Information about the interactions between yeast biocontrol agents, pathogens, and plant hosts (mainly harvested fruit) is needed to better understand biochemical and molecular processes involved in the biocontrol system [10, 15]. 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 9674 unigenes, half of which could be annotated based on homology to genes in the NCBI database (Figures 1 and 2). 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 (Mf-Pdig) or after coming into direct contact with the fungal mycelia of P. digitatum (Mf-Pdig). Our analysis identified more than 250 genes that were differentially expressed and that may potentially affect the biocontrol activity of M. fructicola (Figure 4). 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 16.5% 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 [14, 16, 17]. In the current study, several genes involved in oxidation – reduction activity (GO: 0055114) and implicated in oxidative stress response, such as peroxidases and reductases, were up-regulated in the Mf-fruit interaction (Additional file 5). These results could be associated with the generation and detoxification of reactive oxygen species (ROS).
M. fructicola cells produce ROS as in response to contact with fruit tissue [7, 11]. 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 (SOD1) gene expression showed a transient induction in Mf-fruit samples at 12 and 24 h (Figure 8E). 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 (30 min at 40°C) improved the tolerance of M. fructicola to subsequent high temperature (45°C, 20–30 min) and oxidative stress (0.4 mol L-1) and increased the biocontrol efficacy of the yeast. On the other hand, lower or unaltered expression levels of SOD (Figure 8E) and low levels of ROS in Mf-Pdig interaction (data not shown) 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 (HSP) 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 HSP78 and HSP104 were down regulated in both types of interactions compared to the control (yeast grown in NYDB) (Additional file 5). However, the time course study of HSP78 expression by RT-qPCR revealed significantly higher levels of transcript after 12 h, 24 and 48h in both interactions compared to its level at 5 h (Figure 8F). 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 (ABC) transporters, such as YHK8, YOR1 and SNQ2, were upregulated in the MF-Pdig interaction (Additional file 6). YOR1 and SNG2 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 STB5, a transcription factor involved in regulating multidrug resistance and oxidative stress response, was transiently upregulated in the Mf-Pdig interaction at 24 h (Figure 8C). 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 (e.g. ROS, phytoalexines, toxins) 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 .
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 [23–25]. Jijakli and Lepoivre  indicated that P. guillermondii, a yeast biocontrol agent, exhibits a high level of β-1,3-glucanase activity that could function to degrade the cell walls of postharvest pathogens. P. membranefaciens had higher β-1,3-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 [16, 25, 28]. The ability of yeasts to produce exo-ß-1,3-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 (GLU) and chitinase (CHI) genes are up regulated when yeast cells were in contact with P. digitatum mycelia (Mf-Pdig). When yeast cells were in contact with grapefruit peel tissue (Mf-fruit), however, only CHI was up-regulated. The fact that CHI expression is induced in both exposures to fruit peel tissues and to fungal mycelia (Figure 8B) may indicate that chitinases are involved in several biological interactions and not specifically in pathogen cell wall degradation.
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 (FET3), iron permease (FTR1), and an iron transporter (SIT1) all of which were up-regulated in the Mf-fruit interaction (Additional file 6). 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 (2% O2 and 3% CO2) . M. fructicola, similar to M. pulcherrima, produces a red pigment that is believed to be associated with the binding of iron (unpublished data). Iron permease (CaFTR1), 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 (FgFtr1 and FgFtr2) 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, Zap1 . Zap1 has been identified as a regulator of yeast-hypha balance in biofilms through intercellular signaling. Zap1 promotes accumulation of farnesol, an inhibitor of hypha formation in yeasts. Zap1 promotes accumulation of a postulated diffusible yeast cell inhibitor via the control of the zinc transporter, ZTR2. Zap1 activates the expression of the zinc transporter homologs, ZRT1 and ZRT2 (zinc regulated transporter). Overexpression of ZRT2, improves the growth of the zap1_/zap1_ mutant on a low-zinc medium .
In our study two transporters, ZTR3 and ZTR2, were up-regulated in the Mf-fruit interaction and may be related to low zinc levels on fruit peels. Interestingly, ZTR2 was down-regulated in cells interacting with fungal mycelia. Zap1 is required for full production of farnesol. Farnesol is believed to be generated from the ergosterol biosynthetic intermediate, farnesyl pyrophosphate [32, 33]. 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 (ERG) are oppositely regulated by Zap1 in C. albicans and S. cerevisiae. ERG is positively regulated by ScZAP1 in S. cerevisiae and negatively regulated by CaZAP1 in C. albicans. The apparently opposite roles of Zap1 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; ERG1, ERG 11, ERG5, PSD and DAP1 (Additional file 7). 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 ERG1, leads to increased sensitivity to a number of drugs, including fluconazole and cycloheximide.