Search

Background Maternally Expressed Gene 3 (MEG3) is expressed at low levels in placental villi during preeclampsia; however, its roles in unexplained recurrent spontaneous abortion (URSA) remain unclear. In this study, we aimed to explore the relationship between MEG3 and URSA. Methods The differentially expressed lncRNAs (MEG3) and its downstream genes (RASA1) were identified using bioinformatics analysis of Genomic Spatial Event (GSE) database. The expression levels of MEG3 in embryonic villis (with gestational ages of 49–63 days) and primary trophoblasts were determined using quantitative RT-PCR assay. A mouse model of Embryo implantation, Cell Counting Kit-8 (CCK-8), flow cytometry, and Transwell migration assays were performed to determine the implantation, proliferative, apoptotic, and invasive capacities of trophoblast. The level of phosphorylated core proteins in the RAS-MAPK pathway were analyzed using Western blot assay. The mechanisms of MEG3 in the regulation of RASA1 were studied by RNA pulldown, RNA immunoprecipitation (RIP), DNA pulldown, and chromatin immunoprecipitation (ChIP) assays. Results MEG3 had a low expression level in embryonic villis of 102 URSA patients compared with those of 102 normal pregnant women. MEG3 could promote proliferation and invasion, inhibit the apoptosis of primary trophoblast of URSA patients (PT-U cells), as well as promote embryo implantation of mouse. Besides, MEG3 also promoted the phosphorylation of rapidly accelerated fibrosarcoma (Raf), mitogen-activated protein kinase kinase (MEK), and extracellular-signal-regulated kinase (ERK) proteins. The results of RNA pull down and RIP assays showed that MEG3 bound with the enhancer of zeste homolog 2 (EZH2). The DNA pulldown assay revealed that MEG3 could bind to the promoter sequence of the RAS P21 Protein Activator 1 (RASA1) gene. Further, the ChIP assay showed that MEG3 promoted the binding of EZH2 to the promoter region of the RASA1 gene. Conclusions The inactivation of MEG3 in embryonic villi association with URSA; MEG3 inhibited the expression of RASA1 by mediating the histone methylation of the promoter of RASA1 gene by EZH2, thereby activating the RAS-MAPK pathway and enhancing the proliferative and invasive capacities of trophoblasts.

The liver is one of vital organs of the human body, and it plays an important role in the metabolism and detoxification. Moreover, fetal liver is one of the hematopoietic places during ontogeny. Understanding how this complex organ develops during embryogenesis will yield insights into how functional liver replacement tissue can be engineered and how liver regeneration can be promoted. Here, we combine the advantages of single-cell RNA sequencing and Spatial Transcriptomics (ST) technology for unbiased analysis of fetal livers over developmental time from 8 post-conception weeks (PCW) and 17 PCW in humans. We systematically identified nine cell types, and defined the developmental pathways of the major cell types. The results showed that human fetal livers experienced blood rapid growth and immigration during the period studied in our experiments, and identified the differentially expressed genes, and metabolic changes in the developmental process of erythroid cells. In addition, we focus on the expression of liver disease related genes, and found that 17 genes published and linked to liver disease mainly expressed in megakaryocyte and endothelial, hardly expressed in any other cell types. Together, our findings provide a comprehensive and clear understanding of the differentiation processes of all main cell types in the human fetal livers, which may provide reference data and information for liver disease treatment and liver regeneration.

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1&ndash, 6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice&rsquo, s defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae, conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.

Rice blast, caused by Magnaporthe oryzae (M. oryzae), is one of the most destructive diseases in cultivated rice. The Wuling Mountain Area is marked as the key area for the national prevention and control of rice blast disease in China and is the ideal area for M. oryzae research according to its unique natural condition. In this study, a simple, low-cost and effective marker system including RAPD, REMAP, rep-PCR and Avr genes was developed for genetic diversity analysis of 108 M. oryzae isolates collected from the Wuling Mountain Area during 2012–2014. The results showed significant changes in the genetic structures of M. oryzae populations over the 3 years. However, there was no remarkably simple relationship between the genetic lineages and pathotypes (physiological races) of M. oryzae. The main mechanisms of M. oryzae to overcome host genes in the natural conditions of the Wuling Mountain Area might be point mutations or small deletions of gene sequences.

Due to its unique natural conditions, the Wuling Mountain region is a key area of prevention and control of rice blast disease in China and an ideal area for investigation of its causal fungus, Magnaporthe oryzae (M. oryzae) research. A total of 237 M. oryzae isolates collected from the Wuling Mountain region in 2017–2018 were used for the study of genetic diversity and pathogenicity dynamics. A set of differential varieties (DVs), including 7 Chinese DVs, 12 Japanese DVs and 21 monogenic lines, were established, which differentiate the 237 isolates in this study into different physiological races. Cluster analysis of the combined molecular marker system (RAPD, REMAP, rep-PCR and the avirulence (Avr) gene) results showed that Baiyang and Jianshi had the greatest genetic diversity, which is consistent with the pathogenic results. The proportion of the two mating types was roughly equal each year; however, the dominance of the mating type between the two years was opposite. Furthermore, four isolates contained both mating types, which were assumed to be dual maters. Isolates with amplified bands of Avr genes (Avr-Pizt and Avr-Pita) and corresponding pathogenicity were selected for Sanger sequencing. The result showed that point mutations and small indels resulted in the loss of avirulence function, indicating that mutations are potential mechanism for the rich pathogenic and genetic diversity of M. oryzae in the Wuling Mountain region.

Rice blast resistance caused by Resistance (R) genes, most of which encode coiled-coil (CC), nucleotide-binding (NB), and leucine-rich-repeat (LRR) domains, is race-specific. Panicle blast 1 (Pb1) is a panicle blast resistance gene that confers a durable non-race-specific blast resistance to rice. Despite being non-R, Pb1 also encodes a CC-NB-LRR protein. Pb1 specifically interacts with rice WRKY45, a transcription factor (TF) in the rice salicylic acid signaling pathway, through its CC domain, and that this interaction is important for the blast resistance conferred by Pb1. Additionally, barley MLA10, an R protein for Blumeria graminis, specifically interacts with HvWRKY1/2. Here, we investigated whether the blast resistance between conferred by R genes and non-R Pb1 genes is mediated by their interaction with WRKY TFs. We performed targeted yeast two-hybrid and glutathione S-transferase (GST)-pull-down assays, which showed that all the five rice R proteins of the CC-NB-LRR type tested, Pi36, Pib, Pita, Pit, and Piz-t, interacted with WRKY45 and WRKY66 through their CC domains. However, elicitor-triggered immunity due to these R genes was not affected by the knockdown of these WRKY genes. The results suggest that R proteins can potentially transduce defense signals by interacting with WRKY TFs but the potentials are masked by HR-mediated elicitor-triggered immunity responses.

Blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most devastating diseases of rice due to the high variation of the pathogen. Breeding high-yielding rice cultivar with durable resistance to rice blast is a priority in Southern China and in places where rice cultivation is an important branch in farming. Effectiveness and accuracy of resistant cultivar breeding largely depend on the development of markers specific to the target gene. In silico prediction of the resistance (R) Pi39 gene content of the interval was made and hence a candidate gene was identified to develop a perfect insertion–deletion (InDel) based marker for Pi39 gene selection. The Pi39 gene was successfully introgressed in two elite cultivars using both foreground (the InDel) and background (genome-wide microsatellites) genotypic and phenotypic selection. Five selected BC3F3 progeny lines were recovered and showed a high level of blast resistance. At least 97.5% of their genome was inherited from their recurrent par...

Rice blast resistance (R) genes-mediated resistance response depends on various resistance-related genes involved in incompatible interactions. In this work, the expression profiles of innate rice immunity related genes were examined in the mediated resistance response of true/field resistance genes. Three sets of rice near-isogenic lines (NILs) were used: the resistant NILs carrying true resistance genes in the genetic background of the susceptible cultivar Nipponbare (NB), NB-Pib, NB-Pizt, NB-Pik and NB-Pita2; NILs bearing field resistance genes pi21 in the susceptible cultivar Aichiasahi (AA) AA-pi21, Kahei (KHR). The marker gene OsWRKY45 of salicylic acid (SA) signalling was upregulated in all tested cultivars. And, JAmyb (marker gene of jasmonic acid signalling) showed higher upregulation in the resistance lines with nucleotide-binding sites and leucine-rich repeat (NBS-LRR) R genes Pib, Pizt, Pik, Pita2 and Pikahei than in NB and KHS. SalT of abscisic acid (ABA) signalling may be involved in the R/Avr interaction, including Pizt, Pik, pi21 and Pikahei. However, SalT was shown to negatively regulate Pib/AvrPib interaction. OsPR1b and PBZ1 were differentially expressed and strongly activated at a later stage by 48 h post-inoculation. Interestingly, there was evidence that OsPR1b and PBZ1 played an important role in the pi21-mediated response. It was shown that OsRAR1 could be upregulated in the true resistance line NB-Pita2 and the field resistance line KHR, while OsSGT1 and OsHSP90 could be upregulated in all tested lines. The involvement of these genes illustrated the complexity of the downstream signalling pathways in the mediated resistance response of true/field resistance genes.

Hormone crosstalk is pivotal in plant–pathogen interactions. Here, we report on the accumulation of cytokinins (CK) in rice seedlings after infection of blast fungus Magnaporthe oryzae and its potential significance in rice–M. oryzae interaction. Blast infection to rice seedlings increased levels of N6-(Δ2-isopentenyl) adenine (iP), iP riboside (iPR), and iPR 5′-phosphates (iPRP) in leaf blades. Consistent with this, CK signaling was activated around the infection sites, as shown by histochemical staining for β-glucuronidase activity driven by a CK-responsive OsRR6 promoter. Diverse CK species were also detected in the hyphae (mycelium), conidia, and culture filtrates of blast fungus, indicating that M. oryzae is capable of production as well as hyphal secretion of CK. Co-treatment of leaf blades with CK and salicylic acid (SA), but not with either one alone, markedly induced pathogenesis-related genes OsPR1b and probenazole-induced protein 1 (PBZ1). These effects were diminished by RNAi-knockdown of OsNPR1 or WRKY45, the key regulators of the SA signaling pathway in rice, indicating that the effects of CK depend on these two regulators. Taken together, our data imply a coevolutionary rice–M. oryzae interaction, wherein M. oryzae probably elevates rice CK levels for its own benefits such as nutrient translocation. Rice plants, on the other hand, sense it as an infection signal and activate defense reactions through the synergistic action with SA.

Plant basic leucine zipper (bZIP) proteins play an essential role in the genes expression and regulation in higher plants. They have been shown to regulate diverse plant specific phenomena, including germination, floral induction and development, seed maturation, photomorphogenesis, biotic and abiotic stresses. Resistance response mediated by the rice blast resistance gene Pi36 is a typical signal transduction, in which 12 OsbZIP genes were differentially expressed by microarray analyses. To understand the potential function of OsbZIP genes during the defense responses against rice blast, the expression analysis of these OsbZIP genes, in response to the blast fungus inoculation and the related defense signal molecules induction, were further conducted using real-time fluorescent quantitative polymerase chain reaction (PCR) technique. Our data indicates that among the 12 OsbZIP genes, the expression level eight tested genes were differentially regulated and maintained to 96 h points post inoculation in rice resistant and susceptible cultivars during Magnaporthe oryzae infection, and all of them were also significantly up-regulated by one or several kinds of exogenous plant hormones stresses. Although, these genes were induced only at early time points (1 to 24 h); it is evident that the OsbZIP genes may be involved in different signaling pathway, and play potential important functions in the defense response to rice blast. Keywords: OsbZIP transcription factors, rice blast, resistance response, plant hormones stresses. African Journal of Biotechnology Vol. 12(34), pp. 5294-5302

The resistance gene Pi36 confers a stable and high level of resistance against Chinese isolates of the rice blast pathogen Magnaporthe oryzae. Quantitative real time RT-PCR was used to investigate the expression profiles of various resistance-related genes in both the Pi36/AvPi36 incompatible interactions. Among 17 members of the OsMAPK family, OsMAPK2, OsMAPK4, OsMAPK8 and OsMAPK15 were differentially expressed, as were OsWRKY30, OsWRKY32, OsWRKY64 and OsWRKY67 among the 13 OsWRKY members studied. The induction of the expression of genes from both of these families suggests that both the JA and SA pathways are involved in Pi36-mediated defence. Other genes differentially expressed included the OsbZIP transcription factors, OsNIF1 and OsNIF2, and the NPR1 homologue OsNH2, all of which were reported in the rice blast pathosystem. The involvement of these genes illustrates the complexity of the downstream signalling pathways involved in the Pi36/AvrPi36 interaction.

The resistance (R) gene Pi37, present in the rice cultivar St. No. 1, was isolated by an in silico map-based cloning procedure. The equivalent genetic region in Nipponbare contains four nucleotide binding site–leucine-rich repeat (NBS–LRR) type loci. These four candidates for Pi37 (Pi37-1, -2, -3, and -4) were amplified separately from St. No. 1 via long-range PCR, and cloned into a binary vector. Each construct was individually transformed into the highly blast susceptible cultivar Q1063. The subsequent complementation analysis revealed Pi37-3 to be the functional gene, while -1, -2, and -4 are probably pseudogenes. Pi37 encodes a 1290 peptide NBS–LRR product, and the presence of substitutions at two sites in the NBS region (V239A and I247M) is associated with the resistance phenotype. Semiquantitative expression analysis showed that in St. No. 1, Pi37 was constitutively expressed and only slightly induced by blast infection. Transient expression experiments indicated that the Pi37 product is restricted to the cytoplasm. Pi37-3 is thought to have evolved recently from -2, which in turn was derived from an ancestral -1 sequence. Pi37-4 is likely the most recently evolved member of the cluster and probably represents a duplication of -3. The four Pi37 paralogs are more closely related to maize rp1 than to any of the currently isolated rice blast R genes Pita, Pib, Pi9, Pi2, Piz-t, and Pi36.

The elite rice cultivar Yuejingsimiao 2 (YJ2) is characterized by a high level of grain quality and yield, and resistance against Magnaporthe oryzae. YJ2 showed 100% resistance to four fungal populations collected from Guangdong, Sichuan, Liaoning, and Heilongjiang Provinces, which is a higher frequency than that shown by the well-known resistance (R) gene donor cultivars such as Sanhuangzhan 2 and 28zhan. Segregation analysis for resistance with F(2) and F(4) populations indicated the resistance of YJ2 was controlled by multiple genes that are dominant or recessive. The putative R genes of YJ2 were roughly tagged by SSR markers, located on chromosomes 2, 6, 8, and 12, in a bulked-segregant analysis using genome-wide selected SSR markers with F(4) lines that segregated into 3 resistant (R):1 susceptible (S) or 1R:3S. The recessive R gene on chromosome 8 was further mapped to an interval ≈1.9 cM/152 kb in length by linkage analysis with genomic position-ready markers in the mapping population derived from an F(4) line that segregated into 1R:3S. Given that no major R gene was mapped to this interval, the novel R gene was designated as pi55(t). Out of 26 candidate genes predicted in the region based on the reference genomic sequence of the cultivar Nipponbare, two genes that encode a leucine-rich repeat-containing protein and heavy-metal-associated domain-containing protein, respectively, were suggested as the most likely candidates for pi55(t).

The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silico prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The Pi36-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast R genes Pita, Pib, Pi9, and Piz-t. An RT–PCR analysis showed that Pi36 is constitutively expressed in Kasalath.