Genome-wide identification and immune response analysis of mitogen-activated protein kinase cascades in tea geometrid, Ectropis grisescens Warren (Geometridae, Lepidoptera).

BACKGROUND: Tea geometrid Ectropis grisescens (Geometridae: Lepidoptera), is one of the most destructive defoliators in tea plantations in China. The MAPK cascade is known to be an evolutionarily conserved signaling module, acting as pivotal cores of host-pathogen interactions. Although the chromosome-level reference genome of E. grisescens was published, the whole MAPK cascade gene family has not been fully identified yet, especially the expression patterns of MAPK cascade gene family members upon an ecological biopesticide, Metarhizium anisopliae, remains to be understood. RESULTS: In this study, we have identified 19 MAPK cascade gene family members in E. grisescens, including 5 MAPKs, 4 MAP2Ks, 8 MAP3Ks, and 2 MAP4Ks. The molecular evolution characteristics of the whole Eg-MAPK cascade gene family, including gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication, were systematically investigated. Our results showed that the members of Eg-MAPK cascade gene family were unevenly distributed in 13 chromosomes, and the clustered members in each group shared similar structures of the genes and proteins. Gene expression data revealed that MAPK cascade genes were expressed in all four developmental stages of E. grisescens and were fairly and evenly distributed in four different larva tissues. Importantly, most of the MAPK cascade genes were induced or constitutively expressed upon M. anisopliae infection. CONCLUSIONS: In summary, the present study was one of few studies on MAPK cascade gene in E. grisescens. The characterization and expression profiles of Eg-MAPK cascades genes might help develop new ecofriendly biological insecticides to protect tea trees.

Identification of zona pellucida defects revealed a novel loss-of-function mutation in ZP2 in humans and rats.

Introduction: Human zona pellucida (ZP) plays an important role in reproductive process. Several rare mutations in the encoding genes (ZP1, ZP2, and ZP3) have been demonstrated to cause women infertility. Mutations in ZP2 have been reported to cause ZP defects or empty follicle syndrome. We aimed to identify pathogenic variants in an infertile woman with a thin zona pellucida (ZP) phenotype and investigated the effect of ZP defects on oocyte gene transcription. Methods: We performed whole-exome sequencing and Sanger sequencing of genes were performed for infertilite patients characterized by fertilization failure in routine in vitro fertilization (IVF). Immunofluorescence (IF) and intracytoplasmic sperm injection (ICSI) were used in the mutant oocytes. Single-cell RNA sequencing was used to investigate transcriptomes of the gene-edited (Zp2mut/mut) rat model. Biological function enrichment analysis, quantitative real-time PCR (qRT-PCR), and IF were performed. Results: We identified a novel homozygous nonsense mutation of ZP2 (c.1924C > T, p.Arg642X) in a patient with non-consanguineous married parents. All oocytes showed a thin or no ZP under a light microscope and were fertilized after ICSI. The patient successfully conceived by receiving the only two embryos that developed to the blastocyst stage. The immunofluorescence staining showed an apparently abnormal form of the stopped oocytes. We further demonstrated a total of 374 differentially expressed genes (DEGs) in the transcriptome profiles of Zp2mut/mut rats oocytes and highlighted the signal communication between oocytes and granulosa cells. The pathway enrichment results of DEGs showed that they were enriched in multiple signaling pathways, especially the transforming growth factor-ß (TGF-ß) signaling pathway in oocyte development. qRT-PCR, IF, and phosphorylation analysis showed significantly downregulated expressions of Acvr2b, Smad2, p38MAPK, and Bcl2 and increased cleaved-caspase 3 protein expression. Discussion: Our findings expanded the known mutational spectrum of ZP2 associated with thin ZP and natural fertilization failure. Disruption of the integrity of the ZP impaired the TGF-ß signaling pathway between oocytes and surrounding granulosa cells, leading to increased apoptosis and decreased developmental potential of oocytes.

Molecular Cloning and Characterization of WRKY12, A Pathogen Induced WRKY Transcription Factor from Akebia trifoliata.

WRKY transcription factors (TFs), which are plant-specific TFs, play significant roles in plant defense. Here, a pathogen-induced WRKY gene, named AktWRKY12, which was the homologous gene of AtWRKY12, was isolated from Akebia trifoliata. The AktWRKY12 gene has a total length of 645 nucleotides and an open reading frame (ORF) encoding 214 amino acid polypeptides. The characterizations of AktWRKY12 were subsequently performed with the ExPASy online tool Compute pI/Mw, PSIPRED and SWISS-MODEL softwares. The AktWRKY12 could be classified as a member of WRKY group II-c TFs based on sequence alignment and phylogenetic analysis. The results of tissue-specific expression analysis revealed that the AktWRKY12 gene was expressed in all the tested tissues, and the highest expression level was detected in A. trifoliata leaves. Subcellular localization analysis showed that AktWRKY12 was a nuclear protein. Results showed that the expression level of AktWRKY12 significantly increased in A. trifoliata leaves with pathogen infection. Furthermore, heterologous over-expression of AktWRKY12 in tobacco resulted in suppressed expression of lignin synthesis key enzyme genes. Based on our results, we speculate that AktWRKY12 might play a negative role in A. trifoliata responding to biotic stress by regulating the expression of lignin synthesis key enzyme genes during pathogen infection.

Characterization of the WRKY gene family in Akebia trifoliata and their response to Colletotrichum acutatum.

BACKGROUND: Akebia trifoliata, belonging to the Lardizabalaceae family, is a well-known Chinese traditional medicinal plant, susceptible to many diseases, such as anthracnose and powdery mildew. WRKY is one of the largest plant-specific transcription factor families and plays important roles in plant growth, development and stress response, especially in disease resistance. However, little was known about the numbers, characters, evolutionary relationship and expression of WRKY genes in A. trifoliata in response to plant disease due to lacking of A. trifoliata genome. RESULTS: A total of 42 putative AktWRKY genes were identified based on the full-length transcriptome-sequencing data of A. trifoliata. Then 42 AktWRKY genes were divided into three major groups (Group I-III) based on the WRKY domains. Motif analysis showed members within same group shared a similar motif composition, implying a functional conservation. Tissue-specific expression analysis showed that AktWRKY genes could be detected in all tissues, while few AktWRKY genes were tissue specific. We further evaluated the expression of AktWRKY genes in three varieties in response to Colletotrichum acutatum by qRT-PCR. The expression patterns of AktWRKY genes were similar between C01 and susceptible variety I02, but distinctly different in resistant variety H05. In addition, it showed that more than 64 percentages of AktWRKY genes were differentially expressed during fungal infection in I02 and H05. Furthermore, Gene ontology (GO) analysis showed that AktWRKY genes were categorized into 26 functional groups under cellular components, molecular functions and biological processes, and a predicted protein interaction network was also constructed. CONCLUSIONS: Results of bioinformation analysis and expression patterns implied that AktWRKYs might play multiple function in response to biotic stresses. Our study could facilitate to further investigate the function and regulatory mechanism of the WRKY in A. trifoliata during pathogen response.

The complete chloroplast genome of Stauntonia chinensis and compared analysis revealed adaptive evolution of subfamily Lardizabaloideae species in China.

BACKGROUND: Stauntonia chinensis DC. belongs to subfamily Lardizabaloideae, which is widely grown throughout southern China. It has been used as a traditional herbal medicinal plant, which could synthesize a number of triterpenoid saponins with anticancer and anti-inflammatory activities. However, the wild resources of this species and its relatives were threatened by over-exploitation before the genetic diversity and evolutionary analysis were uncovered. Thus, the complete chloroplast genome sequences of Stauntonia chinensis and comparative analysis of chloroplast genomes of Lardizabaloideae species are necessary and crucial to understand the plastome evolution of this subfamily. RESULTS: A series of analyses including genome structure, GC content, repeat structure, SSR component, nucleotide diversity and codon usage were performed by comparing chloroplast genomes of Stauntonia chinensis and its relatives. Although the chloroplast genomes of eight Lardizabaloideae plants were evolutionary conserved, the comparative analysis also showed several variation hotspots, which were considered as highly variable regions. Additionally, pairwise Ka/Ks analysis showed that most of the chloroplast genes of Lardizabaloideae species underwent purifying selection, whereas 25 chloroplast protein coding genes were identified with positive selection in this subfamily species by using branch-site model. Bayesian and ML phylogeny on CCG (complete chloroplast genome) and CDs (coding DNA sequences) produced a well-resolved phylogeny of Lardizabaloideae plastid lineages. CONCLUSIONS: This study enhanced the understanding of the evolution of Lardizabaloideae and its relatives. All the obtained genetic resources will facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of subfamily Lardizabaloideae.

Genome-wide survey and expression analysis of calcium-dependent protein kinase (CDPK) in grass Brachypodium distachyon.

BACKGROUND: Ca2+ played as a ubiquitous secondary messenger involved in plant growth, development, and responses to various environmental stimuli. Calcium-dependent protein kinases (CDPK) were important Ca2+ sensors, which could directly translate Ca2+ signals into downstream phosphorylation signals. Considering the importance of CDPKs as Ca2+ effectors for regulation of plant stress tolerance and few studies on Brachypodium distachyon were available, it was of interest for us to isolate CDPKs from B. distachyon. RESULTS: A systemic analysis of 30 CDPK family genes in B. distachyon was performed. Results showed that all BdCDPK family members contained conserved catalytic Ser/Thr protein kinase domain, autoinhibitory domain, and EF-hand domain, and a variable N-terminal domain, could be divided into four subgroup (I-IV), based upon sequence homology. Most BdCDPKs had four EF-hands, in which EF2 and EF4 revealed high variability and strong divergence from EF-hand in AtCDPKs. Synteny results indicated that large number of syntenic relationship events existed between rice and B. distachyon, implying their high conservation. Expression profiles indicated that most of BdCDPK genes were involved in phytohormones signal transduction pathways and regulated physiological process in responding to multiple environmental stresses. Moreover, the co-expression network implied that BdCDPKs might be both the activator and the repressor involved in WRKY transcription factors or MAPK cascade genes mediated stress response processes, base on their complex regulatory network. CONCLUSIONS: BdCDPKs might play multiple function in WRKY or MAPK mediated abiotic stresses response and phytohormone signaling transduction in B. distachyon. Our genomics analysis of BdCDPKs could provide fundamental information for further investigation the functions of CDPKs in integrating Ca2+ signalling pathways in response to environments stresses in B. distachyon.

Identification and in Silico Characterization of GT Factors Involved in Phytohormone and Abiotic Stresses Responses in Brachypodium distachyon.

GT factors play critical roles in plant growth and development and in response to various environmental stimuli. Considering the new functions of GT factors on the regulation of plant stress tolerance and seeing as few studies on Brachypodium distachyon were available, we identified GT genes in B. distachyon, and the gene characterizations and phylogenies were systematically analyzed. Thirty-one members of BdGT genes were distributed on all five chromosomes with different densities. All the BdGTs could be divided into five subfamilies, including GT-1, GT-2, GTγ, SH4, and SIP1, based upon their sequence homology. BdGTs exhibited considerably divergent structures among each subfamily according to gene structure and conserved functional domain analysis, but the members within the same subfamily were relatively structure-conserved. Synteny results indicated that a large number of syntenic relationship events existed between rice and B. distachyon. Expression profiles indicated that the expression levels of most of BdGT genes were changed under abiotic stresses and hormone treatments. Moreover, the co-expression network exhibited a complex regulatory network between BdGTs and BdWRKYs as well as that between BdGTs and BdMAPK cascade gene. Results showed that GT factors might play multiple functions in responding to multiple environmental stresses in B. distachyon and participate in both the positive and negative regulation of WRKY- or MAPK-mediated stress response processes. The genome-wide analysis of BdGTs and the co-regulation network under multiple stresses provide valuable information for the further investigation of the functions of BdGTs in response to environment stresses.

The complete chloroplast genome of Holboellia angustifolia (Ranales: Lardizabalaceae), a traditional herbal species.

The complete chloroplast genome of Holboellia  angustifolia was 157,797 bp in length, displayed a typical quadripartite structure, composed of a LSC region of 86,543 bp and a SSC region of 18,972 bp, separated by a pair of IRs of 26,141 bp each. The chloroplast genome contains 130 genes, consisting of 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Fifteen genes have one intron, and 3 genes contain two introns. The overall A/T content in the chloroplast genome of H. angustifolia was 61.31%. Phylogenetic analysis showed that H. angustifolia was closely related to Holboellia latifolia.

Identification, characterization, and expression analysis of auxin response factor (ARF) gene family in Brachypodium distachyon.

Auxin response factors (ARFs) are one type of essential family of transcription factors that bind with auxin response elements (AuxRE), and play vital roles in variety of plant development and physiological processes. Brachypodium distachyon, related to the major cereal grain species, were recently developed to be a good model organism for functional genomics research. So far, genome-wide overview of the ARF gene family in B. distachyon was not available. Here, a systemic analysis of ARF gene family members in B. distachyon was performed. A comprehensive overview of the characterization of the BdARFs was obtained by multiple bioinformatics analyses, including the gene and protein structure, chromosome locations, conserved motifs of proteins, phylogenetic analysis, and cis-elements in promoters of BdARF. Results showed that all BdARFs contained conserved DBD, MR, and CTD could be divided into four classes, Ia, IIa, IIb, and III. Expression profiles of BdARF genes indicated that they were expressed across various tissues and organs, which could be clustered into three main expression groups, and most of BdARF genes were involved in phytohormone signal transduction pathways and regulated physiological process in responding to multiple environmental stresses. And predicted regulatory network between B. distachyon ARFs and IAAs was also discussed. Our genomics analysis of BdARFs could yield new insights into the complexity of the control of BdARF genes and lead to potential applications in the investigation of the accurate regulatory mechanisms of ARFs in herbaceous plants.

Genome-wide survey of heat shock factors and heat shock protein 70s and their regulatory network under abiotic stresses in Brachypodium distachyon.

The heat shock protein 70s (Hsp70s) and heat shock factors (Hsfs) play key roles in protecting plant cells or tissues from various abiotic stresses. Brachypodium distachyon, recently developed an excellent model organism for functional genomics research, is related to the major cereal grain species. Although B. distachyon genome has been fully sequenced, the information of Hsf and Hsp70 genes and especially the regulatory network between Hsfs and Hsp70s remains incomplete. Here, a total of 24 BdHsfs and 29 BdHsp70s were identified in the genome by bioinformatics analysis and the regulatory network between Hsfs and Hsp70s were performed in this study. Based on highly conserved domain and motif analysis, BdHsfs were grouped into three classes, and BdHsp70s divided into six groups, respectively. Most of Hsf proteins contain five conserved domains: DBD, HR-A/B region, NLS and NES motifs and AHA domain, while Hsp70 proteins have three conserved domains: N-terminal nucleotide binding domain, peptide binding domain and a variable C-terminal lid region. Expression data revealed a large number of BdHsfs and BdHsp70s were induced by HS challenge, and a previous heat acclimation could induce the acquired thermotolerance to help seedling suffer the severe HS challenge, suggesting that the BdHsfs and BdHsp70s played a role in alleviating the damage by HS. The comparison revealed that, most BdHsfs and BdHsp70s genes responded to multiple abiotic stresses in an overlapping relationship, while some of them were stress specific response genes. Moreover, co-expression relationships and predicted protein-protein interaction network implied that class A and B Hsfs played as activator and repressors, respectively, suggesting that BdHsp70s might be regulated by both the activation and the repression mechanisms under stress condition. Our genomics analysis of BdHsfs and BdHsp70s provides important evolutionary and functional characterization for further investigation of the accurate regulatory mechanisms among Hsfs and Hsp70s in herbaceous plants.