BACKGROUND
some members of the genus trichoderma, including t. virens, t. harzianum, t. asperellum and t. atroviride, are employed as biocontrol agents of plant pathogens worldwide.
these soil fungi are keen mycoparasites, generally rhizosphere competent, and some have the ability to extensively colonize the outer root layers  <cit> .
in addition to direct parasitism of plant pathogens, interactions with trichoderma enhance plant fitness in response to biotic and abiotic stresses  <cit> .
benefits derived by the host include: a) increased plant growth  <cit> ,  increased resistance to abiotic stresses such as drought and salinity  <cit> ,  induced host defense responses to pathogens  <cit> ;  enhanced nutrient uptake and fertilizer use efficiency  <cit> , and  increased photosynthetic rates  <cit> .
a recent microarray study of two dicots showed that in the plant, genes related to tolerance of oxidative and osmotic stresses are induced by the trichoderma-plant interaction  <cit> .
growth promotion of bean plants has been found to be strain-specific  <cit> .
the combination of close interaction with plants and the ability to tolerate heavy metals make some strains of t. harzianum  <cit>  and t. virens  <cit>  effective agents for soil bioremediation and plant growth promotion.
the trichoderma-plant interaction has been defined as an opportunistic symbiosis  <cit> .
the t. asperellum – cucumber interaction was followed by electron microscopy, and growth is extracellular, with hyphae penetrating the outer root cortex  <cit> .
the interaction between t. harzianum and tomato roots was observed by confocal microscopy with a gfp-expressing strain  <cit> .
in hydroponic cultures during early colonization , hyphae were observed growing between plant cell walls, and by 24 h, the root surface was extensively colonized.
in soil, a switch to yeast-like morphology was observed following colonization.
the fungus, after 48 hours, was mainly extracellular although occasionally intracellular, and in these cases the colonized cell appears to remain viable.
after longer times of interaction in soil  the fungus produced yeast-like cells  <cit> .
in the t. virens – maize interaction, gfp-expressing hyphae are observed on the root surface and growing between cell walls in the epidermis and outer cortex, with no evidence of intracellular growth  <cit> .
the trichoderma-root interaction is not identical to any other previously studied symbiosis, but the interaction is reminiscent in some ways of ectomycorrhizae .
images of trichoderma spp.
colonizing maize  <cit>  or tomato roots  <cit>  show the colonizing mycelia as a loose and relatively sparse network, which is less sharply delineated than the massive em mantle  <cit> .
in both symbioses, mycelia penetrate the root apoplast, but it is not clear how similar are the distributions within the root.
the ectomycorrhizal fungus laccaria bicolor secretes a small protein , highly expressed during colonization of tree roots and needed to establish the symbiosis  <cit> .
missp <dig> is imported into plant cells where it interacts with a transcriptional repressor to antagonize jasmonate-induced gene expression  <cit> .
in arbuscular mycorrhizae, a plant nucleus-targeted effector counteracts the immune response by interacting with a specific plant transcription factor, allowing establishment of the biotrophic interaction  <cit> .
piriformospora indica, like trichoderma virens, has a wide host range, interacting with roots of monocots and dicots.
a recent transcriptomic study showed that this basidiomycete root endophyte tailors the expression of its genome to the host plant and to the stage of the mutualistic interaction  <cit> .
the genomes of the cellulose degrader t. reesei and two mycotrophic, plant-interacting species, t. atroviride and t. virens, have been published  <cit> .
the sequences have provided the tools for a genome-wide view of trichoderma-fungal and trichoderma-plant interactions  <cit> .
several studies have addressed the transcriptome of different trichoderma species, in interaction with a particular plant host  <cit> .
studies of trichoderma transcriptomes in interaction with plant roots, using arrays designed from trichoderma ests, showed regulation of genes related to redox reactions, transport, lipid metabolism and detoxification  <cit> ; small secreted and cell surface proteins, proteases, endochitinase ech <dig>  and novel genes that could be related to nitric oxide biosynthesis, xenobiotic detoxification, and development  <cit> ; and a predominance of carbohydrate metabolism-genes  <cit> .
these studies, which indicate that interaction with the plant host programs expression of many genes in the fungal partner, employed several trichoderma species and times of interaction with the plant host.
here, we compared the same trichoderma strain in interaction with two host plants under the same conditions, to identify host-specific transcriptomic signatures.
although the trichoderma-root interaction represents a distinct type of symbiosis, the similarities to other fungal-plant symbioses led us to the working hypotheses that  the fungal transcriptome should depend on the host plant species, providing a molecular basis for the wide host range; and,  small secreted proteins  and other secreted proteins may be important for the mutualistic interaction and for induction of disease resistance in colonized plants.
in this study we used oligonucleotide microarrays designed from the predicted protein-encoding gene models of t. virens  <cit>  to ask what genes are up-regulated at the transcriptional level, comparing the interaction with either tomato or maize roots.
the results of comparing two plant hosts, monocot and dicot, under the same growth conditions suggest that a different repertoire of genes is expressed in response to different hosts and provide us with a better understanding of this interaction.
such studies would be helpful for crop-specific application of t. virens for maximizing the benefits derived from this type of plant-microbe interactions.
this study would also be helpful in isolating novel promoters for driving expression of desirable trichoderma genes  in the rhizosphere-competent species.
methods
bioassay: seedling and fungal culture
for plant interactions, maize seeds  were treated with 70% ethanol for 5 min, rinsed with water, soaked in 10% hydrogen peroxide solution for 2 hours, rinsed with water, and germinated on moist sterile filter papers to screen for contamination .
after four to five days incubation at 27°c, five seedlings of similar root length were selected and placed on plastic screens suspended in jars containing 250 ml of half strength murashige and skoog  with vitamins and  <dig> % sucrose.
the seedlings were grown in a temperature-controlled growth room under fluorescent light   for three days at 23-25°c.
tomato seedlings were prepared by treating seeds  with 70% ethanol for 20 min, rinsing with water, soaking in 10% bleach solution for 5 min, rinsing in water, and germinated on ms supplemented with  <dig> % agar.
after seven days, five seedlings of same root length were selected for placing in the hydroponic system.
t. virens gv29- <dig> was isolated from a sandy loam soil cultivated with cotton plants in texas, and is deposited at the fungal genetics stock center .
conidia were harvested from 7-days old plates using a smear loop and final spore concentration was determined by dilution using a hemocytometer.
vogel’s minimal medium with  <dig> % sucrose was inoculated with  <dig> ×  <dig> conidia/ml, and the culture was incubated at 150 rpm on a rotary shaker for 48 hours.
the fungal biomass was harvested by filtering through miracloth and rinsed with sterile water.
one g of fungal tissue was added to each jar, and the seedlings  were further incubated for 72 hours in the presence of the fungus.
the hydroponic culture system is illustrated in figure  <dig> figure  <dig>
hydroponic system.
experimental design for the hydroponic system for maize-trichoderma co-culture at 72 h time point .
a seven-day-old maize seedlings grown aseptically in ms medium in culture chambers, inoculated with a fungal preparation of t. virens gv <dig>  and incubated for 72 h .
the strain expresses gfp under control of a constitutive promoter, and panels b, f show merged fluorescence and bright field images.
two controls were included: hydroponic systems containing only t. virens growing in ms in the absence of maize seedlings , and control plants growing in ms without t. virens
.
f and g, maize roots inoculated with t. virens at  <dig> conidia/ml ms medium.
microscope images b, d, and f were taken at 10x magnification .
sample collection and rna preparation
four treatments, with three biological replicates per treatment, were collected: maize roots with t. virens ; tomato roots with t. virens ; maize or tomato roots without fungus ; and t. virens without plant roots .
root systems from the same jar were harvested as an individual sample after 72 hours root-fungus interaction.
samples were gently washed with distilled water to remove clumps of fungal mycelia that adhered only loosely to the roots.
the roots with remaining fungal mycelia were excised from the seedlings, frozen in liquid nitrogen, and extracted directly or stored at −80°c.
control root samples without t. virens were grown and harvested as described above.
for axenic culture, t. virens was grown under the same conditions in the hydroponic culture containers, but without plants.
total rna was extracted from 100 mg of ground tissue  with tri reagent  following the manufacturer’s instructions.
the quality of the rna extracts was evaluated using the agilent  <dig> bioanalyzer and rna  <dig> nano kit  according to the manufacturer’s instructions.
samples with electropherograms exhibiting sharp 18s and 28s rrna peaks and showing no evidence of degradation were retained.
the transcript of the t. virens histone h <dig> gene was easily detectable by rt-pcr in cdna samples from t. virens and in interaction with maize or tomato, but not in control plant samples .
microarray conditions and analysis
the microarray platform was described in  <cit> .
a custom microarray was designed  from the complete set of filtered transcript models  and printed in 15 k arrays .
the array consists of  <dig> probes, including  <dig> agilent control spots, and technical replicates for  <dig> probes.
<dig> probes had the potential for cross-hybridization; these are not among the genes of interest found in this study.
the probes on the array are 60-mer sense oligonucleotides, identified by protein id numbers from v <dig>  of the database .
in some cases, update of the gene models resulted in new id numbers in v <dig> .
the new gene models were then identified here by blast search of the v <dig>  database with the v <dig>  sequences, and the corresponding v <dig>  numbers are given in the additional file 1: table s4; additional file 2: table s1; additional file 3: table s2; additional file 4: table s <dig>  to 1 μg total rna, rna was added from a spike-in kit .
each array hybridization corresponds to a single biological replicate.
in this design there are no technical replicate array hybridizations.
the array contains replicated  oligonucleotide probes, corresponding to  <dig> of the unique probes.
inspection of the data usually showed very similar signal values between duplicate array spots.
these were analysed independently by the array software .
cdna synthesis was primed with oligo dt, and the double-stranded template was used for amplification and labelling by in vitro transcription using the messageampii kit from ambion .
amplified rna  was labelled with cy <dig> and hybridized onto the custom microarrays.
three biological replicate samples were used for m + tv and t + tv, and five for tv alone.
microarray hybridization and washing steps were performed following the agilent protocol for single-channel arrays.
the arrays were scanned at 10% laser power to avoid signal saturation.
agilent’s feature extraction software was used to extract the data.
microarray signals were normalized to allow comparison of samples with different rna amounts, using the spike-in data.
first, the log <dig> microarray data were normalized so that signals for one of the spike-ins  with a log relative concentration of  <dig>  had the same values across all samples.
next, the log <dig> expression data were normalized by linearly interpolating to concentrations using the ten spike-in measurements for each sample and subsequently normalizing to the 75th percentile signal intensity.
the data were converted from log <dig> to linear values, and analyzed for significant differences between plant interaction and control using cybert  <cit>  http://cybert.ics.uci.edu/ with the options: bayes window  <dig> and bayes weight 8; baseline subtraction  <dig> ; cutoff benjamini-hochberg  at p <  <dig> .
for cluster analysis, data, as log <dig> of the microarray signal or log <dig> of the signal normalized by dividing by the average control  signal, were processed using genesis, http://genome.tugraz.at/  with default options for hierarchical clustering.
to compare est entries in the gene lists from  <cit>  and  <cit>  with our gene lists, trichoest id numbers were converted to european nucleotide archive numbers and used to search the database .
the resulting nucleotide sequences were used to search trichoderma virens g <dig>  v <dig>  at jgi , using blastx.
the t. virens genomic probes in the array of  <cit>  are identified by t. virens v <dig>  id numbers and directly identify probes on our array.
annotations were from the t. virens website .
the available annotations are listed in additional file 3: table s <dig>  along with a consensus description from which the functional categories in figure 2d were determined.
in addition, go terms for the same set of significantly regulated genes  were independently identified using blast2go .
the go terms for a control set of  <dig> sequences, starting with protein id  <dig> , were analysed in the same way.figure  <dig>
clustering and functional categories of significantly regulated genes.
a) cluster analysis  of data from individual experiments.
the log <dig> values for each microarray signal divided by the corresponding average control  signal are plotted in the heat map, clustered by relative expression level and experiment.
b) a portion of the cluster analysis, annotated by protein id number.
this magnified portion of  shows a cluster of genes up-regulated in response to both maize and tomato roots.
yellow dots indicate  <dig> of the  <dig> genes significantly up-regulated in response to both plant hosts .
c) expression patterns of genes significantly up-regulated in response to both plant hosts.
patterns are plotted for the  <dig> genes identified by cybert analysis.
d) pie chart of functional categories for the set of genes shown in a. for simplicity large categories were used; the “metabolism” category includes, for example, a large number of predicted proteins with reductase, dehydrogenase, and other enzymatic activities, as well as some secreted hydrolytic enzymes with the exception of glycoside hydrolases which are listed separately.
see additional file 3: table s <dig> for the complete annotated list.
real time qpcr
cdna for qpcr was synthesized starting from 2 μg of rna, which were treated with dnase , and then used for reverse transcription with random primers with the high capacity cdna reverse transcription kit , following the manufacturer’s protocol.
abundance of transcripts was measured by real-time qpcr reactions performed in an applied biosystems  <dig> cycler; approximately 15 ng of cdna were used as template.
the 15 μl reaction volume included  <dig>  μl of 2x absolute sybr green rox mix  and  <dig> nm final concentration of specific primers for the gene of interest.
assays were run in duplicate for each the 4– <dig> biological replicate samples, using the following protocol: initial activation at 95°c for 10 min; 40 cycles of 95°c for 15 s, 60°c for 60 s, followed by a gradual increase in temperature from 60°c to 95°c during the dissociation stage.
ct values were calculated using the applied biosystems software, and transcript abundance was calculated in microsoft excel from ct values and normalized to the tubulin  signal.
standard curves were measured for dilution series of pooled cdna samples, and calculated using the applied biosystems software; the slopes for each primer pair are given in table  <dig> table  <dig>
primer pairs used for qpcr amplification
id v <dig>
consensus annotation
forward primer
reverse primer
amplicon
slope
90504
46158
86039
17705
19757
42536
51095
51211
54057
56652
88010
the slope of the calibration line is given in the last column, measured from a dilution series of pooled cdna samples, using abi software .
the protein model for the small secreted protein  id  <dig> is id  <dig> in v <dig>  of the database.
reporter gene construct
we chose the cbh <dig> glycoside hydrolase gene  because the transcript is abundant in samples from root cultures inoculated with t. virens.
furthermore, this gene was strongly induced in interaction of t. virens with tomato roots in an earlier study  <cit> .
this suggested that the upstream region includes strong expression signals that could be used to design a construct to report gene expression in response to trichoderma-root interactions.
a 2510 bp region upstream of the predicted translation start was amplified from t. virens g <dig>  genomic dna, using the forward primer p90504f, gtagaactgaaaagcttcggtcaatc and reverse primer p90504r, caatttctgatccatggtgtacaatctttg.
the forward primer contains two mismatches, creating a hindiii site.
the reverse primer contains two mismatches, converting the atg start codon into a ncoi site.
the product was cloned into ptz57r/t  to obtain plasmid p <dig>  a 960 bp ncoi/sali fragment from ggfp  containing the gfp coding sequence was ligated  to p <dig>  which was digested with ncoi and sali, and the ligation products transformed to e. coli hit-dh5α .
five μg of the resulting plasmid, p90504-gfp, were co-transformed together with 5 μg of pucatph  to protoplasts of t. virens g <dig> , using the peg protocol as described by  <cit> .
after regeneration overnight, transformation plates were overlayed with 600 μg/ml hygromycin b  in 1% agar .
colonies reaching the surface of the overlay were transferred to pda  with 100 μg/ml hygromycin b. about  <dig> hygromycin-resistant colonies were screened by inoculating wells containing excised maize root sections in  <dig> x ms medium with  <dig> % sucrose.
after three days incubation, the wells were observed under a binocular fluorescence microscope .
three isolates that showed gfp expression were identified and one, designated c <dig>  was used for experiments.
RESULTS
microarray analysis of trichoderma-root interactions
the hydroponic culture system used to follow trichoderma-root interactions is illustrated in figure  <dig>  at the time of harvesting, adhering fungal mycelia were clearly visible on the roots of both plant hosts, as shown for maize in figure 1g.
for comparison of the fungal transcriptome in interaction with roots to that of t. virens alone, the fungus was cultured under the same conditions but without plants .
total rna for preparation of probes for microarray hybridization was prepared from material from each treatment: t. virens alone , in interaction with maize  and with tomato .
complete microarray data are provided in additional file 2: table s <dig> and are available at geo, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse <dig>  the data sets were analyzed in pairs using cybert  for significant differences in transcript abundance: m + tv compared to tv, t + tv compared to tv, and m + tv compared to t + tv.
at a benjamini-hochberg cutoff value of p <  <dig> , the levels of  <dig> transcripts were significantly up-regulated in interaction with maize, and  <dig> in interaction with tomato .
a panel of  <dig> genes, highlighted in the protein id list in additional file 3: table s <dig>  was chosen for qpcr validation.
the primer pairs used are given in table  <dig>  beta tubulin  was the reference gene for qpcr .
the expression patterns of these genes measured by qpcr or microarray hybridization were generally similar .
an exception is  <dig>  where the t. virens control signal was underestimated by qpcr compared to microarray hybridization.
the calibration slope for this primer pair indicates below-optimal efficiency , perhaps accounting for the discrepancy.
in general, the correlation between qpcr and microarray signals holds well, over several orders of magnitude .
the other exception is gh <dig>  which showed no induction in interaction with maize on the microarray, as well as in one set of qpcr experiments.
this transcript, however, was strongly up-regulated in a second set of qpcr experiments .
this is in contrast to the other  <dig> genes in the panel which showed remarkably consistent patterns in experiments performed in two different laboratories and at different times during this study.
cluster analysis was performed, according to experiment and gene name, with the group of transcript levels showing significant regulation in either root interaction .
the significantly regulated genes cluster according to experiment , but some genes deviate from the pattern in some experiments .
the sources of this variation might be sampling effects  or differences between hydroponic chambers in the time course of the trichoderma-root interaction.
as for id  <dig> , time course experiments on specific genes or the entire transcriptome may be needed to resolve these differences.
thus the number of significantly regulated genes may be an underestimate, and we limit the analysis to robustly regulated transcripts.figure  <dig>
qpcr validation of microarray data.
a) qpcr signals normalized to tubulin , and corresponding microarray data, normalized to total signal as described in methods.
bars indicate means of 3– <dig> replicates with standard error; the m + tv and t + tv samples for qpcr were independent of those used for the microarrays.
*,**,** significant at p <  <dig> ,  <dig> ,  <dig>  respectively, by ttest, one-tailed.
b) microarray data corresponding to the transcripts shown in .
*,**,** significant at p <  <dig> ,  <dig> ,  <dig>  respectively, by ttest, one-tailed.
c) correlation between qpcr and microarray signals, plotted from the data in .
the line is a power-law least squares fit  to the combined data set , which appears linear on this log-log plot.
the r <dig> value is shown on the graph.
expression of gfp under control of the upstream region from glycoside hydrolase gene  <dig>  a) roots excised from hydroponic co-cultures of p90504:gfp line c <dig> were photographed under white light or for gfp fluorescence, in a binocular microscope.
b) confocal images of roots from hydroponic co-cultures of p90504:gfp line c <dig> or wt.
green channel: gfp fluorescence, excitation 488 nm, bandpass filter 500–550 nm.
red channel: propidium iodide, excitation 561 nm, filter 575 nm long pass.
the images are projections of five  or  <dig>  z-stack  slices.
propidium iodide stains plant cell walls in the root epidermis and outer cortex layers, as well as some plant and fungal nuclei in some cells.
plant cell autofluorescence  is visible in some areas.
genes regulated in interaction with both plant hosts
the expression of 14 t. virens genes was significantly up-regulated in interaction with both maize and tomato.
of these,  <dig> were found previously, in interaction with tomato .
a cluster of genes co-regulated in interaction with both host plants is shown in figure 2b, annotated by jgi protein model id number.
seven expression patterns of genes belonging to the set significantly up-regulated in both root interactions  cluster together .
the  <dig> genes significantly up-regulated in interaction with both maize and tomato are mostly found on different scaffolds.
in the two cases where a pair of genes was found on the same scaffold, the distance between them was very large .
similar expression patterns in the three conditions , as evident in figure 2c, is therefore not reflected by co-localization in the genome.
ten of the  <dig> genes in this list are annotated as glycoside hydrolases.
seven of the eight genes found to be up-regulated by interaction with both maize and tomato in this study  and with tomato  degrade the root cell walls.
genes up-regulated in interaction with either or both plant hosts
looking at the available annotations for the larger list of genes significantly up-regulated in interaction with either plant host, glycoside hydrolases are again prominent .
another class includes transporters .
there are several predicted transcription factors and other regulatory proteins, and some small secreted proteins.
two genes are related to iron acquisition: fe permease ftr <dig>  and a ferric reductase .
a similar picture, in general, was independently obtained by go term analysis .
the highest-represented biological processes are polysaccharide catabolism, transmembrane transport, and oxidation-reduction .
an identical go term analysis using a randomly chosen set of sequences  resulted in a much more diverse representation of biological processes.
oxidation-reduction is also highly represented in the list obtained from the consensus annotation , although this is not obvious in figure 2d where oxidation-reduction is included in “metabolism”.
of the fourteen genes demonstrating significant up-regulation in response to both maize and tomato roots, nine are annotated as glycoside hydrolases.
all the glycosyl hydrolases  were grouped according to gh families from the annotation , and the total expression  plotted to obtain a semi-quantitative view of the relative contribution of each family, at the transcript level .
the plot indicates that the expression pattern is different for the interaction with maize or tomato.figure  <dig>
representation of glycoside hydrolase  families.
the bars represent the sum of average transcript abundance  for each family.
the error bars indicate sem for families with a single member represented; for families with two or more members, the sem values were combined using the sum of squares rule, to provide an approximate measure of variability.
different cell wall composition of the two plant hosts might be expected to play a role in the set of fungal genes expressed.
the predicted substrates of the fungal glycoside hydrolase families represented  do not appear, however, to be related in any direct way to the composition of maize vs tomato cell walls.
as an example, we note that dicot walls are more pectin-rich than those of monocots  <cit> .
the endopolygalacturonase encoded by transcript  <dig> is the unique member of family gh <dig> expressed in response to interaction with roots.
if the product of  <dig> is indeed the major induced pectin-degrading enzyme, one might have expected higher expression in tomato where the substrate is more abundant.
this pattern was found  <cit>  upon comparison of two colletotrichum species.
one species is a pathogen of brassicaceae, and one of maize.
c. higginsianum in the necrotrophic phase of its interaction with arabidopsis was found to up-regulate a greater number of pectin-degrading enzyme genes than c. graminicola on maize  <cit> .
our data show, however, up-regulation of the gh <dig> gene  <dig> in interaction with both tomato and maize .
dicot and grass cell walls both contain pectin, and the expression of enzyme genes will not always correspond directly with substrate levels.
the predicted ortholog  of  <dig> in t. harzianum is thpg <dig>  silencing of thpg <dig> in t. harzianum showed that it is required for colonization of arabidopsis roots  <cit> .
this could be tested for t. virens on tomato and maize.
the question of how gh family expression might be related to host cell wall composition will need to be addressed at the protein, in addition to transcript, level.
when and where each cazyme is expressed in the root tissue is also important.
genes differentially regulated in interaction with maize or tomato
the overall distribution of gene classes in the list of up-regulated genes is similar for the two plant hosts .
inspection of the microarray data showed that expression in the maize and tomato interactions often followed the same pattern, yet did not pass the significance filter in one or the other data set.
we therefore studied the set of genes whose transcript levels differ significantly at p <  <dig>  between maize and tomato, and are also significantly regulated relative to the t. virens control.
this gene list, which is the intersection of the cybert results from the above two criteria , is given in additional file 4: table s <dig>  transcripts corresponding to  <dig> gene models differed significantly in their expression levels between maize and tomato and also between the control and at least one plant interaction, with ratios of transcript abundance on tomato vs on maize ranging from  <dig>  to  <dig>  .
thus, we have identified genes that can serve as specific reporters for the interaction of t. virens with one of the two plant hosts.
in contrast to the genes significantly up-regulated in interaction with both hosts, of which half had been found previously, only five of the transcripts in additional file 4: table s <dig>  had been identified previously in the t. virens – tomato interaction  <cit>  or are homologs of genes up-regulated in the t. harzianum – tomato interaction  <cit> .figure  <dig>
venn diagram of the number of genes differentially expressed in
t. virens
interactions with maize
vs
tomato roots.
the intersection of the two sets indicates transcripts that were significantly different  in tomato compared to maize, and also significantly up-regulated compared to control  in either tomato  or maize  interactions.
expression patterns of root-regulated
t. virens
genes that are preferentially expressed in interaction with tomato  or maize .
maize-specific expression of three genes, oxidoreductases  <dig>   <dig> and ssp  <dig>  was confirmed by qpcr .
the intracellular invertase tvinv   <cit>  is specific to the maize interaction, though obviously one cannot exclude induction on tomato at a different time point from the 72 h sampled here.
the tomato-specific up-regulated transcripts  remain to be further characterized.
genes encoding small secreted cysteine-rich proteins  in the t. virens genome have been annotated based on the following criteria:  <dig> amino acids or less, at least four cysteine residues, predicted signal peptide for secretion, and lack of annotated enzyme activity  <cit> .
several genes belonging to this  class were up-regulated in interaction with maize or tomato .
up-regulation of transcript  <dig> is specific to maize, and this was validated by qpcr .
this gene in v <dig>  of the t. virens sequence database corresponds to id  <dig> in v <dig> ; in v <dig>  the protein model was incomplete, lacking the n-terminus, and thus was not included in the original ssp list compiled by kubicek et al.
<cit> .
we added the complete model  <dig> to the previous clustering analysis, and found that it belongs to cluster  <dig>  a ssp family of unknown function  <cit> .
id  <dig> is a predicted class ii hydrophobin , significantly up-regulated in response to tomato roots , and showing an up-regulated trend in the average transcript abundance in interaction with maize .
a class ii hydrophobin was induced in t. harzianum by the presence of tomato plants  <cit> .
this protein shows homology to a class ii hydrophobin from t. virens , related only more distantly to  <dig>  both t. virens proteins show the same core structure containing eight cysteine residues arranged in a conserved motif.
the ceratoplatanin family gene sm <dig>  encoding a ssp which is an inducer of systemic resistance, was up-regulated in interaction with cotton  <cit> .
samolski et al.
<cit>  found a  <dig> -fold induction in interaction with tomato, while here there was only a  <dig> -fold  increase in response to tomato roots.
the cell wall protein qid <dig>  <cit>  is unfortunately not on the microarray because the t. virens ortholog was missing from the v <dig>  gene models.
thus, direct comparison with expression of ssp genes in previous studies is not straightforward, probably due to different plant hosts and conditions.figure  <dig>
expression patterns of small secreted proteins.
the transcripts indicated by v <dig>  protein id numbers were significantly up-regulated  in either maize or tomato .
transcript levels for id  <dig>  and id  <dig> were measured also by qpcr, and the data shown here are also plotted in figure 3b, where these genes are labelled ssp and hyd <dig>  respectively.
the “cluster 3” ssp  seems a promising candidate for functional analysis as a maize-interaction-specific small secreted protein.
functional significance of regulated genes
the strong induction of genes for enzymes with the potential to degrade plant cell walls is interesting, as the interaction between t. virens and these two hosts is considered mutualistic.
this finding is consistent with previous reports  <cit> .
in the mutualistic interaction of barley with the basidiomycete piriformospora indica –, hydrolytic enzymes and transporters are strongly induced following the transition from biotrophy to saprotrophic growth on dead root cortex cells.
there is relatively little induction of these genes in the biotrophic p. indica-arabidopsis interaction  <cit> .
while a full meta-analysis is beyond the scope of this study, the similarity between p. indica – barley and the t. virens interactions studied here suggests that the t. virens – root interactions have a limited necrotrophic aspect, like p. indica – barley and in contrast to p. indica - arabidopsis.
if so, cell death should be observed, and there is preliminary evidence for this from scattered propidium iodide staining of plant and fungal nuclei .
furthermore, this similarity between p. indica – barley and the two t. virens interactions points to parallel evolution of a mutualistic, isr-promoting, fungal-root interaction distinct from mycorrhizae, in basidiomycota and ascomycota.
the genes identified here and in previous studies can provide specific reporters for the t. virens-root interaction.
to test this concept we constructed a reporter in which gfp was expressed under control of the predicted upstream regulatory region from the  <dig> gene.
transgenic lines expressing this construct showed strong gfp fluorescence in interaction with roots  and root sections .
the protein sequence of  <dig>  belonging to glycoside hydrolase  family gh <dig>  is nearly identical  to the t. virens t <dig> gene cbhi  which is up-regulated in interaction with tomato  <cit> .
in hydroponic cultures, fluorescent hyphae could be detected in the outer root layers, while externally adhering hyphae showed fluorescence in or on some hyphal compartments .
we have not confirmed penetration into root cortex cells under the conditions studied here, though gfp-expressing hyphae are closely associated with maize cells .
higher-resolution localization of t. virens hyphae in the root cortex will require a line expressing gfp at high, constitutive levels, like the t. harzianum line used in a recent study of the role of salicylic acid in limiting penetration into arabidopsis roots  <cit> .
in the t. harzianum- tomato root interaction there is evidence for intracellular hyphae or yeast-like cells  <cit> .
if the t. virens – root interaction  most resembles the late-stage  p. indica- barley interaction, induction of hydrolytic enzymes might reflect saprotrophic on dead root cells.
the majority of root cells at three days interaction, however, appear living, since their nuclei do not stain with propidium iodide .
the genome of the arbuscular mycorrhizal fungus rhizophagus irregularis lacks gh families predicted to degrade plant cell wall polysaccharides  <cit> .
in contrast, these are induced in t. virens interacting with roots .
two pectate lyase  genes are up-regulated in ectomycorrhiza, as is a gh <dig> gene .
visualization of the time course and location of expression of the gh genes during colonization should help to understand how t. virens fits into the biotroph – saprotroph – pathogen continuum.
trichoderma spp.
interact with both monocots and dicots.
different patterns of gene expression might facilitate this wide  host range.
in support of this view, we found that a number of genes were expressed specifically in response to either maize or tomato .
one of these is invertase tvinv  <cit> , expressed more than 100-fold higher on maize than on tomato.
several small secreted proteins are expressed preferentially in each interaction.
short-chain oxidoreductases appear to be characteristic of the maize interaction.
identification of the substrates of these predicted proteins, if they indeed have enzyme activity, might help identify their  role in the trichoderma-maize root interaction.
in general, there are not enough functional data to fully understand why a particular set of genes is induced in the maize or tomato mutualism.
the existence of specific reporters of each interaction, though, demonstrates specific transcriptomic signatures for the interaction with each plant.
CONCLUSIONS
this study provides a better knowledge of the crosstalk of trichoderma virens, an agriculturally relevant biocontrol agent, with plants.
genes differentially expressed during interaction with plant roots encode proteins belonging to several functional classes including enzymes, transporters and small secreted proteins.
among them, glycoside hydrolases and transporters are highlighted by their abundance and suggest an important factor in the metabolism of host cell walls during colonization of the outer root layers.
activity of the cbh <dig> promoter was reported by gfp fluorescence in a transgenic t. virens line on roots, providing a first spatial view of how the plant re-programs the fungal transcriptome.
host-specific gene expression may contribute to the ability of t. virens to colonize the roots of a wide range of plant species.
the host-specific gene list obtained here will facilitate functional experiments to test this hypothesis.
availability of supporting data
the microarray data sets supporting the results of this article are available in the geo repository, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse <dig>
additional files
additional file 1: table s <dig>  this file lists the sequences of the oligonucleotide probes printed on the t. virens microarray.
additional file 2: table s <dig>  microarray data from five biological replicates of t. virens alone , three of tv cocultured with maize roots  and three with tomato roots  are given as a text file.
the first column  indicates the protein model id from the t. virens genome , which defines a unique oligonucleotide probe on the array.
probes that were printed n times on the array appear in the table as an independent entries .
the microarrays were designed from the v <dig>  database, which contained  <dig>  gene models.
if the intron/exon structure and/or the automated annotation have been updated in v <dig>  , the protein id number in v <dig>  is different.
in this case, corresponding models for genes of interest were identified in the v <dig>  database by blastp search with the protein model from v <dig> .
the alignments were inspected to see that they covered the predicted coding sequence, and the best hit, usually at an e value of  <dig> , was taken as the corresponding v <dig>  sequence.
additional file 3: table s <dig>  annotated list of genes significantly regulated in interaction with maize or tomato.
significantly regulated genes were identified using cybert .
the cutoff for significance was an adjusted benjamini-hochberg probability of p <  <dig> .
column  <dig> indicates the t. virens v <dig>  protein id number for each gene  model.
the corresponding v <dig>  id numbers, found by blastp search of v <dig>  with the v <dig>  sequence, are given in column  <dig>  “both” indicates genes significantly up-regulated in interaction with maize and tomato; “maize” and “tomato” lists include those identified by cybert as significantly up-regulated with either plant host, but not both.
additional file 4: table s <dig>  annotated list of genes significantly regulated in interaction with maize or tomato , which also satisfy the criterion of  significantly different from  at an adjusted benjamini-hochberg probability of p <  <dig> , calculated using cybert.
in the bar graph at the right, the ratios are plotted on a log <dig> scale , ordered according to the gene list .
additional file 5: figure s <dig>  go term analysis for significantly regulated transcripts.
the list of genes from additional file 3: table s <dig>  was analyzed by blast2go as described in the methods.
an arbitrarily chosen list of similar size  was analyzed in the same way, for comparison.
the pie charts indicate the biological process score distribution.
maria e morán-diez and naomi trushina contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
memd designed and performed the biological experiments, rna isolation, and participated in writing the manuscript.
nt carried out the microarray hybridizations and data analysis.
nll and bah performed gene expression assays, biological experiments and participated in statistical analysis.
lr and bah constructed the reporter lines and performed microscopy.
pkm, cmk and bah conceived of the study, participated in its design and coordination and drafted the manuscript.
all authors read and approved the final manuscript.
authors’ information
m.e.m.-d. and n.t.
contributed equally to this study, which was co-directed by c.m.k.
and b.a.h.

 some members of the genus trichoderma, including t. virens, t. harzianum, t. asperellum and t. atroviride, are employed as biocontrol agents of plant pathogens worldwide. these soil fungi are keen mycoparasites, generally rhizosphere competent, and some have the ability to extensively colonize the outer root layers. in addition to direct parasitism of plant pathogens, interactions with trichoderma enhance plant fitness in response to biotic and abiotic stresses. benefits derived by the host include: a) increased plant growth, increased resistance to abiotic stresses such as drought and salinity, induced host defense responses to pathogens; enhanced nutrient uptake and fertilizer use efficiency, and increased photosynthetic rates. a recent microarray study of two dicots showed that in the plant, genes related to tolerance of oxidative and osmotic stresses are induced by the trichoderma-plant interaction. growth promotion of bean plants has been found to be strain-specific. the combination of close interaction with plants and the ability to tolerate heavy metals make some strains of t. harzianum and t. virens effective agents for soil bioremediation and plant growth promotion. the trichoderma-plant interaction has been defined as an opportunistic symbiosis. the t. asperellum – cucumber interaction was followed by electron microscopy, and growth is extracellular, with hyphae penetrating the outer root cortex. the interaction between t. harzianum and tomato roots was observed by confocal microscopy with a gfp-expressing strain. in hydroponic cultures during early colonization, hyphae were observed growing between plant cell walls, and by 24 h, the root surface was extensively colonized. in soil, a switch to yeast-like morphology was observed following colonization. the fungus, after 48 hours, was mainly extracellular although occasionally intracellular, and in these cases the colonized cell appears to remain viable. after longer times of interaction in soil the fungus produced yeast-like cells. in the t. virens – maize interaction, gfp-expressing hyphae are observed on the root surface and growing between cell walls in the epidermis and outer cortex, with no evidence of intracellular growth. the trichoderma-root interaction is not identical to any other previously studied symbiosis, but the interaction is reminiscent in some ways of ectomycorrhizae. images of trichoderma spp. colonizing maize or tomato roots show the colonizing mycelia as a loose and relatively sparse network, which is less sharply delineated than the massive em mantle. in both symbioses, mycelia penetrate the root apoplast, but it is not clear how similar are the distributions within the root. the ectomycorrhizal fungus laccaria bicolor secretes a small protein, highly expressed during colonization of tree roots and needed to establish the symbiosis. missp is imported into plant cells where it interacts with a transcriptional repressor to antagonize jasmonate-induced gene expression. in arbuscular mycorrhizae, a plant nucleus-targeted effector counteracts the immune response by interacting with a specific plant transcription factor, allowing establishment of the biotrophic interaction. piriformospora indica, like trichoderma virens, has a wide host range, interacting with roots of monocots and dicots. a recent transcriptomic study showed that this basidiomycete root endophyte tailors the expression of its genome to the host plant and to the stage of the mutualistic interaction. the genomes of the cellulose degrader t. reesei and two mycotrophic, plant-interacting species, t. atroviride and t. virens, have been published. the sequences have provided the tools for a genome-wide view of trichoderma-fungal and trichoderma-plant interactions. several studies have addressed the transcriptome of different trichoderma species, in interaction with a particular plant host. studies of trichoderma transcriptomes in interaction with plant roots, using arrays designed from trichoderma ests, showed regulation of genes related to redox reactions, transport, lipid metabolism and detoxification; small secreted and cell surface proteins, proteases, endochitinase ech and novel genes that could be related to nitric oxide biosynthesis, xenobiotic detoxification, and development; and a predominance of carbohydrate metabolism-genes. these studies, which indicate that interaction with the plant host programs expression of many genes in the fungal partner, employed several trichoderma species and times of interaction with the plant host. here, we compared the same trichoderma strain in interaction with two host plants under the same conditions, to identify host-specific transcriptomic signatures. although the trichoderma-root interaction represents a distinct type of symbiosis, the similarities to other fungal-plant symbioses led us to the working hypotheses that the fungal transcriptome should depend on the host plant species, providing a molecular basis for the wide host range; and, small secreted proteins and other secreted proteins may be important for the mutualistic interaction and for induction of disease resistance in colonized plants. in this study we used oligonucleotide microarrays designed from the predicted protein-encoding gene models of t. virens to ask what genes are up-regulated at the transcriptional level, comparing the interaction with either tomato or maize roots. the results of comparing two plant hosts, monocot and dicot, under the same growth conditions suggest that a different repertoire of genes is expressed in response to different hosts and provide us with a better understanding of this interaction. such studies would be helpful for crop-specific application of t. virens for maximizing the benefits derived from this type of plant-microbe interactions. this study would also be helpful in isolating novel promoters for driving expression of desirable trichoderma genes in the rhizosphere-competent species. methods bioassay: seedling and fungal culture for plant interactions, maize seeds were treated with 70% ethanol for 5 min, rinsed with water, soaked in 10% hydrogen peroxide solution for 2 hours, rinsed with water, and germinated on moist sterile filter papers to screen for contamination. after four to five days incubation at 27°c, five seedlings of similar root length were selected and placed on plastic screens suspended in jars containing 250 ml of half strength murashige and skoog with vitamins and % sucrose. the seedlings were grown in a temperature-controlled growth room under fluorescent light for three days at 23-25°c. tomato seedlings were prepared by treating seeds with 70% ethanol for 20 min, rinsing with water, soaking in 10% bleach solution for 5 min, rinsing in water, and germinated on ms supplemented with % agar. after seven days, five seedlings of same root length were selected for placing in the hydroponic system. t. virens gv29- was isolated from a sandy loam soil cultivated with cotton plants in texas, and is deposited at the fungal genetics stock center. conidia were harvested from 7-days old plates using a smear loop and final spore concentration was determined by dilution using a hemocytometer. vogel’s minimal medium with % sucrose was inoculated with × conidia/ml, and the culture was incubated at 150 rpm on a rotary shaker for 48 hours. the fungal biomass was harvested by filtering through miracloth and rinsed with sterile water. one g of fungal tissue was added to each jar, and the seedlings were further incubated for 72 hours in the presence of the fungus. the hydroponic culture system is illustrated in figure  figure hydroponic system. experimental design for the hydroponic system for maize-trichoderma co-culture at 72 h time point. a seven-day-old maize seedlings grown aseptically in ms medium in culture chambers, inoculated with a fungal preparation of t. virens gv and incubated for 72 h. the strain expresses gfp under control of a constitutive promoter, and panels b, f show merged fluorescence and bright field images. two controls were included: hydroponic systems containing only t. virens growing in ms in the absence of maize seedlings, and control plants growing in ms without t. virens. f and g, maize roots inoculated with t. virens at conidia/ml ms medium. microscope images b, d, and f were taken at 10x magnification. sample collection and rna preparation four treatments, with three biological replicates per treatment, were collected: maize roots with t. virens; tomato roots with t. virens; maize or tomato roots without fungus; and t. virens without plant roots. root systems from the same jar were harvested as an individual sample after 72 hours root-fungus interaction. samples were gently washed with distilled water to remove clumps of fungal mycelia that adhered only loosely to the roots. the roots with remaining fungal mycelia were excised from the seedlings, frozen in liquid nitrogen, and extracted directly or stored at −80°c. control root samples without t. virens were grown and harvested as described above. for axenic culture, t. virens was grown under the same conditions in the hydroponic culture containers, but without plants. total rna was extracted from 100 mg of ground tissue with tri reagent following the manufacturer’s instructions. the quality of the rna extracts was evaluated using the agilent bioanalyzer and rna nano kit according to the manufacturer’s instructions. samples with electropherograms exhibiting sharp 18s and 28s rrna peaks and showing no evidence of degradation were retained. the transcript of the t. virens histone h gene was easily detectable by rt-pcr in cdna samples from t. virens and in interaction with maize or tomato, but not in control plant samples. microarray conditions and analysis the microarray platform was described in. a custom microarray was designed from the complete set of filtered transcript models and printed in 15 k arrays. the array consists of probes, including agilent control spots, and technical replicates for probes. probes had the potential for cross-hybridization; these are not among the genes of interest found in this study. the probes on the array are 60-mer sense oligonucleotides, identified by protein id numbers from v of the database. in some cases, update of the gene models resulted in new id numbers in v. the new gene models were then identified here by blast search of the v database with the v sequences, and the corresponding v numbers are given in the additional file 1: table s4; additional file 2: table s1; additional file 3: table s2; additional file 4: table s to 1 μg total rna, rna was added from a spike-in kit. each array hybridization corresponds to a single biological replicate. in this design there are no technical replicate array hybridizations. the array contains replicated oligonucleotide probes, corresponding to of the unique probes. inspection of the data usually showed very similar signal values between duplicate array spots. these were analysed independently by the array software. cdna synthesis was primed with oligo dt, and the double-stranded template was used for amplification and labelling by in vitro transcription using the messageampii kit from ambion. amplified rna was labelled with cy and hybridized onto the custom microarrays. three biological replicate samples were used for m + tv and t + tv, and five for tv alone. microarray hybridization and washing steps were performed following the agilent protocol for single-channel arrays. the arrays were scanned at 10% laser power to avoid signal saturation. agilent’s feature extraction software was used to extract the data. microarray signals were normalized to allow comparison of samples with different rna amounts, using the spike-in data. first, the log microarray data were normalized so that signals for one of the spike-ins with a log relative concentration of had the same values across all samples. next, the log expression data were normalized by linearly interpolating to concentrations using the ten spike-in measurements for each sample and subsequently normalizing to the 75th percentile signal intensity. the data were converted from log to linear values, and analyzed for significant differences between plant interaction and control using cybert http://cybert.ics.uci.edu/ with the options: bayes window and bayes weight 8; baseline subtraction; cutoff benjamini-hochberg at p < . for cluster analysis, data, as log of the microarray signal or log of the signal normalized by dividing by the average control signal, were processed using genesis, http://genome.tugraz.at/ with default options for hierarchical clustering. to compare est entries in the gene lists from and with our gene lists, trichoest id numbers were converted to european nucleotide archive numbers and used to search the database. the resulting nucleotide sequences were used to search trichoderma virens g v at jgi, using blastx. the t. virens genomic probes in the array of are identified by t. virens v id numbers and directly identify probes on our array. annotations were from the t. virens website. the available annotations are listed in additional file 3: table s along with a consensus description from which the functional categories in figure 2d were determined. in addition, go terms for the same set of significantly regulated genes were independently identified using blast2go. the go terms for a control set of sequences, starting with protein id, were analysed in the same way.figure clustering and functional categories of significantly regulated genes. a) cluster analysis of data from individual experiments. the log values for each microarray signal divided by the corresponding average control signal are plotted in the heat map, clustered by relative expression level and experiment. b) a portion of the cluster analysis, annotated by protein id number. this magnified portion of shows a cluster of genes up-regulated in response to both maize and tomato roots. yellow dots indicate of the genes significantly up-regulated in response to both plant hosts. c) expression patterns of genes significantly up-regulated in response to both plant hosts. patterns are plotted for the genes identified by cybert analysis. d) pie chart of functional categories for the set of genes shown in a. for simplicity large categories were used; the “metabolism” category includes, for example, a large number of predicted proteins with reductase, dehydrogenase, and other enzymatic activities, as well as some secreted hydrolytic enzymes with the exception of glycoside hydrolases which are listed separately. see additional file 3: table s for the complete annotated list. real time qpcr cdna for qpcr was synthesized starting from 2 μg of rna, which were treated with dnase, and then used for reverse transcription with random primers with the high capacity cdna reverse transcription kit, following the manufacturer’s protocol. abundance of transcripts was measured by real-time qpcr reactions performed in an applied biosystems cycler; approximately 15 ng of cdna were used as template. the 15 μl reaction volume included  μl of 2x absolute sybr green rox mix and nm final concentration of specific primers for the gene of interest. assays were run in duplicate for each the 4– biological replicate samples, using the following protocol: initial activation at 95°c for 10 min; 40 cycles of 95°c for 15 s, 60°c for 60 s, followed by a gradual increase in temperature from 60°c to 95°c during the dissociation stage. ct values were calculated using the applied biosystems software, and transcript abundance was calculated in microsoft excel from ct values and normalized to the tubulin signal. standard curves were measured for dilution series of pooled cdna samples, and calculated using the applied biosystems software; the slopes for each primer pair are given in table  table primer pairs used for qpcr amplification id v consensus annotation forward primer reverse primer amplicon slope 90504 46158 86039 17705 19757 42536 51095 51211 54057 56652 88010 the slope of the calibration line is given in the last column, measured from a dilution series of pooled cdna samples, using abi software. the protein model for the small secreted protein id is id in v of the database. reporter gene construct we chose the cbh glycoside hydrolase gene because the transcript is abundant in samples from root cultures inoculated with t. virens. furthermore, this gene was strongly induced in interaction of t. virens with tomato roots in an earlier study. this suggested that the upstream region includes strong expression signals that could be used to design a construct to report gene expression in response to trichoderma-root interactions. a 2510 bp region upstream of the predicted translation start was amplified from t. virens g genomic dna, using the forward primer p90504f, gtagaactgaaaagcttcggtcaatc and reverse primer p90504r, caatttctgatccatggtgtacaatctttg. the forward primer contains two mismatches, creating a hindiii site. the reverse primer contains two mismatches, converting the atg start codon into a ncoi site. the product was cloned into ptz57r/t to obtain plasmid p a 960 bp ncoi/sali fragment from ggfp containing the gfp coding sequence was ligated to p which was digested with ncoi and sali, and the ligation products transformed to e. coli hit-dh5α. five μg of the resulting plasmid, p90504-gfp, were co-transformed together with 5 μg of pucatph to protoplasts of t. virens g, using the peg protocol as described by. after regeneration overnight, transformation plates were overlayed with 600 μg/ml hygromycin b in 1% agar. colonies reaching the surface of the overlay were transferred to pda with 100 μg/ml hygromycin b. about hygromycin-resistant colonies were screened by inoculating wells containing excised maize root sections in x ms medium with % sucrose. after three days incubation, the wells were observed under a binocular fluorescence microscope. three isolates that showed gfp expression were identified and one, designated c was used for experiments.  microarray analysis of trichoderma-root interactions the hydroponic culture system used to follow trichoderma-root interactions is illustrated in figure  at the time of harvesting, adhering fungal mycelia were clearly visible on the roots of both plant hosts, as shown for maize in figure 1g. for comparison of the fungal transcriptome in interaction with roots to that of t. virens alone, the fungus was cultured under the same conditions but without plants. total rna for preparation of probes for microarray hybridization was prepared from material from each treatment: t. virens alone, in interaction with maize and with tomato. complete microarray data are provided in additional file 2: table s and are available at geo, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse the data sets were analyzed in pairs using cybert for significant differences in transcript abundance: m + tv compared to tv, t + tv compared to tv, and m + tv compared to t + tv. at a benjamini-hochberg cutoff value of p < , the levels of transcripts were significantly up-regulated in interaction with maize, and in interaction with tomato. a panel of genes, highlighted in the protein id list in additional file 3: table s was chosen for qpcr validation. the primer pairs used are given in table  beta tubulin was the reference gene for qpcr. the expression patterns of these genes measured by qpcr or microarray hybridization were generally similar. an exception is where the t. virens control signal was underestimated by qpcr compared to microarray hybridization. the calibration slope for this primer pair indicates below-optimal efficiency, perhaps accounting for the discrepancy. in general, the correlation between qpcr and microarray signals holds well, over several orders of magnitude. the other exception is gh which showed no induction in interaction with maize on the microarray, as well as in one set of qpcr experiments. this transcript, however, was strongly up-regulated in a second set of qpcr experiments. this is in contrast to the other genes in the panel which showed remarkably consistent patterns in experiments performed in two different laboratories and at different times during this study. cluster analysis was performed, according to experiment and gene name, with the group of transcript levels showing significant regulation in either root interaction. the significantly regulated genes cluster according to experiment, but some genes deviate from the pattern in some experiments. the sources of this variation might be sampling effects or differences between hydroponic chambers in the time course of the trichoderma-root interaction. as for id, time course experiments on specific genes or the entire transcriptome may be needed to resolve these differences. thus the number of significantly regulated genes may be an underestimate, and we limit the analysis to robustly regulated transcripts.figure qpcr validation of microarray data. a) qpcr signals normalized to tubulin, and corresponding microarray data, normalized to total signal as described in methods. bars indicate means of 3– replicates with standard error; the m + tv and t + tv samples for qpcr were independent of those used for the microarrays. *,**,** significant at p < ,, respectively, by ttest, one-tailed. b) microarray data corresponding to the transcripts shown in. *,**,** significant at p < ,, respectively, by ttest, one-tailed. c) correlation between qpcr and microarray signals, plotted from the data in. the line is a power-law least squares fit to the combined data set, which appears linear on this log-log plot. the r value is shown on the graph. expression of gfp under control of the upstream region from glycoside hydrolase gene a) roots excised from hydroponic co-cultures of p90504:gfp line c were photographed under white light or for gfp fluorescence, in a binocular microscope. b) confocal images of roots from hydroponic co-cultures of p90504:gfp line c or wt. green channel: gfp fluorescence, excitation 488 nm, bandpass filter 500–550 nm. red channel: propidium iodide, excitation 561 nm, filter 575 nm long pass. the images are projections of five or z-stack slices. propidium iodide stains plant cell walls in the root epidermis and outer cortex layers, as well as some plant and fungal nuclei in some cells. plant cell autofluorescence is visible in some areas. genes regulated in interaction with both plant hosts the expression of 14 t. virens genes was significantly up-regulated in interaction with both maize and tomato. of these, were found previously, in interaction with tomato. a cluster of genes co-regulated in interaction with both host plants is shown in figure 2b, annotated by jgi protein model id number. seven expression patterns of genes belonging to the set significantly up-regulated in both root interactions cluster together. the genes significantly up-regulated in interaction with both maize and tomato are mostly found on different scaffolds. in the two cases where a pair of genes was found on the same scaffold, the distance between them was very large. similar expression patterns in the three conditions, as evident in figure 2c, is therefore not reflected by co-localization in the genome. ten of the genes in this list are annotated as glycoside hydrolases. seven of the eight genes found to be up-regulated by interaction with both maize and tomato in this study and with tomato degrade the root cell walls. genes up-regulated in interaction with either or both plant hosts looking at the available annotations for the larger list of genes significantly up-regulated in interaction with either plant host, glycoside hydrolases are again prominent. another class includes transporters. there are several predicted transcription factors and other regulatory proteins, and some small secreted proteins. two genes are related to iron acquisition: fe permease ftr and a ferric reductase. a similar picture, in general, was independently obtained by go term analysis. the highest-represented biological processes are polysaccharide catabolism, transmembrane transport, and oxidation-reduction. an identical go term analysis using a randomly chosen set of sequences resulted in a much more diverse representation of biological processes. oxidation-reduction is also highly represented in the list obtained from the consensus annotation, although this is not obvious in figure 2d where oxidation-reduction is included in “metabolism”. of the fourteen genes demonstrating significant up-regulation in response to both maize and tomato roots, nine are annotated as glycoside hydrolases. all the glycosyl hydrolases were grouped according to gh families from the annotation, and the total expression plotted to obtain a semi-quantitative view of the relative contribution of each family, at the transcript level. the plot indicates that the expression pattern is different for the interaction with maize or tomato.figure representation of glycoside hydrolase families. the bars represent the sum of average transcript abundance for each family. the error bars indicate sem for families with a single member represented; for families with two or more members, the sem values were combined using the sum of squares rule, to provide an approximate measure of variability. different cell wall composition of the two plant hosts might be expected to play a role in the set of fungal genes expressed. the predicted substrates of the fungal glycoside hydrolase families represented do not appear, however, to be related in any direct way to the composition of maize vs tomato cell walls. as an example, we note that dicot walls are more pectin-rich than those of monocots. the endopolygalacturonase encoded by transcript is the unique member of family gh expressed in response to interaction with roots. if the product of is indeed the major induced pectin-degrading enzyme, one might have expected higher expression in tomato where the substrate is more abundant. this pattern was found upon comparison of two colletotrichum species. one species is a pathogen of brassicaceae, and one of maize. c. higginsianum in the necrotrophic phase of its interaction with arabidopsis was found to up-regulate a greater number of pectin-degrading enzyme genes than c. graminicola on maize. our data show, however, up-regulation of the gh gene in interaction with both tomato and maize. dicot and grass cell walls both contain pectin, and the expression of enzyme genes will not always correspond directly with substrate levels. the predicted ortholog of in t. harzianum is thpg silencing of thpg in t. harzianum showed that it is required for colonization of arabidopsis roots. this could be tested for t. virens on tomato and maize. the question of how gh family expression might be related to host cell wall composition will need to be addressed at the protein, in addition to transcript, level. when and where each cazyme is expressed in the root tissue is also important. genes differentially regulated in interaction with maize or tomato the overall distribution of gene classes in the list of up-regulated genes is similar for the two plant hosts. inspection of the microarray data showed that expression in the maize and tomato interactions often followed the same pattern, yet did not pass the significance filter in one or the other data set. we therefore studied the set of genes whose transcript levels differ significantly at p <  between maize and tomato, and are also significantly regulated relative to the t. virens control. this gene list, which is the intersection of the cybert results from the above two criteria, is given in additional file 4: table s transcripts corresponding to gene models differed significantly in their expression levels between maize and tomato and also between the control and at least one plant interaction, with ratios of transcript abundance on tomato vs on maize ranging from to. thus, we have identified genes that can serve as specific reporters for the interaction of t. virens with one of the two plant hosts. in contrast to the genes significantly up-regulated in interaction with both hosts, of which half had been found previously, only five of the transcripts in additional file 4: table s had been identified previously in the t. virens – tomato interaction or are homologs of genes up-regulated in the t. harzianum – tomato interaction.figure venn diagram of the number of genes differentially expressed in t. virens interactions with maize vs tomato roots. the intersection of the two sets indicates transcripts that were significantly different in tomato compared to maize, and also significantly up-regulated compared to control in either tomato or maize interactions. expression patterns of root-regulated t. virens genes that are preferentially expressed in interaction with tomato or maize. maize-specific expression of three genes, oxidoreductases and ssp was confirmed by qpcr. the intracellular invertase tvinv is specific to the maize interaction, though obviously one cannot exclude induction on tomato at a different time point from the 72 h sampled here. the tomato-specific up-regulated transcripts remain to be further characterized. genes encoding small secreted cysteine-rich proteins in the t. virens genome have been annotated based on the following criteria: amino acids or less, at least four cysteine residues, predicted signal peptide for secretion, and lack of annotated enzyme activity. several genes belonging to this class were up-regulated in interaction with maize or tomato. up-regulation of transcript is specific to maize, and this was validated by qpcr. this gene in v of the t. virens sequence database corresponds to id in v; in v the protein model was incomplete, lacking the n-terminus, and thus was not included in the original ssp list compiled by kubicek et al.. we added the complete model to the previous clustering analysis, and found that it belongs to cluster a ssp family of unknown function. id is a predicted class ii hydrophobin, significantly up-regulated in response to tomato roots, and showing an up-regulated trend in the average transcript abundance in interaction with maize. a class ii hydrophobin was induced in t. harzianum by the presence of tomato plants. this protein shows homology to a class ii hydrophobin from t. virens, related only more distantly to both t. virens proteins show the same core structure containing eight cysteine residues arranged in a conserved motif. the ceratoplatanin family gene sm encoding a ssp which is an inducer of systemic resistance, was up-regulated in interaction with cotton. samolski et al. found a -fold induction in interaction with tomato, while here there was only a -fold increase in response to tomato roots. the cell wall protein qid is unfortunately not on the microarray because the t. virens ortholog was missing from the v gene models. thus, direct comparison with expression of ssp genes in previous studies is not straightforward, probably due to different plant hosts and conditions.figure expression patterns of small secreted proteins. the transcripts indicated by v protein id numbers were significantly up-regulated in either maize or tomato. transcript levels for id and id were measured also by qpcr, and the data shown here are also plotted in figure 3b, where these genes are labelled ssp and hyd respectively. the “cluster 3” ssp seems a promising candidate for functional analysis as a maize-interaction-specific small secreted protein. functional significance of regulated genes the strong induction of genes for enzymes with the potential to degrade plant cell walls is interesting, as the interaction between t. virens and these two hosts is considered mutualistic. this finding is consistent with previous reports. in the mutualistic interaction of barley with the basidiomycete piriformospora indica –, hydrolytic enzymes and transporters are strongly induced following the transition from biotrophy to saprotrophic growth on dead root cortex cells. there is relatively little induction of these genes in the biotrophic p. indica-arabidopsis interaction. while a full meta-analysis is beyond the scope of this study, the similarity between p. indica – barley and the t. virens interactions studied here suggests that the t. virens – root interactions have a limited necrotrophic aspect, like p. indica – barley and in contrast to p. indica - arabidopsis. if so, cell death should be observed, and there is preliminary evidence for this from scattered propidium iodide staining of plant and fungal nuclei. furthermore, this similarity between p. indica – barley and the two t. virens interactions points to parallel evolution of a mutualistic, isr-promoting, fungal-root interaction distinct from mycorrhizae, in basidiomycota and ascomycota. the genes identified here and in previous studies can provide specific reporters for the t. virens-root interaction. to test this concept we constructed a reporter in which gfp was expressed under control of the predicted upstream regulatory region from the gene. transgenic lines expressing this construct showed strong gfp fluorescence in interaction with roots and root sections. the protein sequence of belonging to glycoside hydrolase family gh is nearly identical to the t. virens t gene cbhi which is up-regulated in interaction with tomato. in hydroponic cultures, fluorescent hyphae could be detected in the outer root layers, while externally adhering hyphae showed fluorescence in or on some hyphal compartments. we have not confirmed penetration into root cortex cells under the conditions studied here, though gfp-expressing hyphae are closely associated with maize cells. higher-resolution localization of t. virens hyphae in the root cortex will require a line expressing gfp at high, constitutive levels, like the t. harzianum line used in a recent study of the role of salicylic acid in limiting penetration into arabidopsis roots. in the t. harzianum- tomato root interaction there is evidence for intracellular hyphae or yeast-like cells. if the t. virens – root interaction most resembles the late-stage p. indica- barley interaction, induction of hydrolytic enzymes might reflect saprotrophic on dead root cells. the majority of root cells at three days interaction, however, appear living, since their nuclei do not stain with propidium iodide. the genome of the arbuscular mycorrhizal fungus rhizophagus irregularis lacks gh families predicted to degrade plant cell wall polysaccharides. in contrast, these are induced in t. virens interacting with roots. two pectate lyase genes are up-regulated in ectomycorrhiza, as is a gh gene. visualization of the time course and location of expression of the gh genes during colonization should help to understand how t. virens fits into the biotroph – saprotroph – pathogen continuum. trichoderma spp. interact with both monocots and dicots. different patterns of gene expression might facilitate this wide host range. in support of this view, we found that a number of genes were expressed specifically in response to either maize or tomato. one of these is invertase tvinv, expressed more than 100-fold higher on maize than on tomato. several small secreted proteins are expressed preferentially in each interaction. short-chain oxidoreductases appear to be characteristic of the maize interaction. identification of the substrates of these predicted proteins, if they indeed have enzyme activity, might help identify their role in the trichoderma-maize root interaction. in general, there are not enough functional data to fully understand why a particular set of genes is induced in the maize or tomato mutualism. the existence of specific reporters of each interaction, though, demonstrates specific transcriptomic signatures for the interaction with each plant.  this study provides a better knowledge of the crosstalk of trichoderma virens, an agriculturally relevant biocontrol agent, with plants. genes differentially expressed during interaction with plant roots encode proteins belonging to several functional classes including enzymes, transporters and small secreted proteins. among them, glycoside hydrolases and transporters are highlighted by their abundance and suggest an important factor in the metabolism of host cell walls during colonization of the outer root layers. activity of the cbh promoter was reported by gfp fluorescence in a transgenic t. virens line on roots, providing a first spatial view of how the plant re-programs the fungal transcriptome. host-specific gene expression may contribute to the ability of t. virens to colonize the roots of a wide range of plant species. the host-specific gene list obtained here will facilitate functional experiments to test this hypothesis. availability of supporting data the microarray data sets supporting the results of this article are available in the geo repository, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse additional files additional file 1: table s this file lists the sequences of the oligonucleotide probes printed on the t. virens microarray. additional file 2: table s microarray data from five biological replicates of t. virens alone, three of tv cocultured with maize roots and three with tomato roots are given as a text file. the first column indicates the protein model id from the t. virens genome, which defines a unique oligonucleotide probe on the array. probes that were printed n times on the array appear in the table as an independent entries. the microarrays were designed from the v database, which contained gene models. if the intron/exon structure and/or the automated annotation have been updated in v, the protein id number in v is different. in this case, corresponding models for genes of interest were identified in the v database by blastp search with the protein model from v. the alignments were inspected to see that they covered the predicted coding sequence, and the best hit, usually at an e value of, was taken as the corresponding v sequence. additional file 3: table s annotated list of genes significantly regulated in interaction with maize or tomato. significantly regulated genes were identified using cybert. the cutoff for significance was an adjusted benjamini-hochberg probability of p < . column indicates the t. virens v protein id number for each gene model. the corresponding v id numbers, found by blastp search of v with the v sequence, are given in column “both” indicates genes significantly up-regulated in interaction with maize and tomato; “maize” and “tomato” lists include those identified by cybert as significantly up-regulated with either plant host, but not both. additional file 4: table s annotated list of genes significantly regulated in interaction with maize or tomato, which also satisfy the criterion of significantly different from at an adjusted benjamini-hochberg probability of p < , calculated using cybert. in the bar graph at the right, the ratios are plotted on a log scale, ordered according to the gene list. additional file 5: figure s go term analysis for significantly regulated transcripts. the list of genes from additional file 3: table s was analyzed by blast2go as described in the methods. an arbitrarily chosen list of similar size was analyzed in the same way, for comparison. the pie charts indicate the biological process score distribution. maria e morán-diez and naomi trushina contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions memd designed and performed the biological experiments, rna isolation, and participated in writing the manuscript. nt carried out the microarray hybridizations and data analysis. nll and bah performed gene expression assays, biological experiments and participated in statistical analysis. lr and bah constructed the reporter lines and performed microscopy. pkm, cmk and bah conceived of the study, participated in its design and coordination and drafted the manuscript. all authors read and approved the final manuscript. authors’ information m.e.m.-d. and n.t. contributed equally to this study, which was co-directed by c.m.k. and b.a.h. 
the transcriptomes for t. virens alone were compared with fungus-inoculated tomato or maize roots by hybridization on microarrays of unique oligonucleotides designed from the predicted protein-coding gene models. host-specific gene expression may contribute to the ability of t. virens to colonize the roots of a wide range of plant species. transcript levels of genes were modulated by interaction with roots. average transcript levels for these genes ranged from fold higher on tomato than on maize to fold for the most tomato-specific gene. average transcript levels for these genes were to fold higher on maize than on tomato. among them, glycoside hydrolases and transporters are highlighted by their abundance and suggest an important factor in the metabolism of host cell walls during colonization of the outer root layers. ten of these were expressed higher in the fungus in co-culture with tomato roots than with maize. the trichoderma-root interaction is hosted by a wide range of plant species, including monocots and dicots. to test the hypothesis that gene expression by the fungal partner in this beneficial interaction is modulated by the plant, trichoderma virens was co-cultured with maize or tomato in a hydroponic system allowing interaction with the roots. members of the fungal genus trichoderma directly antagonize soil-borne fungal pathogens, and an increasing number of species are studied for their potential in biocontrol of plant pathogens in agriculture. some species also colonize plant roots, promoting systemic resistance. based on the relevant role of trichoderma virens as a biological control agent this study provides a better knowledge of its crosstalk with plants in a host-specific manner. the differentially expressed genes encode proteins belonging to several functional classes including enzymes, transporters and small secreted proteins. of the genes up-regulated on either or both host plants, differed significantly in their expression levels between maize and tomato. glycoside hydrolases and transporters were highly represented among transcripts induced by co-culture with roots.
 members of the fungal genus trichoderma directly antagonize soil-borne fungal pathogens, and an increasing number of species are studied for their potential in biocontrol of plant pathogens in agriculture. some species also colonize plant roots, promoting systemic resistance. the trichoderma-root interaction is hosted by a wide range of plant species, including monocots and dicots.  to test the hypothesis that gene expression by the fungal partner in this beneficial interaction is modulated by the plant, trichoderma virens was co-cultured with maize or tomato in a hydroponic system allowing interaction with the roots. the transcriptomes for t. virens alone were compared with fungus-inoculated tomato or maize roots by hybridization on microarrays of unique oligonucleotides designed from the predicted protein-coding gene models. transcript levels of genes were modulated by interaction with roots. almost all were up-regulated. glycoside hydrolases and transporters were highly represented among transcripts induced by co-culture with roots. of the genes up-regulated on either or both host plants, differed significantly in their expression levels between maize and tomato. ten of these were expressed higher in the fungus in co-culture with tomato roots than with maize. average transcript levels for these genes ranged from fold higher on tomato than on maize to fold for the most tomato-specific gene. the other host-specific transcripts were expressed more strongly in co-culture with maize than with tomato. average transcript levels for these genes were to fold higher on maize than on tomato.  based on the relevant role of trichoderma virens as a biological control agent this study provides a better knowledge of its crosstalk with plants in a host-specific manner. the differentially expressed genes encode proteins belonging to several functional classes including enzymes, transporters and small secreted proteins. among them, glycoside hydrolases and transporters are highlighted by their abundance and suggest an important factor in the metabolism of host cell walls during colonization of the outer root layers. host-specific gene expression may contribute to the ability of t. virens to colonize the roots of a wide range of plant species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 