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Children born to mothers who drink during pregnancy are at risk for growth retardation, memory, learning, and cognitive deficits under a lifelong disability known as Fetal Alcohol Spectrum Disorders (FASD), which occurs at a high rate in the US (~1/100 live births) and worldwide. There are three outstanding features of FASD. (a) Humans have been associated with alcohol consumption dated as far back as 7000 BC (McGovern et al., 2004) and there is no sign of waning. (b) FASD can range across a large spectrum of severity, from the more severe Fetal Alcohol Syndrome (FAS) (encompassing facial, brain, and gross deformity) to the hard to detect, subtle mental dysfunctions. The Centers for Disease Control and Prevention (CDC) indicates that approximately 7–8% of pregnant women consume alcohol in US, but diagnosed FASD occurs in a much smaller percentage (CDC, 2012). There is strong evidence to indicate that genetic makeup is a major contributing factor to the differential vulnerability to FASD. (c) Alcohol's deleterious effect has recently been found to go beyond cellular toxicity, to affect epigenetics. The epigenetic chemical code, methylation and acetylation written on top of genomic base elements (e.g., DNA cytosine and histone tails), can confer 3D DNA packaging and fundamentally alter gene transcription. Alcohol has recently been recognized to have strong influences on methylation and acetylation (see Resendiz et al., 2014a) via alcohol metabolism. Furthermore, current evidence points to alcohol's influence on the interaction of genetic and epigenetic factors. These fascinating new views are the center of this eBook, which includes new data and an in-depth discussion of the recent findings and expert opinions. It is hoped that by elucidating the genetic x epigenetic (GxE) interaction at the center of fetal alcohol exposure, new insight will lead the community of scientists toward a greater understanding of this disease, and lay a foundation for prospective new treatments and interventions. FASD is, theoretically, an avoidable disease. Unfortunately, there are many misconceptions and a lack of general public awareness of the profound causality of alcohol on developmental dysfunction. This eBook includes a public awareness effort with an Opinion from Singh et al. (2014), challenging the research community to convey the message that “at no time during pregnancy is alcohol 100% safe to drink.” Is the differential vulnerability of mother and offspring to alcohol derived from the genetic difference rendered by the physiological environment (e.g., differential maternal placenta) or the fetal genotypes themselves? The forum begins with a study to tease out this entanglement. Gilliam (2014) analyzes this question by experimentally transferring the fertilized blastocysts of the alcohol sensitive C57BL/6N (B6) mice to that of alcohol resistant DBA/2 (D2) dams, and vice versa. The finding is intriguing, and the result is supported by another design directly exposing the embryos of B6 and D2 (with identical alcohol conditions) in an incubator thereby bypassing maternal influence (e.g., Ogawa et al., 2005; Chen et al., 2011) and further by in vivo study between lines of genetically diverse mice (see Loucks and Carvan, 2004; Anthony et al., 2010; Downing et al., 2012). These studies indicated that abnormal neurodevelopment depends not only on the dose and pattern of alcohol exposure but also on interactions among environmental, genetic, and maternal factors. Which genes underlie the abnormal development featured in FASD? The Smith et al. (2014) chapter has a succinct discussion of the multiple genomic factors that can increase vulnerability to facial deficits through pathways such as calcium-mediated neural crest apoptosis. Additionally, multiple genomic factors acting on different genetic backgrounds (e.g., in W98S vs. W98D chicks) may contribute to the differential sensitivity of the ethanol-imposed apoptosis. What differences lie within the genome that really impact the genetic contribution to alcohol vulnerability and resistance? To ultimately answer this question requires an interrogation of the entire genome at single nucleotide resolution. The paper “Genomic signatures in the differential vulnerability to fetal alcohol in B6 and D2 mice” did just that (Lossie et al., 2014). The single nucleotide variant (SNV) analysis captured ~900 genes on promoter regions where transcription factors bind (e.g., Eya2, Csmd3) that may affect transcription and on non-coding regions that may result in missense mutations leading to abnormal protein formation. How does alcohol utilize GxE to turn neuroprogenitor cells away from their normal course? Goldowitz et al. (2014) used a B6XD2 mice crossing to demonstrate that alcohol exposure alters the γH2AX histone to mediate differential cerebral cortical apoptosis between genetic lines. It appears the neuroprogenitor cells used an intricate epigenetic program to guide neuronal differentiation and maturation. Resendiz et al. (2014b) present a timely and in-depth discussion of the normal neurodevelopmental epigenetic program and how alcohol deregulates this program. Alcohol exposure alters gene promoter methylation, histone modification, and deregulates non-coding RNA that challenges canonical gene expression and can result in an observable phenotype. Imprinted genes are known to play a particularly important role in human growth and development. Masemola et al. (2015) examined human blood cells to show that an FAS cohort in South Africa undergo CpG methylation changes in imprinting control regions (ICRs) (e.g., lower KvDMR1 and PEG3 DMR) that control allele specific gene expression. If such changes are also carried in the brain, they may contribute to neurodevelopmental abnormalities seen in FASD. As discussed in Kleiber et al. (2014), genetics and epigenetics are intertwined and changes to this dynamic may last into adulthood, contributing to FASD (Lo and Zhou, 2014). These GxE interactions are also elucidated through examples presented in the previously introduced papers (Lossie et al., 2014; Smith et al., 2014). The complex interplay of genetic and epigenetic factors are also showcased in the altered hypothalamic-pituitary-axis axis, which may account for causality in mental impairment and cancer (Mead and Sarkar, 2014). Additionally, DNA methylation changes, if carried through the germ line, may affect multiple generations subsequent to alcohol exposure (Mead and Sarkar, 2014). Such complex transmission may be established through imprinting of parental genes, which defies Mendelian inheritance (Tunc-Ozcan et al., 2014). Further, validations of transgenerational effects of alcohol exposure are needed, and are in progress. In summary, this eBook reveals that FASD has a genetic propensity perturbed by an environmental input which may in part be registered through epigenetics. Abnormal epigenetic marks may be accumulated over time or even generations. This emphasizes our original message that no specific level of drinking is ever safe during pregnancy. Finally, treatment and prevention of FASD would be best addressed by taking both genetic and epigenetic factors into consideration.

Epigenetic regulation is essential for growth and development in eukaryotic organisms (Henikoff et al., 2008; Suzuki and Bird, 2008) and is also responsible for the establishment, maintenance, and reversal of non-genetic cellular memory that records developmental and environmental cues, including those arising from biotic and abiotic stress (Bonasio et al., 2010). A series of recent stimulating papers, show that biotic stress can trigger a transgenerational epigenetic response in plants, where DNA methylation seems to play a central role. Plants sense and respond to environmental cues by a repertoire of mechanisms that regulate gene expression in order to maximize chances of survival in hostile environments. In addition to preformed defense traits, plants have evolved inducible defenses against microbial pathogens, herbivores, and even other plants that involve the regulation of gene expression for the synthesis of defensive secondary metabolites and specific proteins (Walling, 2000; Howe and Jander, 2008; Mithofer and Boland, 2012). Plants rely on the innate immunity of each cell and on systemic signals emanating from infection sites (Jones and Dangl, 2006). Plant hormones play essential roles during systemic defense signaling (Robert-Seilaniantz et al., 2011). Salicylic acid (SA) primarily triggers resistance against biotrophic and hemibiotrophic pathogens whereas a combination of jasmonic acid (JA) and ethylene (ET) signaling activates resistance against necrotrophic pathogens (Glazebrook, 2005; Robert-Seilaniantz et al., 2011). SA acts as an endogenous signal involved in systemic acquired resistance (SAR), an inducible resistance against a broad spectrum of pathogens including viruses, bacteria, and fungi that cause necrosis through rapid programmed cell death of infected cells, known as the hypersensitive response (Durrant and Dong, 2004). SAR induces resistance in the systemic (uninoculated) plant organs in response to local infection (Vlot et al., 2009). Recent evidence shows that NON-EXPRESSOR of PATHOGENESIS-RELATED GENES 3 (NPR3) and NPR4 are SA receptors that bind SA with different affinities and regulate degradation of the transcription cofactor NPR1 in a SA-dependent manner (Fu et al., 2012). In addition to their role during plant development, JA and JA-related compounds, including methyl-jasmonate (MeJA) and jasmonate-isoleucine conjugate (JA-Ile), play essential roles during endogenous regulation of plant resistance to mechanical wounding and herbivory by modulating global changes in gene expression (Creelman and Mullet, 1997; Sheard et al., 2010). Priming (or sensitization) refers to the enhanced ability for the quicker and more effective activation of specific cellular defense responses upon previous exposure to biotic or abiotic stress (Conrath et al., 2002, 2006). Defense pathways and priming can also be induced by application of chemical stimuli including the non-protein amino acid beta-amino-butyric acid (BABA) (Zimmerli et al., 2000). In addition to plant hormones, small RNAs and genes involved in the biogenesis of small RNAs are also components of the plant defense strategies against herbivory and microbial pathogens (Pandey et al., 2008; Ruiz-Ferrer and Voinnet, 2009; Katiyar-Agarwal and Jin, 2010). Here we comment on a series of papers that provide evidence on transgenerational epigenetic effects during biotic stress. Rasmann et al. (2012) report transgenerational priming responses (TPR) in tomato and Arabidopsis induced by caterpillar herbivory (Helicoverpa zea and Pieris rapae) and application of MeJA. TPR results in a reduction of up to ~40% in caterpillar weight gain and this effect persists after one stress free generation in Arabidopsis (Rasmann et al., 2012). Experiments with the coronatine insentitive1 (coi1-1) and the triple dicer-like2/dicer-like3/dicer-like4 (dcl2/dcl3/dcl4) mutants show that the TPR depends on JA perception and on components of the RNA-dependent DNA methylation pathway involved in de novo DNA methylation (RdDM) (Law and Jacobsen, 2010; Rasmann et al., 2012). In order to assay transgenerational effects in response to the biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (PstDC3000) in Arabidopsis, Luna et al. (2012) recurrently inoculated a set of parental lines (P0) with increasing doses of PstDC3000. Fitness of progeny (P1) from PstDC3000 infected plants did not differ statistically from control plants progeny (C1) (Luna et al., 2012). However, P1 plants showed a statistically significant reduction in the colonization and disease symptoms after inoculation with: (1) the oomycete pathogen Hyaloperonospora arabidopsidis and (2) PstDC3000-lux (a bioluminescent strain of PstDC3000). Once established, this TPR can be maintained over one stress-free generation. Similar experiments employing parental lines in an npr1-1 mutant background showed that colonization reduction by H. arabidopsidis depends on a functional NPR1 gene. Consistent with an NPR1-dependent priming of SA-inducible defense, a faster and stronger induction of SA-inducible defense genes such as PATHOGENESIS-RELATED GENE 1 (PR1), WRKY6, WRKY53, and WRKY70 was also observed in P1 plants (Luna et al., 2012). Antagonistic effects between JA and SA pathways were confirmed by inoculating P1 plants with the nectrotrophic fungus Alternaria brassicicola (an inducer of JA-dependent defense response). P1 plants contained similar endogenous levels of JA, JA-Ile, JA-precursor, and SA to C1 plants, but showed increased hyphal colonization and a reduction in the expression of JA-inducible genes, including PLANT DEFENSINE 1.2 (PDF1.2). The priming effects on SA and JA inducible genes correlate with the NPR1-dependent deposition of epigenetic marks characteristic of active chromatin (H3K9ac; acetylation of Lys-9 on Histone 3) in the case of PR-1, WRKY6 and WRKY53; and repressive chromatin (H3K27me3; trimethylation of Lys-27 on Histone 3) in the case of PDF1.2. Although a role for chromatin modifications mediating the TPR wasn't ruled out, assays with the domains rearranged methyltransferase 1 and 2/cytosine methyltransferase 3 (drm1/drm2/cmt3) triple mutant, affected in DNA methylation, showed that this mutants mimics the TPR observed in P1 plants and indicates that components of the RdDM pathway are required for TPR (Luna et al., 2012). Similar experiments from Slaughter et al. (2012) using either BABA or an avirulent Pst strain also showed a TPR against PstDC3000 and H. arabidopsidis, and an IBS1 (IMPAIRED BABA-INDUCED STERILITY RESPONSE 1)-dependent increased expression of PR1, PR2, and PR5. However, in sharp contrast to Luna et al. and Rasmann et al., the TPR was only maintained in the immediate progeny which might be the result of differences in experimental approaches (recurrent vs. single inoculation/stimulation) or actually represent biological differences in the molecular mechanism(s) involved (Slaughter et al., 2012). High resolution genomewide profiling of the DNA methylation landscape in Arabidopsis from Dowen et al. (2012) shows that global disruption of establishment and maintenance of DNA methylation in a set of mutants including drm1/drm2/cmt3 and methyltransferase 1 (met1) enhances resistance to bacteria and induces widespread dynamic changes in methylation. Distinct patterns of differentially methylated regions (DMR) can be observed when wild type plants are exposed to SA or either virulent or avirulent Pst strains. SA treatment uncovered a class of 21-nt siRNAs particularly evident at transposable elements (TEs)–associated DMRs and in many cases, SA-induced DMR associate with reprogramming of TEs and neighboring genes (Dowen et al., 2012). Taken together, this set of papers unveiled a TPR during biotic stress in plants where DNA methylation and components of the RdDM pathway play a major role. Some open questions: what is the molecular nature of the TPR signal? What cells are responsible for sensing and transmitting the biotic stress memory? What is the impact of plant's age and intensity of the biotic stress on the TPR? Can the TPR behave as a paramutation-like phenomena?