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Transcriptomic de novo analysis of pitaya (Hylocereus polyrhizus) canker disease caused by Neoscytalidium dimidiatum
Min Xu1,
Cheng-Li Liu1,
Juan Luo2,
Zhao Qi1,
Zhen Yan1,
Yu Fu1,
Shuang-Shuang Wei1 &
Hua Tang ORCID: orcid.org/0000-0001-9891-16941
Canker disease caused by Neoscytalidium dimidiatum is the most serious disease that attacks the pitaya industry. One pathogenic fungus, referred to as ND8, was isolated from the wild-type red-fleshed pitaya (Hylocereus polyrhizus) of Hainan Province. In the early stages of this disease, stems show little spots and a loss of green color. These spots then gradually spread until the stems became rotten due to infection by various strains. Canker disease caused by Neoscytalidium dimidiatum poses a significant threat to pitaya commercial plantations with the growth of stems and the yields, quality of pitaya fruits. However, a lack of transcriptomic and genomic information hinders our understanding of the molecular mechanisms underlying the pitaya defense response.
We investigated the host responses of red-fleshed pitaya (H. polyrhizus) cultivars against N. dimidiatum using Illumina RNA-Seq technology. Significant expression profiles of 23 defense-related genes were further analyzed by qRT-PCR. The total read length based on RNA-Seq was 25,010,007; mean length was 744, the N50 was 1206, and the guanine-cytosine content was 44.48%. Our investigation evaluated 33,584 unigenes, of which 6209 (18.49%) and 27,375 (81.51%) were contigs and singlets, respectively. These unigenes shared a similarity of 16.62% with Vitis vinifera, 7.48% with Theobroma cacao, 6.6% with Nelumbo nucifera and 5.35% with Jatropha curcas. The assembled unigenes were annotated into non-redundant (NR, 25161 unigenes), Kyoto Encyclopedia of Genes and Genomes (KEGG, 17895 unigenes), Clusters of Orthologous Groups (COG, 10475 unigenes), InterPro (19,045 unigenes), and Swiss-Prot public protein databases (16,458 unigenes). In addition, 24 differentially expressed genes, which were mainly associated with plant pathology pathways, were analyzed in-depth.
This study provides a basis for further in-depth research on the protein function of the annotated unigene assembly with cDNA sequences.
Pitaya, also known as dragon fruit, is an important tropical and subtropical fruit tree. At present, there are two known species of pitaya: the red-fleshed pitaya (Hylocereus polyrhizus) and the white-fleshed pitaya (Hylocereus undatus). However, the most cultivated and commercialized species is the red-fleshed pitaya with its red pulp and pericarp (H. polyrhizus) that is enriched with anthocyanins [1, 2]. Pitaya belongs to the Cactaceae and its native tropical zone is Latin America, which includes Mexico, and Central and South America [3]. Currently, it is also extensively grown throughout tropical and subtropical regions worldwide [4], especially in Asian countries, such as Malaysia, Vietnam, Thailand, Philippines and China. Presently, the main areas of pitaya cultivation (especially the red dragon fruits) include the Hainan, Guangxi, Guangdong, Yunnan, Fujian and Taiwan provinces of China.
Pitaya has been proven to be very adaptable to environmental conditions and disease pressures. However, the rapid spread of stem canker, a disease caused by Neoscytalidium dimidiatum among red-fleshed pitaya (H. polyrhizus), has become a serious threat to some commercial plantations (Fig. 1). The disease was first reported in Taiwan province of China, and features a distinctive canker on stems and fruits, and quickly spread to most commercial cultivation areas [5]. Shortly thereafter, it was discovered and reported in Guangdong Province of China, Malaysia, Israel, and Florida with similar characteristics [6,7,8,9,10]. Various studies have reported the incidence of canker disease caused by Neoscytalidium dimidiatum and have provided basic information relating to the identity and isolation of pathogenic strains.
The impact of the canker disease on the pitaya stems and fruits. a Diseased stems in pitaya plantation; b Diseased fruits in pitaya plantation
Generally, two main recognition systems are associated with the response to an attack by the microorganism. One system recognizes the pathogen-associated molecular patterns (PAMPs) via pattern-recognition receptors (PRRs), which are localized on the membrane and activate an innate immune response, or more specifically PAMP-triggered immunity (PTI) [11]. The PAMPs are structural molecules, such as bacterial peptidoglycan and flagellin, oomycete glucans, lipopolysaccharide harpin, xylanase and chitin [12, 13]. In contrast, PRRs are cell surface-localized receptors, most of which are receptor-like kinases (RLKs) or receptor-like proteins (RLPs). The RLKs contain extracellular, transmembrane, and cytoplasmic kinase domains, whereas the RLPs lack the kinase domain [14]. A burst of reactive oxygen species (ROS) is a typical reaction in early cellular PTI events; activation of the mitogen-activated protein kinase (MAPK) cascade and expression of immune-related genes are also associated with such events [15]. The second system is mediated by intracellular immune receptors, which directly or indirectly recognize virulence effectors of pathogens that are secreted within host cells, thereby inducing effector-triggered immunity (ETI). In the second system, pathogen effectors are recognized by intracellular receptors, which are referred to as resistance (R) proteins, and mainly include leucine-rich repeats (LRR) or the LRR-like family, toll/interleukin-1-receptor (TIR), and serine/threonine kinases (S/TK) to activate the ETI response [14, 16]. Compared with PTI, the activation of ETI by receptors localized to the nucleus appears to be more directly associated with transcriptional regulation of the expression of defense genes [17]. However, some ambiguous theories exist regarding PTI and ETI, as it also appears that both can be robust or weak, depending on the interaction, defense signaling pathways, receptors and environmental conditions [13].
The RNA-Sequencing (RNA-Seq) technique is an excellent method that permits quick and extensive comprehension of the transcriptional level of genes in a variety of plant species using next-generation sequencing technologies [18]. Here, to investigate the genes associated with the defense response of pitaya to canker disease caused by Neoscytalidium dimidiatum in PTI and ETI systems, we used RNA-Seq technology to monitor the transcriptomic profiles of red-fleshed pitaya (H. polyrhizus) in response to fungal invasion. A comparison was made between normal and diseased stem tissuses during transcriptomic analysis, and the significant differentially expressed genes (DEGs) were analyzed in detail. These DEGs were annotated in public protein databases, suggesting that they may play important roles in PTI and ETI systems in response to the invasion of Neoscytalidium dimidiatum. The aim of the present study was to identify the key genes that encode steps in the plant pathological pathways of pitaya. This article provides a solid basis for a better understanding of the transcriptomic response of the red-fleshed pitaya (H. polyrhizus) to canker disease.
Sequencing, de novo assembly and annotation
Healthy and diseased stem tissues of red-fleshed pitaya (H. polyrhizus) (Fig. 2) were collected from the orchard of Ledong County, Hainan Province. Three normal samples (N1, N2, and N3), and three diseased samples (D1, D2, and D3), were used for transcriptional sequencing. However, N1 and D2 examples were abandoned because the results were not consistent. The unigenes assembled from the four remaining samples were then merged. In total, we evaluated 33,584 unigenes, of which 6209 (18.49%) and 27,375 (81.51%) were contigs and singlets, respectively. The total read length was 25,010,007, mean length was 744 bp, the N50 was 1206, and the guanine-cytosine (GC) content was 44.48%, based on RNA-Seq. The range of the unigene lengths in the four samples was 497–702 bp. Details relating to the assembled information from each sample and the merged results are shown in Table 1. In total, 1608 DEGs, with a fold change ≥2.00 or a fold change ≤0.5, and a false discovery rate (FDR) ≤ 0.001, were monitored. On average, the number of unigenes from D samples was 45.7% higher than other samples, reflecting a significant increase in the number of sequences resulting from fungal infection.
Pitaya tissues used for de novo transcriptomic analysis. a Normal stems; b Diseased stems
Table 1 Results of assembled samples
Owing to the lack of a reference genome for H. polyrhizus, the assembled unigenes were blast searched into the Non-redundant (NR), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), InterPro, and SwissProt public protein databases using search tools. The species with the greatest number of H. polyrhizus unigenes were Vitis vinifera (16.62%), Theobroma cacao (7.48%), Nelumbo nucifera (6.6%), and Jatropha curcas (5.35%), as shown in Fig. 3. Creation of a Venn diagram (Fig. 4), showed that there were 7305 unigenes annotated in all databases, 25,161 unigenes annotated in the NR database, 17,895 unigenes annotated in the KEGG database, 10,475 unigenes annotated in the COG database, 19,045 unigenes annotated in the InterPro database, and 16,458 Unigenes annotated in the SwissProt database. These findings provide a basis for further in-depth research on protein function of the annotated unigenes, which were assembled with cDNA sequences.
Species distribution of all unigenes. The x-axis shows the species of annotated unigenes while the y-axis shows the number of unigenes
Venn diagram of unigenes annotated in NR, KEGG, COG, InterPro, and SwissProt protein databases
Gene ontology (GO) functional annotation of DEGs
Based on GO functional annotation, approximately 1608 DEGs were classified into three essential categories, namely biological process, cellular component, and molecular function. The unigenes we identified covered a wide range of functional GO categories. Of the 1608 DEGs classified, most were associated with catalytic activity (173 DEGs, 10.75%), binding (134 DEGs, 8.33%), and transporter (30 DEGs, 1.87%) aspects of molecular function. The biological processes were associated with cellular processes (160 DEGs, 9.95%), metabolic processes (157 DEGs, 9.76%), and single-organism processes (124 DEGs, 7.71%). The cellular component processes were mainly associated with the membrane (134 DEGs, 8.33%), cell (107 DEGs, 6.65%), membrane part (106 DEGs, 6.59%), and cell part (104 DEGs, 6.47%) aspects (Fig. 5). Analysis of the GO terms of pitaya transcripts showed that these genes might have been associated with kinases (catalytic activity) and receptors (cellular and metabolic processes, and the membrane).
Gene Ontology (GO) annotation of differentially expressed genes (DEGs). The x-axis shows the number of genes while the y-axis shows gene function annotation of GO categories
DEGs associated with plant-pathogen interactions
Twenty-four DEGs were associated with plant-pathogen interaction pathways; of these, 23 were up-regulated and one was down-regulated. These unigenes play different roles in the plant-pathogen interaction pathways. Unigene13603_All and Unigene11517_All were annotated in the cyclic nucleotide-gated channel (CNGC) family, which mediates Ca2+ influx from cellular stores in plants [19]. The CNGCs are reportedly Ca2+-permeable channels that interact with the ubiquitous Ca2+ sensor calmodulin, and play important roles in the response to plant hormones, biotic and abiotic stresses, and plant immunity [20,21,22]. Unigene12122_All, Unigene16412_All, and CL2472.Contig2_All were annotated in the calcium-dependent protein kinase family (CDPK). The CDPKs are crucial sensors of changes in calcium concentration and play multiple roles in plant development and growth, abscisic acid (ABA)-mediated processes, jasmonic acid (JA) biosynthesis, plant tolerance to stress, and plant fungal stimuli interaction [23,24,25,26,27,28]. The CDPKs are phosphorylated by phosphatase and participate in the hypersensitive response (HR). Furthermore, another calcium-related gene, Unigene7838_All, was annotated in two types of Ca2+-sensing proteins, calmodulin and calmodulin-like (CaM/CML) (Fig. 6). The CaM/CML protein family plays complex and versatile roles in modulating cellular responses to various stimuli, particularly biotic stresses in grapevine [29]. The ROS produced by respiratory burst oxidase homologs are associated with multiple signal transduction pathways in diverse biological processes in plants [30]. In our research, an up-regulated Unigene (13565_All) was annotated in the respiratory burst oxidase homologs (Rboh) membrane-bound enzyme family, which is also known as the NADPH oxidases [31]. In tomato plants, Rboh activity and H2O2 signaling are important components of melatonin-induced stress tolerance [32]. This unigene may therefore encode the most critical enzyme of pitaya associated with ROS production under conditions of Neoscytalidium dimidiatum infection.
Differentially expressed genes (DEGs) associated with plant-pathogen interactions in H. polyrhizus. The original plant-pathogen interaction pathway was downloaded from the KEGG database (https://www.genome.jp/dbget-bin/www_bget?map04626). Genes in red frames were up-regulated, those in green frames were down-regulated, and those in black frames showed no change in regulation
A plant-pathogen interaction pathway (Fig. 6) showed that the flagellin-sensing 2 (FLS2) family was significantly up-regulated, and included Unigene13867_All, Unigene9087_All, CL1260.Contig2_All, Unigene13234_All, Unigene24332_All, and Unigene15298_All. Furthermore, FLS2 is a well-known PRR kinase that recognizes a conserved 22 amino acid N-terminal sequence of the bacterial flagellin protein (flg22) [33]. Previous research has shown that FLS2 contains three domains, which include an extracellular LRR domain, a transmembrane domain and a cytoplasmic kinase domain [34]. The FLS2 receptor complex is directly coupled with Arabidopsis heterotrimeric G proteins to regulate immune signaling via both pre-activation and post-activation mechanisms [35]. Our transcriptome data showed that LRR receptor-like serine/threonine-protein kinases (LRR-RLKs) were differentially expressed and included Unigene13867_All, CL1043.Contig1_All, and Unigene9087_All. The LRR-RLKs play pivotal roles in plant growth, development, cellular signal transduction, brassinosteroid (BR) and ABA signal pathways, and responses to biotic and abiotic stresses [36]. The unigenes we detected that were associated with FLS2 may play important roles in the HR and defense-related gene induction in response to fungal infection.
Moreover, three LRR classes of plant disease resistance genes PRS [37] (Unigene8050_All, Unigene15133_All, and Unigene14674_All); a resistance PBS gene (Unigene24332_All) that encodes a serine/threonine-protein kinase [38]; a pto-interacting protein gene PTI (Unigene24688_All) that encodes a serine/threonine kinase [39]; and a receptor for the fungal elicitor ethylene-inducing xylanase (EIX) that binds with EIX to induce the HR in tobacco and tomato [40] (Unigene24332_All), were significantly up-regulated in pathogen interaction pathways. These DEGs may participate in the HR, defense-related gene induction or programmed cell death (Fig. 6). The expression profiles of DEGs in four samples asociated with plant-pathogen interactions are presented in the pheatmap show in Fig. 7, and in Table 2.
Heatmap of differentially expressed genes (DEGs) related to the defense response. Red indicates up-regulated expression, whereas green indicates down-regulated expression
Table 2 Expression profiles of unigenes associated with plant pathological interactions
Predicted expression of transcription factors (TFs)
Transcription factors (TFs) are proteins that, when in contact with RNA polymerase, can confirm a transcription initiation complex and participate in the transcription initiation process. Generally, a TF contains a transcriptional activation/repression domain and a transcriptional binding domain [41]. As a regulator, a TF regulates the expression of genes downstream of a pathway by binding with DNA sequences either by itself or by interacting with other TFs or proteins. TFs are associated with the significant mechanisms of the regulation of disease resistance genes and receptors for cellular localization and activation, as well as DNA binding and transcription activities [42]. In the merged data of all samples in the present study, a total of 832 transcription factors were predicted, and the majority of the TF families were categorized as MYB (95, 11.42%); MYB-related (77, 9.25%); APETALA2/ethylene-responsive element binding protein (AP2-EREBP, 64, 7.69%); C3H (56, 6.73%); WRKY (52, 6.25%); bHLH (42, 5.05%); NAC (37, 4.45%); and GRAS (34, 4.09%), among others (Fig. 8).
Differentially expressed genes (DEGs) and transcription factor (TF) family classification. The x-axis shows the number of genes while the y-axis shows TF family classification
The TFs make essential contributions to the processes of growth, development, flowering, and biotic and abiotic stresses in the plant. For instance, the family of MYB factors include the conserved MYB DNA-binding domain and appears to be involved in the regulation of the cell cycle in animals, plants and other higher eukaryotes [43]. The AP2-EREBP genes contained the AP2 family, RAV (related to ABI3/MP) family, and ERF (ethylene response factor) family, based on the number of AP2 domains and sequence similarity [44]. The AP2-EREBP gene reportedly plays roles in controlling flower and seed developmental processes, regulatory networks in response to hormones, pathogen attacks and environmental signals [45, 46]. Moreover, NAC TFs reportedly also take part in the plant-pathogen interactions of Arabidopsis, potato, maize, and wheat in response to wounding or pathogen attack as a transcriptional regulator [47]. In our plant pathology interaction pathways, WRKYs (Unigene9452_All, Unigene2169_All, Unigene14674_All, Unigene2538_All, and Unigene16508_All) were significantly and differentially expressed. The WRKY proteins can interact with themselves, VQ-domain proteins, chromatin remodeling proteins, calmodulin-like proteins or some essential MAP kinases [48]. The unigenes identified in the present study may act as downstream products of the MAPK pathway to activate the expression of defense-related genes, or recognize the fungal effector, which may result in the induction of defense-related genes or programmed cell death (Fig. 6). The differentially expressed TFs under investigation may play essential roles in growth, development, leaf senescence and responses to biotic and abiotic stimuli.
qRT-PCR verification
To validate the RNA-Seq data, 23 unigenes (Table 3) that were significantly expressed, most of which were associated with phytopathology, were selected for additional qRT-PCR analysis. These unigenes included one ethylene-responsive TF (Unigene12062_All); two LRR receptor-like serine/threonine-protein kinases (Unigene13867_All and Unigene9087_All); one salicylic acid-binding protein (CL2401.Contig2_All); two CDPK-related kinases (Unigene12122_All and Unigene16412_All); three disease resistance proteins (Unigene2329_All, Unigene8050_All, and Unigene15133_All); two RLPs (Unigene24332_All and Unigene15298_All); four WRKY TFs (Unigene9452_All, Unigene2169_All, Unigene2538_All, and Unigene16508_All); one auxin response factor (Unigene27293_All); one NADPH-related protein (Unigene24688_All); one lipoxygenase protein (CL1613.Contig2_All); and five differentially expressed proteins (CL2068.Contig2_All, CL1097.Contig1_All, CL1813.Contig1_All, Unigene2331_All, and Unigene9233_All). These genes were either significantly up- or down-regulated in diseased tissues. The results of the qRT-PCR assay showed that 20 of the 23 genes selected for qRT-PCR (three biological replicates) conformed to the RNA-Seq data, with an accuracy of 87%. These results (Fig. 9) indicated that the RNA-Seq findings were both stable and reliable.
Table 3 Gene expression in normal (N) and diseased (D) tissues based on qRT-PCR
Validation of gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Gene expression levels were measured by qRT-PCR and compared with RNA-Seq results. Histograms represent the fold changes of genes (D3/N3) by qRT-PCR, whereas line charts represent gene expression according to the log2 ratio (fragments per kilobase of transcript per million mapped reads (FPKM) of D/FPKM of N) in RNA-Seq. All genes selected for qRT-PCR analysis were analyzed in three biological replicates. Bars represent the ± SEΔCт of three experiments. Details of the genes selected for qRT-PCR are provided in Table 3
The cultivation of pitaya is an emerging industry with a bright future and high commercial value, especially for red-fleshed pitaya (H. polyrhizus). However, canker disease caused by Neoscytalidium dimidiatum presents the most devastating attacks on pitaya plantations at present [9]. The most efficient solution to prevent this disease is to cut off the infected tissues and bury them in soil away from the plantation. In addition, molecular breeding is the best way to acquire disease-resistant cultivars; however, this is not easily done with pitaya. Moreover, additional tasks need to be completed to verify which cultivar is the most resistant among a variety of pitaya species.
Plant immunity is a complicated process that is effected mainly in response to extracellular or intracellular receptors that recognize PAMPs or effectors in PTI and ETI systems [13, 14]. The defense response mechanisms and roles of the receptors have been previously reported in Arabidopsis, tomato, and rice, among others [34, 35, 37, 39, 40, 49, 50], but has been scarcely described in pitaya. In our transcriptome results, higher expression levels of unigenes were detected in H. polyrhizus and 24 DEGs associated with the plant-pathogen interaction pathway. Most of these genes are signal receptors, protein kinases, and TFs, which may play critical roles in the plant-pathogen interaction networks.
Calcium acts as an important conserved second messenger in plant immune and stress responses [51]. Calcium is sensed by Ca2+-binding proteins in signaling networks, including calmodulin (CaM), calmodulin-like (CML), CDPKs, and calcineurin B-like proteins [19, 52]. The CNGCs are calmodulin-permeable cation transport channels that have the potential to integrate signals from cyclic nucleotide and Ca2+ signaling pathways [53]. In our transcriptome analysis, seven unigenes (Table 2) related to Ca2+ levels and signaling pathways were annotated and showed significant changes in expression levels in diseased red-fleshed pitaya. These genes included two CNGCs related genes (Unigene13603_All and Unigene11517_All); one CaM/CML (Unigene7838_All); three CDPK genes (Unigene12122_All, Unigene16412_All, and CL2472.Contig2); and one Rboh family gene (Unigene13565_All). These genes act as receptors or kinases in the HR, cell wall reinforcement, and stomatal closure (Fig. 6).
Pathogenesis-related (PR) proteins comprise one of the major sources of plant-derived allergens, and are generally induced by various types of pathogens, such as viruses, bacteria and fungi [54]. In pepper, PR4b reportedly interacts with LRR proteins and PR4c, as a plasma membrane-localized cysteine protease inhibitor, to trigger cell death and the defense signaling response [55, 56]. In the present study, we also monitored four differentially expressed PR homologous proteins, which were annotated in the SwissProt and InterPro public protein databases. Among these four proteins, two were up-regulated (Unigene9313_All and Unigene11745_All), and the other two were down-regulated (CL1813.Contig1_All and CL1271.Contig2_All). Further work, involving protein function verification and genetic transformation, is now being carried out to investigate their roles in the response to the fungal infection of H. polyrhizus.
A total of 832 TFs were monitored in the present study. The TF proteins are key factors in the regulatory networks that control development, metabolism, and the responses to biotic and abiotic stresses in numerous plant species. In our RNA-Seq data, MYB and MYB-related family genes comprised the largest group, which accounted for 20.67%. A MYB domain protein, COLORED1 (C1), which is associated with anthocyanin synthesis, was first identified in maize [57]. The MYB TF family is a major player in drought and cold responses [58, 59], the phenylpropanoid metabolic pathway [60], and JA and ABA signal transduction pathways [61, 62]. The NAC TFs comprise a large protein family, with 138 putative NAC genes in Arabidopsis, as listed in the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.cbi.pku.edu.cn/) [63]. However, 37 (4.45%) putative NAC genes in H. polyrhizus were identified based on our RNA-Seq data. The NAC family of genes have a highly conserved N-terminal DNA-binding domain and a variable C-terminal domain. The NAC TFs participate in the response to drought, salinity, and/or low temperatures and regulate JA-signaled defense responses in various plants [64, 65]. In addition, 52 WRKY superfamily TF genes were monitored in the present study and five unigenes (Unigene 9452_All, Unigene 14674_All, Unigene 2538_All, Unigene 2169_All, and Unigene 16508_All) were differentially expressed upstream in red pitaya. The WRKY genes are quite common in plants, which contain a conserved N-terminal sequence of WRKYGQK, along with a Zn finger-like motif (C2H2 type or C2HC type) [66]. The trend in qRT-PCR expression levels of four WRKY unigenes (Unigene 9452_All, Unigene 2169_All, Unigene 16508_All, and Unigene 2538_All) were consistent with the RNA-Seq data (Fig. 10 and Table 3). The Unigene 14674_All was an unnamed protein product, which was annotated to both WRKY and RRS1-R gene families. Pathway results suggested that the five differentially expressed WRKY unigenes were important candidate genes of H. polyrhizus involved in plant pathology interactions (Fig. 6).
Bioinformatics analysis of the de novo transcriptome study processes
Our findings may pave a fundamental step to a better understanding of the defense responses of pitaya. We have provided hypotheses for the functions of the genes identified, according to public protein annotation databases. However, specific details of the roles of these genes in pitaya defense responses pose a relevant and interesting challenge. Thus, further research is now required to clarify how significant DEGs regulate pitaya defense processes via genetic transformation, when infected by Neoscytalidium dimidiatum.
At present, transgenic research is the best method with which to clarify the functions of a gene. However, this is difficult to achieve in tropical and subtropical non-model plants. Three major difficulties are associated with transgenic research in pitaya. Firstly, there have been problems associated with the establishment of a tissue culture system. Secondly, we need to consider the stability of the tissue culture system and the resolution of issues if stability is unfavorable. Finally, the period required to screen a disease-resistant cultivar is relatively long. Nevertheless, in spite of the difficulties faced in the study of non-model plants, transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4 were reported recently following a 3-year field trial [67]. That was a major breakthrough for the banana industry and could also imply a brighter future for the pitaya industry. Thus, the problems associated with transgenic pitaya should be solved eventually with further broad-scale research.
Plant materials and culture conditions
Healthy and diseased stem tissues of red-fleshed pitaya (H. polyrhizus) were collected from the orchard of Ledong County, Hainan Province. These plants had been growing for approximately five years in a plantation under natural conditions. Fungal spores were isolated from diseased tissues, and morphological and molecular analyses were conducted to determine the species of Neoscytalidium dimidiatum 8 (ND8). The article about isolation of ND8 has been published in Australasian Plant Pathology [68]. The collected stem tissues were divided into two groups with similar characteristics in triplicate within each group. The stems were cut into pieces and immediately frozen in liquid nitrogen. The three replicates of the normal group were named N1, N2, and N3, whereas the three replicates of the diseased group were named D1, D2, and D3. Three biological replicates, each consisting of four stem cutting bases, were frozen in liquid nitrogen and stored at − 80 °C until further analysis. Sample tissues were transferred to Shenzhen BGI Tech Company (Shenzhen, China) for transcriptome de novo assembly and sequencing using an Illumina HiSeq system.
Total RNA extraction
Tissues (100 mg) were placed into a precooled mortar to be ground into a powder with liquid nitrogen. The ground stem powder was collected into a precooled 2 mL microcentrifuge tube containing 2 mL RNase-free cetyltrimethylammonium bromide (CTAB) extracting solution, which contained 0.1% diethyl pyrocarbonate (DEPC), 2.5% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), and 4% polyvinylpyrrolidone (PVP). The tubes were then placed in a water bath at 65 °C for 30 min to induce cell lysis, and centrifuged at 13,000 rpm for 5 min at 25–28 °C. The residue was discarded, and the supernatant was transferred into a clean 2 mL microcentrifuge tube. Trichloromethane (1 mL) was added to the supernatant solution, mixed lightly, and centrifuged at 13000 rpm for 15 min at 4 °C. The supernatant was then transferred into a clean 2 mL microcentrifuge tube, and this procedure was repeated once more. Subsequently, 8 M lithium chloride was added with RNase-free water to 0.6 volume of the supernatant and left overnight. The mixture was centrifuged for 30 min at 13000 rpm and 4 °C to obtain an RNA precipitate. RNase-free ethyl alcohol (75%) was used to wash the RNA precipitate twice. Finally, RNA was dissolved in RNase-free ddH2O and stored at − 80 °C until further use.
Construction of the cDNA library and de novo RNA-Seq
Poly (A) mRNA from the total RNA was enriched by poly-T oligo-attached magnetic beads (Invitrogen, Thermo Fisher Scientific, CN), according to the manufacturer's protocol. Purified mRNA was then divided into short fragments by mixing with fragmentation buffer. First strand cDNA was synthesized using a random hexamer primer. Buffer, dNTPs, RNase H, and DNA polymerase I were then added to the fragmented mRNA templates to synthesize second-strand cDNA. The double-stranded cDNA was purified using magnetic beads. End repair, and the addition of 3′-end single nucleotide A (adenine), were then performed. Finally, sequencing adaptors were ligated to the fragments, which were enriched by PCR amplification. During the quality control step, a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and a StepOnePlus Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA) were used to qualify and quantify sequences for the sample libraries. The constructed library products were finally prepared for sequencing using a HiSeq 2000 system (Illumina, San Diego, CA, USA) at the Beijing Genomics Institute (BGI).
Filtering of raw reads and the assembly of clean reads
Before any further analysis could be conducted, we needed to consider quality control of the data. In addition, low-quality sequences were filtered out of the raw data to reduce data noise. We defined "dirty" raw reads as those that contained adapter sequence, high levels of unknown bases, and low-quality reads. These needed to be removed before data analysis. Filtering steps were as follows: 1) Reads with adapters were removed; 2) reads in which unknown bases were more than 10% were removed; and 3) low-quality reads were removed (the percentage of low-quality bases is usually over 50% in a read, and we defined a low-quality base to be one with a sequencing quality of no more than 5). After filtering, the remaining reads were referred to as "clean reads" and used for downstream bioinformatics analysis. Clean data were obtained and stored in the FASTQ format after filtering.
Clean reads were assembled using the Trinity paired-end assembly method [69]. The assembled clean reads, also referred to as transcripts, were clustered and this eliminated any redundancy in obtaining unigenes using the TIGR Gene Indices clustering tools (TGICL) [70]. For the study of multi-samples, the previous step using the TGICL for downstream analysis of the unigenes was repeated. The processed unigenes were referred to as "All-Unigenes." All-Unigenes were divided into two parts; one part included clusters of a few unigenes with more than 70 similarities among them (starting with "CL," followed by the gene family ID); and the other included singletons (starting with "Unigene"). The clean data arising from high-throughput sequencing (RNA-Seq) results, and the processed files, have been submitted to the Gene Expression Omnibus (GEO) database at the GenBank website with accession number GSE119976. The process of preliminary bioinformatics analysis was performed according to the flow diagram in Fig. 10.
Functional annotation and the detection of differentially expressed unigenes
After assembly, H. polyrhizus pitaya All unigenes (contigs and singletons) were functionally aligned to the public protein databases, NR, GO, KEGG, COG, InterPro, and SwissProt. All unigenes were annotated according to their sequence similarity to previously annotated genes in these public databases. The phyper function of R software (https://www.r-project.org) was used for enrichment analysis, according to the results of GO and KEGG pathway gene annotations in functional analyses. The p-value was corrected by the FDR. We considered that unigenes with a FDR ≤ 0.01 were significantly expressed in the GO and KEGG pathways. Moreover, NOIseq [71] and PassionDis [72] methods were used to test DEGs. A fold change ≥2.00 or fold change ≤0.5, with a Probability ≥0.8 in NOIseq, and fold change ≥2.00 or fold change ≤0.5, with a FDR ≤ 0.001 in PassionDis of unigenes were used to define DEGs in the normal and diseased libraries. According to the results of the DEGs test, the pheatmap function of R software was used for hierarchical clustering analysis.
Verification of DEGs by quantitative reverse transcription PCR (qRT-PCR)
To investigate the stability and accuracy of RNA-Seq, 23 DEGs that were possibly associated with plant pathology, were selected for real-time quantitative RT-PCR (qRT-PCR). Total RNA extraction was performed as described in previous steps. Following treatment with DNase I (Thermo Fisher Scientific, USA), first strand cDNA synthesis was conducted according to the manufacturer's protocols using the PrimeScript™ 1st strand cDNA synthesis kit (code No.6210A, Takara, Japan). qRT-PCR was performed using a 20-μL reaction system, containing 10 μL of SYBR® Premix Ex TaqII (Tli RNase Plus) (2×), 2 μL cDNA templates, 0.8 μL PCR forward primer (10 μM), 0.8 μL PCR reverse primer (10 μM), 0.4 μL ROX reference Dye II (50×) and 6 μL of nuclease-free water. The qRT-PCR assays (three technical replicates) were carried out using a 7500 Applied Biosystems qRT-PCR System (Life Tech, USA). The primers of all genes selected for qRT-PCR were designed using Primer Premier 6 software (www.premierbiosoft.com). The pitaya ubiquitin gene (UBQ) was used as an internal reference control for data normalization. The primers for UBQ were as follows: F: 5′-TGAATCATCCGACACCA-3′; and R: 5′-TCCTCTTCTTAGCACCACC-3′ [73]. Reactions were carried out under the following conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. Output data were generated using the Applied Biosystems 7500 software version 2.0.6 (ABI, America). All reactions were performed with two independent biological replicates, and the expression levels calculated for each sample were based on three technical replicates. The 2-ΔΔCT method was used for qRT-PCR data analysis, in which PCT. (test) = CT (target, test) - CT (reference, check); ΔCT (calibrator) = CT (target, calibrator) - CT (reference, calibrator); and ΔΔCT = ΔCT (test) - ΔCT (calibrator). The expression ratio of the experimental and control groups was 2-ΔΔCT.
ABA:
abscisic acid
brassinosteroid
CaM/CML:
calmodulin and calmodulin-like
CDPK:
CNGC:
cyclic nucleotide-gated channel
COG:
Clusters of Orthologous Groups
DEGs:
differentially expressed genes
EIX:
ethylene-inducing xylanase
ERF:
ethylene response factor
ETI:
effector-triggered immunity
FLS2:
flagellin-sensing 2
GC:
guanine-cytosine
hypersensitive response
jasmonic acid
leucine-rich repeats
LRR-RLKs:
LRR receptor-like serine/threonine-protein kinases
Non-redundant
PAMPs:
pathogen-associated molecular patterns
Pathogenesis-related
PRRs:
pattern-recognition receptors
PTI:
PAMP-triggered immunity
Rboh:
respiratory burst oxidase homologs
RLKs:
receptor-like kinases
RLPs:
receptor-like proteins
RNA-Seq:
S/TK:
serine/threonine kinases
TF:
TIR:
toll/interleukin-1-receptor
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We would like to thank the Charlesworth Author Services for language editing services.
This work was supported by the Key Research and Development Project of Hainan Province (ZDYF2018080), the Crop Science Postgraduate Innovation Project of Hainan University Tropical Agriculture and Forestry College (ZWCX2018007), the Natural Science Foundation of China (NSFC; 31360364) and the Innovation and Research Project of the General Institutes of Higher Education of Hainan Province in 2018 (Hys2018–18).
The datasets generated during the current study, in the form of raw sequenece reads from the four transcriptome samples of pitaya (Hylocereus polyrhizus) under normal and diseased conditions are available at the NCBI GEO database with the accession number GSE119976.
Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Institute of Tropical Agriculture and Forestry, Hainan University, No.58 Renmin Avenue, Haikou, 570228, Hainan, People's Republic of China
Min Xu, Cheng-Li Liu, Zhao Qi, Zhen Yan, Yu Fu, Shuang-Shuang Wei & Hua Tang
University of Sanya, No.191 Yingbin Avenue Xueyuan Road, Sanya, 572000, Hainan, People's Republic of China
Juan Luo
Min Xu
Cheng-Li Liu
Zhao Qi
Zhen Yan
Yu Fu
Shuang-Shuang Wei
Hua Tang
TH conceived the study and participated in the design of all experiments. XM performed the qRT-PCR analysis, statistical analysis and drafted the manuscript. LCL, QZ, YZ and FY carried out the statistical analysis. LJ and WSS participated in data analysis and discussion about the experiments. All authors read and approved the final manuscript.
Correspondence to Hua Tang.
The orchard of Ledong Country, Hainan Province clarified the permissions granted to collect H.polyrhizus plant materials for research in this article.
Xu, M., Liu, CL., Luo, J. et al. Transcriptomic de novo analysis of pitaya (Hylocereus polyrhizus) canker disease caused by Neoscytalidium dimidiatum. BMC Genomics 20, 10 (2019). https://doi.org/10.1186/s12864-018-5343-0
Red-fleshed pitaya
Transcriptomic
Canker disease


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The Importance of the Constitution and how it relates to the Supreme Court; and the soon confirmation of Justice Neil Gorsuch to the Supreme Court
Capitalism, Communism, Government Mountain
LISTEN NOW! (Or Watch Full Program!) When individuals are running for a potential seat in our leadership, promises are made. When it comes time to elect the candidate for a designated position, we take into consideration what decisions and actions were promised would be carried out. Before President Trump became our current President, he promised to appoint a Supreme Court Justice that would abide by the Constitution. President Trump has fulfilled this promise and nominated Judge Neil Gorsuch to fill the present vacancy in the Supreme Court.
Judge Gorsuch understands the immense responsibility that is being presented to him. "The Supreme Court's work is vital not just to a region of the Country, but to the whole, vital to the protection of the people's liberties under law; and to the continuity of our Constitution, the greatest charter of human liberty the world has ever known." He also understands the Constitution's original intent for the Judicial Branch; not make law, but to interpret those that have already been made. "I respect to the fact that in our legal order, it is for the Congress and not the courts to write new laws. It is the role of judges to apply not alter, the work of the people's representatives."
It is always a vital decision to decide who will be the next President of the United States. What some may not know, is that this decision also brings additional appointees in other areas of government; namely, The Supreme Court. Whoever is elected as President, will also appoint Supreme Court Justices when vacancies present themselves. Rick Joyner and Lt. Gen (Ret.) Boykin discussed the very importance of this matter. Boykin points out, "The most enduring impact of any presidential administration is the Supreme Court Justices." The reason being is the length of time that a Supreme Court Justice will serve compared to the time a President will serve. Boykin continues, "…so when you have presidents appointing Supreme Court justices, they will be there long after that president has left office and they will continue making decisions that would be aligned with that President." Think about the vital decisions that are brought before the Supreme Court and the importance of those who will decide the moral direction that our Country will go. It is important to have a Supreme Court Justice who will not insert their preference, or political leanings, when deciding on a particular court case, but to decide solely based on what is alignment with the Constitution.
The reason that Washington exists is because it is spelled out in the Constitution. Without the awareness of what is said within the Constitution, one would be unaware of just how far we have gone from its original intentions. Instead of laws being created by Congress, in the Legislative Branch, we are seeing laws being created from the Judicial Branch, The Supreme Court. Our freedoms and liberties are not being taken away, we are giving them away. This is only possible because we are not aware of how operations were initially intended to be carried out. When we start giving up our liberties for comfort and convenience, we will find ourselves in a reality that results in Communism.
As Professor of Jurisprudence at Princeton University, Robert George often asks his students how the framers of the Constitution sought to "preserve liberty and prevent tyranny". As he describes, many of his students get the answer wrong, because their answers "reflects a common misunderstanding of the Constitution." George further explains his response by sharing this important matter; "The Constitution did not envision a national government of general jurisdiction – meaning a government that could do whatever it wanted — but rather, a government of enumerated and delegated powers — a government that had authority over only specific areas of American life. All other powers were to be beyond the scope of the national government and reserved to the States or to the American people themselves." He further points how this being the reason why the 9th and 10th Amendments were added to the Constitution. As he describes, "These amendments reinforced the idea that the national government couldn't just assume powers it had not been specifically granted by the Constitution." It is because we do not understand the original intent of the Constitution that we have unknowingly been allowing other branches to create law. We can see now how the power to make law must be given back to Congress, and it must remain within the hands of Congress.
As President Trump continues to address the American people on what is taking place under his Administration, he shares about the present status of Judge Gorsuch's nomination and how the Senate will be voting on this nomination. President Trump details on how the process of making laws is supposed to be carried out. "Elected representatives from all across the country come together, they host hearings, they listen to the concerns of the people, and then they try to write laws that address those concerns and make life better for all Americans." When processes are not instantaneous, but linger and are drawn out, that means they are simply going through the process of how it's ordered in the Constitution. "It's a process that is meant to take time and energy, to ensure that every new law will better serve our wonderful citizens. That's how our democratic process works. The duty of judges; therefore, is not to rewrite the laws, but to uphold the laws and to apply the Constitution as written."
As we have elected the leaders that are now in authority, it is important that we also continue to support their leadership in prayer. We need to pray for our leaders. So many cases are taken before the Supreme Court. Take into consideration Roe v. Wade, which made abortion legal in the United States. If we want God to move, we cannot allow innocent bloodshed. It makes a difference who will be in the Supreme Court.
Allow your understanding of the Supreme Court to grow as you see how important it is to know how America was designed to be carried out through the words of the Constitution, how laws are made, why it matters who our Supreme Court justices are, and so much more. Also shared in this segment: Legislative Branch, Executive Branch, Judicial Branch, Prager University, democracy, Republic, Conservative, Justice Antonin Scalia, Dredd Scott Decision, Constitutional Law, Federalists, slavery, tyranny, liberties, freedom, Rick Joyner, Senate, bipartisan, and Congress. Greg and John shared in this segment.
Courtesy of Mark-Hayes/Shutterstock.com
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You Are Here: Home » Reviews » Laptops » Basic Laptops » Acer Aspire TC-780-UR11 Desktop Review
Acer Aspire TC-780-UR11 Desktop Review
Acer Aspire TC-780 Desktop | Intel Core i5-7400 Quad-Core 3.0 GHz | Choose Ram & HD Size...
2019 Acer Aspire Flagship Desktop | Intel Core i5-8400 6-Core 2.8 GHz | 8GB DDR4 Memory | 512GB...
There's always a thin line between machines you use for work and play. When it comes to the former, the Acer Aspire TC-780-UR11 suffices as an affordable tower desktop that can handle everything from spreadsheets to word documents, with ease. Although it isn't the most powerful in the room, its close looks and enhanced performance over its predecessors propel it to the top in the category. While it doesn't have a dedicated graphics card, it makes up with a strong Kaby Lake processor, generous storage, relatively good expandability, wireless connectivity, and a varied selection of I/0 ports.
The Aspire TC-780-UR11 maintains the same design and measurements as its predecessor the Acer Aspire AMZKi5. It is still small enough fit on an office desk with limited space. Its midtower chassis measures 15.67 by 6.89 by 17.4 inches (HWD), weighs 18.4 pounds; meaning it can literally fit anywhere. It inherits the black painted chassis with a hairline finish from its predecessor, with a top tray that comes handy when you need to charge or sync your smartphone from one of the USB 3.0 ports.
If you need to do a few upgrades here-and-there down the road, it has some real-estate that will allow for expansions. It has two memory slots, although one of them is occupied by an 8GB memory module, so you can throw in an extra 8GB, but each takes a maximum of 16GB.
The system uses a 300-watt power supply, which is fine for a budget desktop. Other expansion options include a single optical drive and a single hard drive bay, so you'll have to choose between the two, as you have only one SATA port to service an additional drive. Included in the package is a wired keyboard and mouse, you'll have to buy a monitor separately, we tested it with our Editors' Choice the Acer R240HY $109.99 at Amazon and the picture output was impressive.
Connectivity is excellent for a budget system. On the front upper-deck there's a tray-loading DVD burner, which is integral in case you need to rip some CDs or install programs from DVDs, though most installations can be done online. You'll also find two USB 3.0 ports, headphone and microphone jacks and an SD card reader on the top face of the system.
The backside has an audio line in/out port, an HDMI port, one USB 3.0 port and a VGA port for that old LCD you've always wanted to use. Internet connectivity comes via 802.11 ac Wi-Fi and Bluetooth 4.0, alongside a Gigabit Ethernet port. For connectivity dated peripherals such as keyboard, mouse and flash drives, you have four USB 2.0 ports, which adds up to the system's impressive connectivity.
Thankfully, there's a spacious 1TB (7200RPM SATA3) hard drive, so you'll have tons of storage space out of the box for your movies, downloads, videos and other files. If you need more, the Acer Aspire TC-780-UR12 has double the RAM and storage –16GB and 2TB respectively. Recently, we've seen SSDs and hybrid drives trickle into the desktop category, but for a budget PC they'd only serve to bump the price, so we're just safe with the traditional rotating hard drives.
Performance is a highlight here, thanks to the system's seventh-generation (Kaby Lake micro-architecture) Intel Core i7-7700 processor, 8GB system memory and Intel HD 630 graphics. This is a notable bump from early this year's iteration Acer Aspire TC (ATC-780-AMZki5 that was outfitted with an Intel Core i5-7400 processor and the same system memory.
This processor clocks 3.6GHz, but can hit 4.2GHz in turbo mode, which is just enough muscle to do some basic-medium multitasking without stuttering.The system comes with Windows 10 Home (64-bit), a stable CPU and enough RAM that make it ideal for day-to-day computing. Essentially, you will be fine using the Aspire for at least the next five years for day-to-day tasks like editing in office apps, video conferencing, and web browsing.
As expected, you won't be doing much of gaming here, since the Intel HD 630 graphics card won't deliver much. Its 3D gaming score is lackluster compared with dedicated gaming PCs but you should be able to play games like Minecraft smoothly at lower resolution. If you need more than that, you'll have to look upwards for a real gaming rig with a discrete graphics card, presumably the budget CyberpowerPC Gamer Ultra GUA3400A gaming desktop.
Worth the buck
The Acer Aspire TC-780-UR11 is a perfect choice for a budget desktop, especially if you want the option to upgrade in your PC in the future. It lacks a dedicated graphics card like the Lenovo Ideacentre 300, but the Acer model's 1TB memory, Wi-Fi connectivity and seventh-generation Intel Core i7 processor makes up for that. On just about all the other factors, the newer iteration and its predecessor, they are effectively equals, and the Wi-Fi means that both can be used in any room in your home with a Wi-Fi signal. That flexibility, versatility and real-estate to grow into something bigger through upgrades make the Aspire UR11 an easy choice to recommend for anyone in the market for a budget desktop PC that will remain relevant for a few more years.
The Acer Aspire TC-780-UR11 is a budget desktop PC with enough features to keep home/office users satisfied. Plus, it throws in Wi-Fi capability and enough expansion for future expansion.
Nice priced
Good connectivity
8GB system memory
Seventh-generation Intel Core i7 processor
Solid performance numbers
The 300-watt power supply limits upgrades
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On this mild, gently snowy Sunday morning, I am enjoying a cup of chai tea and a slice of freshly baked Date Bread. If this first day of March is anything to go by, this month will come in like a lamb and go out like a lion, although I'll admit I don't really know what that phrase means.
I read two awesome books last week - yes, two! They were both such page-turners that I quite literally flew through each of them in a couple of days. The first was the excellent novel, Big Little Lies, by bestselling Australian author Liane Moriarty. This novel begins with one of the main characters, Madeline, singing out in the car, "I'm forty today… forty!", drawing the word out. Her daughter Chloe responds by singing out, "I'm five… five!" This sets the tone for this family, forty-year-old larger-than-life Madeline, who talks and shops as a way to fill her time, second husband Ed, who does his best to keep out of things and keep the peace, precocious five-year-old Chloe, who is just starting kindergarten, seven-year-old Fred, and fourteen-year-old Abigail, Madeline's daughter from her first marriage. Madeline is friends with beautiful yet distant Celeste, mother of rambunctious twins Josh and Max, wife to Perry, a wealthy, charming hedgefund manager who is away on business trips as much as he is at home. These two women befriend Jane, single mother of Ziggy, the child of a one-night stand that went terribly wrong. Shy, quiet Jane is new to the area, and welcomes the other women's offers of friendship and invitations for coffee at their favourite beachside café. When sweet-natured Ziggy is accused of bullying another child, Amabelle, which he adamantly denies, tensions between kindergarten moms mount. This struggle between women who have known each other in the community for years and those who side with timid newcomer Jane increases as readers are drawn along through the six months leading up to the disastrous school Trivia Night, where, due to strong cocktails and lack of catered food, all hell breaks loose and someone dies. Was it murder or an accident? And who was the victim? The cast of characters in this darkly comic mystery include Madeline's ex-husband Nathan, his younger, new-age wife Bonnie and their waif-like daughter Skye, who is also starting kindergarten at the same school as Chloe, much to Madeline's horror. There is Tom, owner and barista at the café where the ladies frequent, Ms Barnes, the kindergarten teacher, and Mrs Lipman, the principal of the school, along with many others on the school parent council. As the author pulls the reader deeper and deeper into the lives and secrets of each family, we learn that all is not what it seems and everyone has something to hide. I don't usually like "women's fiction", as I'm sure I've mentioned before, and I don't like "chick lit", either. I also don't read a lot of "domestic fiction". But this novel somehow blends all three genres and yet transcends them to become much more than the sum of its parts. It manages to be funny while also presenting the serious issues of bullying and abuse with dignity and respect, offering insight into human nature and revealing what lies beneath relationships that may appear, to the public eye, to be perfect. While reading this novel, which I found to be un-put-down-able, I felt as though I were eavesdropping on the gossip of a group of women I had come to know and like. I would recommend this book to just about anyone.
And I just finished reading the selection for next week's book club meeting, Before I Go To Sleep by S J Watson. I have read this book before, and remember really enjoying it, so I thought it would be something a bit different to add to our book list. I enjoyed it less this time, probably because I remembered the ending so found it less of a mystery than before. This novel tells the story of Christine Lucas, who wakes up each morning with no memory of where she is or why there is a strange man in the bed beside her. When she looks in the bathroom mirror, she is horrified to see an aged face looking back at her, and wonders what has happened. The man in bed, Ben, tells her that she had an accident more than twenty years ago, leaving her with retrograde and anterograde amnesia; that is, she is unable to retrieve most memories from before the accident, and she is also unable to create and store new memories. Her amnesia, however, is unusual, in that she can retain new memories each day, while she is awake, for up to about 24 hours, but these are all erased as soon as she goes to sleep, and she wakes up each morning as blank as a slate. She has no recollection of her husband or her life, and Ben must fill her in every day, leaving tasks written on a blackboard for her to do each day to fill her time. We find out that she has been secretly seeing neurologist Dr Nash for treatment, and he has encouraged her to write in a journal as a way to remind herself of the things she has learned about her past and present life. This journal she keeps hidden from Ben, as she senses that he has been lying to her and keeping things from her deliberately. But she is never sure what is truth and what are lies, and she struggles to piece together her reality and her past in order to live more fully. She asks at one point about halfway through the book, "What are we, if not the accumulation of our memories?" By undergoing treatment with Dr Nash and rereading her journal entries, her memories begin to slowly return, in bits and pieces, flashes that appear in an instant and then are gone. These she cannot retain into the next day, but by writing them down, she is able to slowly form a picture of who she was and is. But who can she trust, when those who claim to love her and want to help her offer contradicting stories about her past? This book really made me think about memory, how much it defines who we are and how much we rely on it in so many ways in our daily lives. This nail-biting read is sure to keep you glued to your seat until the very last page, as the tension builds and more truths are revealed. I don't want to spoil anything, so I'll just say that I was hoping for a different ending than the one in the book. Still, it was a great read, and I would recommend it to those who enjoy reading domestic thrillers (is there such a genre as that?), such as The Silent Wife or Gone Girl, or those who liked the excellent, though confusing, film "Memento", which was about a man who could not form new memories and who gets caught up in a complex web of lies and deception.
That's all for today!


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