI GEO database).