BACKGROUND
considerable research has been devoted in the past two decades to investigate the use of naturally occurring yeasts for managing postharvest diseases on a variety of fruits and vegetables  <cit> .
at present, only two commercial products are available for postharvest use, each having a very small market share of the collective technologies used to manage postharvest diseases.
biosave  used for the control of pome fruit, sweet potato, and potato diseases  <cit> , and “shemer”  <cit>  used commercially for the control of sweet potato and carrot storage diseases  <cit> , strawberry  <cit> , apple  <cit>  and citrus fruit  <cit> .
knowledge about mechanisms of action of yeast biocontrol agents used to manage postharvest diseases is relatively superficial because appropriate methods to study microbial interactions in wounds of fruit has been lacking.
various mechanisms, however, have been described, including antibiosis, production of lytic enzymes, parasitism, induction of host resistance, and competition for limiting nutrients and space  <cit> .
results described in these studies are mostly correlative and provide indirect evidence for possible involvement of one or more of these components in the mechanism of action.
this may be due to the lack of an understanding of the key biochemical and molecular processes occurring within tritrophic interactions  that define the effectiveness of a biocontrol system.
a characteristic response of fruit to pathogen attack or application of a biocontrol agent is the activation of defence responses, including production of inhibitors of cell wall-degrading enzymes of the pathogen, activity of antifungal compounds , active oxygen species, and reinforcement of the cell wall of the host  <cit> .
in relation to yeast biocontrol agents, induction of resistance in fruit tissue has been reported to involve, to a greater or lesser extent, the active elicitation of all of the above responses.
hershkovitz et al.
<cit>  recently reported the first molecular study of the response of citrus fruit to the application of the yeast biocontrol agent, m. fructicola.
results indicated that significant changes in gene expression occur in grapefruit wounded tissue in response to the application of m. fructicola including changes in a variety of genes involved in response to biotic and abiotic stresses, signaling, defence and secondary metabolism.
these findings imply that complex biochemical and molecular processes are involved in the reaction of fruit host tissue to the introduction of yeast cells, which may have the potential to influence the efficacy of the biocontrol agent.
castoria et al.
<cit>  indicated that the ability to tolerate high levels of reactive oxygen species  produced in fruit tissue in response to wounding is an essential characteristic of effective yeast antagonists.
macarisin et al.
<cit>  demonstrated that the antagonistic yeasts, candida oleophila and m. fructicola, actually induce defence-related oxidative responses in intact fruit exocarp tissues and within wounds of apple and citrus, generating greater levels of super oxide anion  and hydrogen peroxide .
torres et al.
<cit>  reported that, in addition to nutrient competition, the biocontrol activity of pantoea agglomerans could be attributed to the ability of the antagonist to induce an oxidative response in orange fruit peel tissue by triggering h2o <dig> production, superoxide dismutase, and catalase activities.
in another study, transient exposure of the yeast m. fructicola to a sublethal heat or oxidative stress increased overall abiotic stress tolerance, providing cross-protection to a range of other more severe abiotic stresses  <cit> .
the study showed that higher levels of trehalose, induced by exposure to a sub-lethal abiotic stress, was associated with an increase in ros scavenging, general stress tolerance, and more rapid growth in apple wounds, collectively resulting in improved biocontrol activity.
larralde-corona et al.
<cit>  investigated the effect of the exposure of the yeast pichia guilliermondii to penicillium digitatum on yeast gene expression by analyzing expressed sequencing tags  obtained by subtractive suppressive hybridization .
the yeast was exposed to starvation by carbon source, actively growing mycelium of p. digitatum separated from yeast cells with membrane and induction by fungal cell walls.
only one est, associated with energy metabolism, was obtained in response to the limited carbon source, seven ests in the separated membrane system, all relating to metabolic networks such as: energy, nitrogen, cell cycle, abc transporters, response to stress and one unknown sequence.
the exposure of the yeast cells to fungal cell walls produced the highest number of ests, a total of  <dig>  including all the metabolic networks mentioned above plus ests associated with signal transduction.
compared to the information available about the response of host tissue to the introduction of yeast biocontrol agents, little is known about global molecular changes taking place in yeast cells in response to an interaction with either fruit tissue or a pathogen's mycelia.
in the current study, we characterized transcriptional changes in m. fructicola cells following their interaction with grapefruit peel tissue and mycelium of p. digitatum using next generation sequencing technology for rna  on the illumina platform.
along with de-novo assembly and characterization of the transcriptome, analysis of global patterns of gene expression and functional categorization were performed.
RESULTS
illumina sequencing, assembly, and annotation
transcriptome analysis of m. fructicola utilizing illumina sequencing generated a total of  <dig> , <dig> reads.
the raw reads were assembled into  <dig>  contigs by trinity software with an average size of  <dig>  bp and median of 349 bp.
the sequences were determined to represent  <dig> unigenes with an average length of  <dig>  bp and median of 385 bp.
the size distribution is shown in figure  <dig>  to annotate these genes, reference sequences were searched using blastx within the non-redundant  ncbi nucleotide database using a cut-off e-value of 10- <dig> .
a total of  <dig> genes  could not be annotated based on matches in the ncbi database .
a high level  of transcripts from the individual libraries could be mapped to the assembled transcriptome using bowtie software.
these transcripts were blasted against the m. fructicola draft genome  with cutoff e-value of 1e- <dig> and identity >85%.
according to this analysis  <dig> unigenes  could be aligned to the genome, indicating high accuracy of our assembly .
the remaining  <dig> unigenes could be a result of erroneous assembly.
as shown in figure  <dig>  42% of sequences had significant homology matches  in the ncbi database, while 58% of the sequences had matches ranging between  <dig> e10- <dig> and  <dig> e10- <dig>  the similarity distribution showed that 11% of the sequences had > 90% homology, followed by 86% of the sequences ranging between  <dig> to 90% homology.
only 3% of the sequences had homology lower than 50% .
species specific distribution indicated that 69% of the sequences matched gene sequences of the yeast clavispora lustiniae, followed by other yeasts debar-yomyces hansenii , scheffersomyces stiptis , meyerozyma guilliermondii , spathaspora passa-lidarum, millerozyma farinose and candida tenuis  .
the phylogenetic analyses presented in figure 2d shown close sequence similarity between m. fructicola and c. lustiniae .
classification of gene ontology
to classify the functions of m. fructicola unigenes, we searched the gene ontology  database and used the wego tool  for plotting go annotation results .
a total of  <dig> sequences out of  <dig>  were categorized into  <dig> functional groups .
in each of the three main categories  of the go classification the 'cell and cell part', 'antioxidant and binding' and 'metabolic and cellular processes' terms were the most dominant.
in contrast, 'metallochapreon', and 'rhythmic process' were least abundant.
transcription profiles
rna-seq technology is a sensitive and reliable tool to study transcriptional changes.
using this technique we identified  <dig> genes differentially expressed  with at least a  <dig> -fold change in yeast cells in response to yeast 24 h after interaction with p. digitatum .
among these,  <dig> were upregulated and  <dig> were downregulated.
in the mf-fruit interaction,  <dig> genes had differential expression where  <dig> were upregulated and  <dig> were downregulated, while  <dig> genes were regulated in both interactions.
de genes in either mf-pdig and mf-fruit interactions or specific to one of these interactions are shown in a two-dimensional hierarchical cluster heat map and venn diagram  additional file  <dig>
functional analysis of differentially expressed genes
all differentially regulated genes were organized in functional categories based on their gene ontology  terms  and were members of at least one of the three principal go branches: biological process, molecular function, and cellular component .
all  <dig> functional groups were found to be over-expressed in mf-pdig interaction compared to all transcribed genes of m. fructicola controls .
we focused our analyses on “biological process” categories.
those categories were organized in three groups: response to stress and stimulus transport , transport  and metabolic processes .
the induced genes in the category of response to stress in mf-pdig interaction are mainly involved in multidrug resistance and resistance to singlet oxygen species  and in oxidation-reduction activity in the mf-fruit interaction  .
the genes encoding enzymes involved in response to heat stress were, such as heat shock proteins hsp <dig>  hsp <dig>  hsp <dig> and sgt <dig>  were repressed, in both of interactions.
transport genes involved in iron transport and uptake , zinc transport  and sulp anion  were upregulated in the mf-fruit interaction while in the mf-pdig interaction, ammonium permease , inorganic phosphate  transporter and low-affinity manganese transporter  were induced .
a glucose sensor  and two glucose transporter  genes were induced in the mf-pdig interaction.
a group of genes associated with transmembrane transport, such as general amino acid permease , oligopeptide transporter , a permease , which mediates uptake of glycerophosphoinositol and glycerophosphocholine as sources for inositol and phosphate, an a sugar transporter , were induced in both interactions.
functional analysis of the metabolic processes category included lipid, cofactor and cellular amino acid metabolic process annotations.
many genes involved in the ergosterol-biosynthesis pathway  were up-regulated in the mf-fruit interaction .
many genes involved in amino acid metabolism  were up-regulated in the mf-pdig interaction.
in the mf-fruit interaction, two groups of genes involved in cellular amino acid metabolism were found to be enriched: one up-regulated  and the second, down-regulated  .
real time pcr  analysis
to confirm the expression profiles obtained from the rna-seq expression data, rt-qpcr analysis was carried out for  <dig> genes selected on the basis of their biological significance: genes involved in the degradation of fungal cell wall, such as glucanse  and chitinase , were selected as were genes related to response to stresses, such as an abc transporter, a transcription factor , a heat shock protein , superoxide dismutase , a serine threonine  kinase, and a signal transducing mek kinase.
although minor variation of transcription levels were obtained,  <dig> of the tested  <dig> genes correlated well with the fold-change, either up or down- obtained in the rna-seq analysis  .
time points included in the rt-qpcr analysis were  <dig>   <dig>   <dig> and 48 h after interaction of the yeast cells with either grapefruit peel or p. digitatum mycelia .
expression level of the sod <dig> gene in rt-qpcr was two-fold higher compare to the rna-seq data set.
transcript levels of glu were induced in m. fructicola cells after  <dig> and 48 h in the mf-pdig interaction .
chi levels were up-regulated in both mf-pdig and mf-fruit interactions after 24 h and 48 h .
stb <dig> expression level displayed transient induction in both mf-fruit and mf-pdig interactions at 24 h , while the ser-thr kinase was transiently repressed in both mf-fruit and mf-pdig interactions at 24 h .
the abc transporter was induced in the mf-pdig interaction at  <dig> and 48 h, while in the mf-fruit interaction it was induced only at 24 h .
expression levels of sod <dig> and mekk were repressed in the mf-pdig interaction at all-time points tested compared to control cells grown in nydb media.
in the mf-fruit interaction these genes showed a transient induction at  <dig> and 24 h .
hsp transcript levels increased following contact with either the fruit peel or p. digitatum mycelia but were down-regulated in comparison to the control, m. fructicola cells grown in nydb .
discussion
information about the interactions between yeast biocontrol agents, pathogens, and plant hosts  is needed to better understand biochemical and molecular processes involved in the biocontrol system  <cit> .
in a previous study, we identified global changes in gene expression in grapefruit peel tissue following the application of the yeast metschnikowia fructicola <cit> .
marked changes in the profiles of genes expressed in fruit tissue were observed in response to application of yeast into wounded fruit tissue.
in the present study, the main objective was to compare changes in gene expression in m. fructicola cells following the interaction of the yeast with either grapefruit peel tissue or p. digitatum mycelia.
although the genome of m. fructicola has not been sequenced, de-novo assembly of the transcriptome of m. fructicola resulted in the identification of a total of  <dig> unigenes, half of which could be annotated based on homology to genes in the ncbi database .
as shown in figure 2c, 69% of the unigene sequences identified in m. fructicola showed highest homology to clavispora lustiniae genes.
thus the rna-seq based transcriptome analysis of m. fructicola generated a large number of newly identified yeast genes, providing a substantial contribution to the existing sequence database.
using rna-seq, we compared transcriptomes of m. fructicola after the yeast were exposed to either grapefruit peel tissue  or after coming into direct contact with the fungal mycelia of p. digitatum .
our analysis identified more than  <dig> genes that were differentially expressed and that may potentially affect the biocontrol activity of m. fructicola .
the number of genes regulated in mf-fruit interaction approximately same that regulated in mf-pdig and the overlap between the two interaction is only  <dig> % of genes co-regulated.
stress related genes
activation of stress-related genes in yeast biocontrol agents induced in response to the pathogen or plant host has been observed in several biocontrol systems  <cit> .
in the current study, several genes involved in oxidation – reduction activity  and implicated in oxidative stress response, such as peroxidases and reductases, were up-regulated in the mf-fruit interaction .
these results could be associated with the generation and detoxification of reactive oxygen species .
m. fructicola cells produce ros as in response to contact with fruit tissue  <cit> .
these studies indicate that ros plays a major role in the initial response of yeast cells to host tissue and act as a signal, inducing an oxidative burst in the fruit tissue which subsequently leads to the induction of resistance mechanisms in the host tissue.
the time course study of superoxide dismutase  gene expression showed a transient induction in mf-fruit samples at  <dig> and 24 h .
these results provide further evidence that yeast cells are undergoing oxidative stress while in contact with fruit tissue and suggest the possibility of an adaptive response.
in this regard, liu et al.
<cit>  demonstrated that a mild heat-shock pretreatment  improved the tolerance of m. fructicola to subsequent high temperature  and oxidative stress  and increased the biocontrol efficacy of the yeast.
on the other hand, lower or unaltered expression levels of sod  and low levels of ros in mf-pdig interaction  indicate that in this type of interaction, yeast cells are not under oxidative stress and hence activation of protective systems against superoxide radicals is not necessary.
high levels of oxidative stress in microorganisms is accompanied by the induction of heat-shock proteins  in an attempt to ameliorate damage to proteins caused by ros  <cit> .
induction of hsps in response to biotic and abiotic stimuli is well documented where they assist in the folding and unfolding, and general stability, of various proteins.
production of hsps in microorganisms is associated with various abiotic stresses, including oxidative stress  <cit> .
in our present study, genes encoding hsp <dig> and hsp <dig> were down regulated in both types of interactions compared to the control  .
however, the time course study of hsp <dig> expression by rt-qpcr revealed significantly higher levels of transcript after 12 h,  <dig> and 48h in both interactions compared to its level at 5 h .
this result is surprising since it was anticipated that hsps would be induced in yeast cells that are in contact with fruit peel tissue or with fungal mycelia considering the stressful environment it represents, such as desiccation due to the dryness of the fruit surface, ros, and phytoalexins.
response to chemical stress
genes belonged to plasma membrane atp-binding cassette  transporters, such as yhk <dig>  yor <dig> and snq <dig>  were upregulated in the mf-pdig interaction .
yor <dig> and sng <dig> genes were induced in s. cerevisiae following exposure to the monoterpene thymol  <cit> .
the function of these transporters in s. cerevisiae and their possible role in yeasts tolerance to monoterpene have yet to be elucidated  <cit> .
in pichia guilliermondii, two abc transporter related proteins involved in the secretion of toxic compounds were induced during its interaction with p. digitatum <cit> .
many of the abc transporters are reported to be tightly regulated by transcription factors  <cit> .
in this regard, the current study showed that stb <dig>  a transcription factor involved in regulating multidrug resistance and oxidative stress response, was transiently upregulated in the mf-pdig interaction at 24 h .
induction of abc transporters in m. fructicola cells may be needed for the yeast to tolerate exposure to fruit surface wounds or to mycelia where many potentially toxic substances  may be present.
this explanation may also be relevant to the ability of yeasts to tolerate high levels of ros in apple fruit wounds which was suggested to be an essential characteristic of a successful biocontrol agent  <cit> .
hydrolytic enzymes
lytic enzymes, such as glucanases and chitinases, involved in degradation of fungal cell walls have been considered to play an important role in the biological control of postharvest pathogens  <cit> .
jijakli and lepoivre  <cit>  indicated that p. guillermondii, a yeast biocontrol agent, exhibits a high level of β- <dig> -glucanase activity that could function to degrade the cell walls of postharvest pathogens.
p. membranefaciens had higher β- <dig> -glucanase and exochitinase activity but less endo-chitinase activity than c. albidus in the presence of fungal cell walls  <cit> .
several studies have demonstrated the ability of yeasts to produce and secrete glucanases and chitinases in culture medium amended with fungal cells walls  <cit> .
the ability of yeasts to produce exo-ß- <dig> -glucanase and chitinase has been suggested to play a role in the firm attachment of yeast cells to fungal hyphae and the partial degradation of fungal mycelia  <cit> .
the current study clearly indicated that glucanase  and chitinase  genes are up regulated when yeast cells were in contact with p. digitatum mycelia .
when yeast cells were in contact with grapefruit peel tissue , however, only chi was up-regulated.
the fact that chi expression is induced in both exposures to fruit peel tissues and to fungal mycelia  may indicate that chitinases are involved in several biological interactions and not specifically in pathogen cell wall degradation.
iron homeostasis
many pathogens utilize secreted siderophores and/or high-affinity uptake systems to acquire iron, and many are able to utilize ferritin, transferrin, lactoferrin, heme and heme-containing proteins for cellular processes related to virulence  <cit> .
iron competition was reported as the main mode of action of m. pulcherrima inhibition of botrytis cinerea, alternaria alternata and p. expansum <cit> .
iron is essential for fungal growth and pathogenesis.
iron metabolism has been well characterized in the model yeast saccharomyces cerevisiae.
there are two different pathways that allow iron uptake from the environment: the reductive iron-uptake pathway mediated by fet3p, a multicopper oxidase, and ftr1p, an iron permease localized on the plasma membrane; and the siderophore-mediated uptake pathway.
in our transcriptional analysis, we identified homologs of transmembrane ferric reductases , iron permease , and an iron transporter  all of which were up-regulated in the mf-fruit interaction .
this information provides a clue to the potential important role of the reductive iron uptake system in the interaction of m. fructicola with fruit tissues.
iron competition was reported as the main mode of action in the ability of m. pulcherrima to inhibit botrytis cinerea, alternaria alternata and p.expansum infection of apples stored at 1°c for 8 months under controlled atmosphere   <cit> .
m. fructicola, similar to m. pulcherrima, produces a red pigment that is believed to be associated with the binding of iron .
iron permease , in candida albicans, was shown to mediate iron acquisition from transferrin and was required for systemic infection  <cit> .
in the plant pathogenic fungus, fusarium graminearum, iron permeases  function in the reductive iron uptake pathway but do not play a major role in pathogenicity  <cit> .
metal ion homeostasis is interdependent, linking iron availability to that of other metals, like zinc.
zinc itself was reported to be a determinant in cell-cell signaling in c. albicans biofilm formation and regulated by the transcription factor, zap <dig>  <cit> .
zap <dig> has been identified as a regulator of yeast-hypha balance in biofilms through intercellular signaling.
zap <dig> promotes accumulation of farnesol, an inhibitor of hypha formation in yeasts.
zap <dig> promotes accumulation of a postulated diffusible yeast cell inhibitor via the control of the zinc transporter, ztr <dig>  zap <dig> activates the expression of the zinc transporter homologs, zrt <dig> and zrt <dig> .
overexpression of zrt <dig>  improves the growth of the zap1_/zap1_ mutant on a low-zinc medium  <cit> .
in our study two transporters, ztr <dig> and ztr <dig>  were up-regulated in the mf-fruit interaction and may be related to low zinc levels on fruit peels.
interestingly, ztr <dig> was down-regulated in cells interacting with fungal mycelia.
zap <dig> is required for full production of farnesol.
farnesol is believed to be generated from the ergosterol biosynthetic intermediate, farnesyl pyrophosphate  <cit> .
c. albicans biofilm growth is associated with the overall upregulation of ergosterol biosynthesis as well as increased resistance to antifungal compounds that target ergosterol  <cit> .
ergosterol biosynthetic genes  are oppositely regulated by zap <dig> in c. albicans and s. cerevisiae.
erg is positively regulated by sczap <dig> in s. cerevisiae and negatively regulated by cazap <dig> in c. albicans <cit> .
the apparently opposite roles of zap <dig> in erg gene regulation in the two organisms may arise from the difference in growth conditions.
in our study we observed up-regulation of a group of genes involved in ergosterol metabolism and signaling; erg <dig>  erg  <dig>  erg <dig>  psd and dap <dig> .
because of the relationship between drug resistance and membrane ergosterol composition, many genes of the ergosterol biosynthetic pathway have been analyzed in c. albicans.
pasrija et al.
<cit>  showed that suppressing the expression of an squalene epoxidase erg <dig>  leads to increased sensitivity to a number of drugs, including fluconazole and cycloheximide.
CONCLUSIONS
the results presented in this study provide the first use of rna-seq, and de novo assembly, to conduct a comprehensive analysis of the transcriptome of m. fructicola cells that are in contact with fruit peel tissue or p. digitatum mycelia.
de-novo assembly of the illumina sequences generated a large number of new hypothetical proteins, which were categorized into  <dig> functional groups, in m. fructicola.
the identification of m. fructicola genes associated with biocontrol interactions will form the basis of understanding the biology of biocontrol systems and mechanisms associated with effective biocontrol agents.

 considerable research has been devoted in the past two decades to investigate the use of naturally occurring yeasts for managing postharvest diseases on a variety of fruits and vegetables. at present, only two commercial products are available for postharvest use, each having a very small market share of the collective technologies used to manage postharvest diseases. biosave used for the control of pome fruit, sweet potato, and potato diseases, and “shemer” used commercially for the control of sweet potato and carrot storage diseases, strawberry, apple and citrus fruit. knowledge about mechanisms of action of yeast biocontrol agents used to manage postharvest diseases is relatively superficial because appropriate methods to study microbial interactions in wounds of fruit has been lacking. various mechanisms, however, have been described, including antibiosis, production of lytic enzymes, parasitism, induction of host resistance, and competition for limiting nutrients and space. results described in these studies are mostly correlative and provide indirect evidence for possible involvement of one or more of these components in the mechanism of action. this may be due to the lack of an understanding of the key biochemical and molecular processes occurring within tritrophic interactions that define the effectiveness of a biocontrol system. a characteristic response of fruit to pathogen attack or application of a biocontrol agent is the activation of defence responses, including production of inhibitors of cell wall-degrading enzymes of the pathogen, activity of antifungal compounds, active oxygen species, and reinforcement of the cell wall of the host. in relation to yeast biocontrol agents, induction of resistance in fruit tissue has been reported to involve, to a greater or lesser extent, the active elicitation of all of the above responses. hershkovitz et al. recently reported the first molecular study of the response of citrus fruit to the application of the yeast biocontrol agent, m. fructicola. results indicated that significant changes in gene expression occur in grapefruit wounded tissue in response to the application of m. fructicola including changes in a variety of genes involved in response to biotic and abiotic stresses, signaling, defence and secondary metabolism. these findings imply that complex biochemical and molecular processes are involved in the reaction of fruit host tissue to the introduction of yeast cells, which may have the potential to influence the efficacy of the biocontrol agent. castoria et al. indicated that the ability to tolerate high levels of reactive oxygen species produced in fruit tissue in response to wounding is an essential characteristic of effective yeast antagonists. macarisin et al. demonstrated that the antagonistic yeasts, candida oleophila and m. fructicola, actually induce defence-related oxidative responses in intact fruit exocarp tissues and within wounds of apple and citrus, generating greater levels of super oxide anion and hydrogen peroxide. torres et al. reported that, in addition to nutrient competition, the biocontrol activity of pantoea agglomerans could be attributed to the ability of the antagonist to induce an oxidative response in orange fruit peel tissue by triggering h2o production, superoxide dismutase, and catalase activities. in another study, transient exposure of the yeast m. fructicola to a sublethal heat or oxidative stress increased overall abiotic stress tolerance, providing cross-protection to a range of other more severe abiotic stresses. the study showed that higher levels of trehalose, induced by exposure to a sub-lethal abiotic stress, was associated with an increase in ros scavenging, general stress tolerance, and more rapid growth in apple wounds, collectively resulting in improved biocontrol activity. larralde-corona et al. investigated the effect of the exposure of the yeast pichia guilliermondii to penicillium digitatum on yeast gene expression by analyzing expressed sequencing tags obtained by subtractive suppressive hybridization. the yeast was exposed to starvation by carbon source, actively growing mycelium of p. digitatum separated from yeast cells with membrane and induction by fungal cell walls. only one est, associated with energy metabolism, was obtained in response to the limited carbon source, seven ests in the separated membrane system, all relating to metabolic networks such as: energy, nitrogen, cell cycle, abc transporters, response to stress and one unknown sequence. the exposure of the yeast cells to fungal cell walls produced the highest number of ests, a total of including all the metabolic networks mentioned above plus ests associated with signal transduction. compared to the information available about the response of host tissue to the introduction of yeast biocontrol agents, little is known about global molecular changes taking place in yeast cells in response to an interaction with either fruit tissue or a pathogen's mycelia. in the current study, we characterized transcriptional changes in m. fructicola cells following their interaction with grapefruit peel tissue and mycelium of p. digitatum using next generation sequencing technology for rna on the illumina platform. along with de-novo assembly and characterization of the transcriptome, analysis of global patterns of gene expression and functional categorization were performed.  illumina sequencing, assembly, and annotation transcriptome analysis of m. fructicola utilizing illumina sequencing generated a total of, reads. the raw reads were assembled into contigs by trinity software with an average size of  bp and median of 349 bp. the sequences were determined to represent unigenes with an average length of  bp and median of 385 bp. the size distribution is shown in figure  to annotate these genes, reference sequences were searched using blastx within the non-redundant ncbi nucleotide database using a cut-off e-value of 10-. a total of genes could not be annotated based on matches in the ncbi database. a high level of transcripts from the individual libraries could be mapped to the assembled transcriptome using bowtie software. these transcripts were blasted against the m. fructicola draft genome with cutoff e-value of 1e- and identity >85%. according to this analysis unigenes could be aligned to the genome, indicating high accuracy of our assembly. the remaining unigenes could be a result of erroneous assembly. as shown in figure  42% of sequences had significant homology matches in the ncbi database, while 58% of the sequences had matches ranging between e10- and e10- the similarity distribution showed that 11% of the sequences had > 90% homology, followed by 86% of the sequences ranging between to 90% homology. only 3% of the sequences had homology lower than 50%. species specific distribution indicated that 69% of the sequences matched gene sequences of the yeast clavispora lustiniae, followed by other yeasts debar-yomyces hansenii, scheffersomyces stiptis, meyerozyma guilliermondii, spathaspora passa-lidarum, millerozyma farinose and candida tenuis. the phylogenetic analyses presented in figure 2d shown close sequence similarity between m. fructicola and c. lustiniae. classification of gene ontology to classify the functions of m. fructicola unigenes, we searched the gene ontology database and used the wego tool for plotting go annotation results. a total of sequences out of were categorized into functional groups. in each of the three main categories of the go classification the'cell and cell part','antioxidant and binding' and'metabolic and cellular processes' terms were the most dominant. in contrast,'metallochapreon', and'rhythmic process' were least abundant. transcription profiles rna-seq technology is a sensitive and reliable tool to study transcriptional changes. using this technique we identified genes differentially expressed with at least a -fold change in yeast cells in response to yeast 24 h after interaction with p. digitatum. among these, were upregulated and were downregulated. in the mf-fruit interaction, genes had differential expression where were upregulated and were downregulated, while genes were regulated in both interactions. de genes in either mf-pdig and mf-fruit interactions or specific to one of these interactions are shown in a two-dimensional hierarchical cluster heat map and venn diagram additional file functional analysis of differentially expressed genes all differentially regulated genes were organized in functional categories based on their gene ontology terms and were members of at least one of the three principal go branches: biological process, molecular function, and cellular component. all functional groups were found to be over-expressed in mf-pdig interaction compared to all transcribed genes of m. fructicola controls. we focused our analyses on “biological process” categories. those categories were organized in three groups: response to stress and stimulus transport, transport and metabolic processes. the induced genes in the category of response to stress in mf-pdig interaction are mainly involved in multidrug resistance and resistance to singlet oxygen species and in oxidation-reduction activity in the mf-fruit interaction. the genes encoding enzymes involved in response to heat stress were, such as heat shock proteins hsp hsp hsp and sgt were repressed, in both of interactions. transport genes involved in iron transport and uptake, zinc transport and sulp anion were upregulated in the mf-fruit interaction while in the mf-pdig interaction, ammonium permease, inorganic phosphate transporter and low-affinity manganese transporter were induced. a glucose sensor and two glucose transporter genes were induced in the mf-pdig interaction. a group of genes associated with transmembrane transport, such as general amino acid permease, oligopeptide transporter, a permease, which mediates uptake of glycerophosphoinositol and glycerophosphocholine as sources for inositol and phosphate, an a sugar transporter, were induced in both interactions. functional analysis of the metabolic processes category included lipid, cofactor and cellular amino acid metabolic process annotations. many genes involved in the ergosterol-biosynthesis pathway were up-regulated in the mf-fruit interaction. many genes involved in amino acid metabolism were up-regulated in the mf-pdig interaction. in the mf-fruit interaction, two groups of genes involved in cellular amino acid metabolism were found to be enriched: one up-regulated and the second, down-regulated. real time pcr analysis to confirm the expression profiles obtained from the rna-seq expression data, rt-qpcr analysis was carried out for genes selected on the basis of their biological significance: genes involved in the degradation of fungal cell wall, such as glucanse and chitinase, were selected as were genes related to response to stresses, such as an abc transporter, a transcription factor, a heat shock protein, superoxide dismutase, a serine threonine kinase, and a signal transducing mek kinase. although minor variation of transcription levels were obtained, of the tested genes correlated well with the fold-change, either up or down- obtained in the rna-seq analysis. time points included in the rt-qpcr analysis were and 48 h after interaction of the yeast cells with either grapefruit peel or p. digitatum mycelia. expression level of the sod gene in rt-qpcr was two-fold higher compare to the rna-seq data set. transcript levels of glu were induced in m. fructicola cells after and 48 h in the mf-pdig interaction. chi levels were up-regulated in both mf-pdig and mf-fruit interactions after 24 h and 48 h. stb expression level displayed transient induction in both mf-fruit and mf-pdig interactions at 24 h, while the ser-thr kinase was transiently repressed in both mf-fruit and mf-pdig interactions at 24 h. the abc transporter was induced in the mf-pdig interaction at and 48 h, while in the mf-fruit interaction it was induced only at 24 h. expression levels of sod and mekk were repressed in the mf-pdig interaction at all-time points tested compared to control cells grown in nydb media. in the mf-fruit interaction these genes showed a transient induction at and 24 h. hsp transcript levels increased following contact with either the fruit peel or p. digitatum mycelia but were down-regulated in comparison to the control, m. fructicola cells grown in nydb. discussion information about the interactions between yeast biocontrol agents, pathogens, and plant hosts is needed to better understand biochemical and molecular processes involved in the biocontrol system. in a previous study, we identified global changes in gene expression in grapefruit peel tissue following the application of the yeast metschnikowia fructicola. marked changes in the profiles of genes expressed in fruit tissue were observed in response to application of yeast into wounded fruit tissue. in the present study, the main objective was to compare changes in gene expression in m. fructicola cells following the interaction of the yeast with either grapefruit peel tissue or p. digitatum mycelia. although the genome of m. fructicola has not been sequenced, de-novo assembly of the transcriptome of m. fructicola resulted in the identification of a total of unigenes, half of which could be annotated based on homology to genes in the ncbi database. as shown in figure 2c, 69% of the unigene sequences identified in m. fructicola showed highest homology to clavispora lustiniae genes. thus the rna-seq based transcriptome analysis of m. fructicola generated a large number of newly identified yeast genes, providing a substantial contribution to the existing sequence database. using rna-seq, we compared transcriptomes of m. fructicola after the yeast were exposed to either grapefruit peel tissue or after coming into direct contact with the fungal mycelia of p. digitatum. our analysis identified more than genes that were differentially expressed and that may potentially affect the biocontrol activity of m. fructicola. the number of genes regulated in mf-fruit interaction approximately same that regulated in mf-pdig and the overlap between the two interaction is only % of genes co-regulated. stress related genes activation of stress-related genes in yeast biocontrol agents induced in response to the pathogen or plant host has been observed in several biocontrol systems. in the current study, several genes involved in oxidation – reduction activity and implicated in oxidative stress response, such as peroxidases and reductases, were up-regulated in the mf-fruit interaction. these results could be associated with the generation and detoxification of reactive oxygen species. m. fructicola cells produce ros as in response to contact with fruit tissue. these studies indicate that ros plays a major role in the initial response of yeast cells to host tissue and act as a signal, inducing an oxidative burst in the fruit tissue which subsequently leads to the induction of resistance mechanisms in the host tissue. the time course study of superoxide dismutase gene expression showed a transient induction in mf-fruit samples at and 24 h. these results provide further evidence that yeast cells are undergoing oxidative stress while in contact with fruit tissue and suggest the possibility of an adaptive response. in this regard, liu et al. demonstrated that a mild heat-shock pretreatment improved the tolerance of m. fructicola to subsequent high temperature and oxidative stress and increased the biocontrol efficacy of the yeast. on the other hand, lower or unaltered expression levels of sod and low levels of ros in mf-pdig interaction indicate that in this type of interaction, yeast cells are not under oxidative stress and hence activation of protective systems against superoxide radicals is not necessary. high levels of oxidative stress in microorganisms is accompanied by the induction of heat-shock proteins in an attempt to ameliorate damage to proteins caused by ros. induction of hsps in response to biotic and abiotic stimuli is well documented where they assist in the folding and unfolding, and general stability, of various proteins. production of hsps in microorganisms is associated with various abiotic stresses, including oxidative stress. in our present study, genes encoding hsp and hsp were down regulated in both types of interactions compared to the control. however, the time course study of hsp expression by rt-qpcr revealed significantly higher levels of transcript after 12 h, and 48h in both interactions compared to its level at 5 h. this result is surprising since it was anticipated that hsps would be induced in yeast cells that are in contact with fruit peel tissue or with fungal mycelia considering the stressful environment it represents, such as desiccation due to the dryness of the fruit surface, ros, and phytoalexins. response to chemical stress genes belonged to plasma membrane atp-binding cassette transporters, such as yhk yor and snq were upregulated in the mf-pdig interaction. yor and sng genes were induced in s. cerevisiae following exposure to the monoterpene thymol. the function of these transporters in s. cerevisiae and their possible role in yeasts tolerance to monoterpene have yet to be elucidated. in pichia guilliermondii, two abc transporter related proteins involved in the secretion of toxic compounds were induced during its interaction with p. digitatum. many of the abc transporters are reported to be tightly regulated by transcription factors. in this regard, the current study showed that stb a transcription factor involved in regulating multidrug resistance and oxidative stress response, was transiently upregulated in the mf-pdig interaction at 24 h. induction of abc transporters in m. fructicola cells may be needed for the yeast to tolerate exposure to fruit surface wounds or to mycelia where many potentially toxic substances may be present. this explanation may also be relevant to the ability of yeasts to tolerate high levels of ros in apple fruit wounds which was suggested to be an essential characteristic of a successful biocontrol agent. hydrolytic enzymes lytic enzymes, such as glucanases and chitinases, involved in degradation of fungal cell walls have been considered to play an important role in the biological control of postharvest pathogens. jijakli and lepoivre indicated that p. guillermondii, a yeast biocontrol agent, exhibits a high level of β- -glucanase activity that could function to degrade the cell walls of postharvest pathogens. p. membranefaciens had higher β- -glucanase and exochitinase activity but less endo-chitinase activity than c. albidus in the presence of fungal cell walls. several studies have demonstrated the ability of yeasts to produce and secrete glucanases and chitinases in culture medium amended with fungal cells walls. the ability of yeasts to produce exo-ß- -glucanase and chitinase has been suggested to play a role in the firm attachment of yeast cells to fungal hyphae and the partial degradation of fungal mycelia. the current study clearly indicated that glucanase and chitinase genes are up regulated when yeast cells were in contact with p. digitatum mycelia. when yeast cells were in contact with grapefruit peel tissue, however, only chi was up-regulated. the fact that chi expression is induced in both exposures to fruit peel tissues and to fungal mycelia may indicate that chitinases are involved in several biological interactions and not specifically in pathogen cell wall degradation. iron homeostasis many pathogens utilize secreted siderophores and/or high-affinity uptake systems to acquire iron, and many are able to utilize ferritin, transferrin, lactoferrin, heme and heme-containing proteins for cellular processes related to virulence. iron competition was reported as the main mode of action of m. pulcherrima inhibition of botrytis cinerea, alternaria alternata and p. expansum. iron is essential for fungal growth and pathogenesis. iron metabolism has been well characterized in the model yeast saccharomyces cerevisiae. there are two different pathways that allow iron uptake from the environment: the reductive iron-uptake pathway mediated by fet3p, a multicopper oxidase, and ftr1p, an iron permease localized on the plasma membrane; and the siderophore-mediated uptake pathway. in our transcriptional analysis, we identified homologs of transmembrane ferric reductases, iron permease, and an iron transporter all of which were up-regulated in the mf-fruit interaction. this information provides a clue to the potential important role of the reductive iron uptake system in the interaction of m. fructicola with fruit tissues. iron competition was reported as the main mode of action in the ability of m. pulcherrima to inhibit botrytis cinerea, alternaria alternata and p.expansum infection of apples stored at 1°c for 8 months under controlled atmosphere. m. fructicola, similar to m. pulcherrima, produces a red pigment that is believed to be associated with the binding of iron. iron permease, in candida albicans, was shown to mediate iron acquisition from transferrin and was required for systemic infection. in the plant pathogenic fungus, fusarium graminearum, iron permeases function in the reductive iron uptake pathway but do not play a major role in pathogenicity. metal ion homeostasis is interdependent, linking iron availability to that of other metals, like zinc. zinc itself was reported to be a determinant in cell-cell signaling in c. albicans biofilm formation and regulated by the transcription factor, zap. zap has been identified as a regulator of yeast-hypha balance in biofilms through intercellular signaling. zap promotes accumulation of farnesol, an inhibitor of hypha formation in yeasts. zap promotes accumulation of a postulated diffusible yeast cell inhibitor via the control of the zinc transporter, ztr zap activates the expression of the zinc transporter homologs, zrt and zrt. overexpression of zrt improves the growth of the zap1_/zap1_ mutant on a low-zinc medium. in our study two transporters, ztr and ztr were up-regulated in the mf-fruit interaction and may be related to low zinc levels on fruit peels. interestingly, ztr was down-regulated in cells interacting with fungal mycelia. zap is required for full production of farnesol. farnesol is believed to be generated from the ergosterol biosynthetic intermediate, farnesyl pyrophosphate. c. albicans biofilm growth is associated with the overall upregulation of ergosterol biosynthesis as well as increased resistance to antifungal compounds that target ergosterol. ergosterol biosynthetic genes are oppositely regulated by zap in c. albicans and s. cerevisiae. erg is positively regulated by sczap in s. cerevisiae and negatively regulated by cazap in c. albicans. the apparently opposite roles of zap in erg gene regulation in the two organisms may arise from the difference in growth conditions. in our study we observed up-regulation of a group of genes involved in ergosterol metabolism and signaling; erg erg erg psd and dap. because of the relationship between drug resistance and membrane ergosterol composition, many genes of the ergosterol biosynthetic pathway have been analyzed in c. albicans. pasrija et al. showed that suppressing the expression of an squalene epoxidase erg leads to increased sensitivity to a number of drugs, including fluconazole and cycloheximide.  the results presented in this study provide the first use of rna-seq, and de novo assembly, to conduct a comprehensive analysis of the transcriptome of m. fructicola cells that are in contact with fruit peel tissue or p. digitatum mycelia. de-novo assembly of the illumina sequences generated a large number of new hypothetical proteins, which were categorized into functional groups, in m. fructicola. the identification of m. fructicola genes associated with biocontrol interactions will form the basis of understanding the biology of biocontrol systems and mechanisms associated with effective biocontrol agents. 
this study provides new insight into the biology of the tritrophic interactions that occur in a biocontrol system such as the use of the yeast, m. fructicola for the control of green mold on citrus caused by p. digitatum. based on homology, sequences were annotated with a gene description, gene ontology, and clustered into functional groups. we performed a transcriptome analysis, using rna-seq technology, to examine the response of m. fructicola with citrus fruit and with the postharvest pathogen, penicillium digitatum. in the antagonist-pathogen interaction, genes related to transmembrane, multidrug transport and to amino acid metabolism were induced. in the antagonist-fruit interaction, expression of genes involved in oxidative stress, iron homeostasis, zinc homeostasis, and lipid metabolism were induced. approximately 50% of the unigenes could be annotated based on homology matches in the ncbi database. an analysis of differential expression when the yeast was interacting with the fruit vs. the pathogen revealed more than genes with specific expression responses. patterns of gene expression in the two interactions were examined at the individual transcript level by quantitative real-time pcr analysis. the yeast metschnikowia fructicola is an antagonist with biological control activity against postharvest diseases of several fruits.
 the yeast metschnikowia fructicola is an antagonist with biological control activity against postharvest diseases of several fruits. we performed a transcriptome analysis, using rna-seq technology, to examine the response of m. fructicola with citrus fruit and with the postharvest pathogen, penicillium digitatum.  more than million sequencing reads were assembled into unigenes. approximately 50% of the unigenes could be annotated based on homology matches in the ncbi database. based on homology, sequences were annotated with a gene description, gene ontology, and clustered into functional groups. an analysis of differential expression when the yeast was interacting with the fruit vs. the pathogen revealed more than genes with specific expression responses. in the antagonist-pathogen interaction, genes related to transmembrane, multidrug transport and to amino acid metabolism were induced. in the antagonist-fruit interaction, expression of genes involved in oxidative stress, iron homeostasis, zinc homeostasis, and lipid metabolism were induced. patterns of gene expression in the two interactions were examined at the individual transcript level by quantitative real-time pcr analysis.  this study provides new insight into the biology of the tritrophic interactions that occur in a biocontrol system such as the use of the yeast, m. fructicola for the control of green mold on citrus caused by p. digitatum. antagonist-fruit interactionantagonist-pathogen interactionbiological agentrna-seq 