Wheat lesion mimic homology gene TaCAT2 enhances plant resistance to biotic and abiotic stresses.

Lesion mimic mutants (LMMs) refer to the spontaneous formation of disease-like spots on leaves without any obvious pathogen infection. The LMM genes can regulate plant immunity, thus promoting the defense of crops against pathogens. However, there is a lack of systematic understanding of the regulatory mechanism of LMMs in wheat. This study identified a wheat LMM TaCAT2, a homolog of the Arabidopsis CAT2. The prediction of the cis-regulatory element revealed that TaCAT2 was involved in the response of plants to various hormones and stresses. RT-qPCR analysis indicated that TaCAT2 was significantly up-regulated by NaCl, drought, and Fusarium graminearum infection. Fluorescence microscopy showed that the TaCAT2 was localized to the peroxisome. Overexpression of TaCAT2 enhanced plant resistance to Phytophthora infestation and F. graminearum by constitutionally activating SA and JA pathways. VIGS of TaCAT2 enhanced the sensitivity of wheat to F. graminearum. Further, TaCAT2 enhanced stress resistance by scavenging the excessive ROS and increasing the activities of antioxidative enzymes. This study lays the basis for the functional identification of TaCAT2 and its applicability in the disease resistance of wheat.

Graphormer supervised de novo protein design method and function validation.

Protein design is central to nearly all protein engineering problems, as it can enable the creation of proteins with new biological functions, such as improving the catalytic efficiency of enzymes. One key facet of protein design, fixed-backbone protein sequence design, seeks to design new sequences that will conform to a prescribed protein backbone structure. Nonetheless, existing sequence design methods present limitations, such as low sequence diversity and shortcomings in experimental validation of the designed functional proteins. These inadequacies obstruct the goal of functional protein design. To improve these limitations, we initially developed the Graphormer-based Protein Design (GPD) model. This model utilizes the Transformer on a graph-based representation of three-dimensional protein structures and incorporates Gaussian noise and a sequence random masks to node features, thereby enhancing sequence recovery and diversity. The performance of the GPD model was significantly better than that of the state-of-the-art ProteinMPNN model on multiple independent tests, especially for sequence diversity. We employed GPD to design CalB hydrolase and generated nine artificially designed CalB proteins. The results show a 1.7-fold increase in catalytic activity compared to that of the wild-type CalB and strong substrate selectivity on p-nitrophenyl acetate with different carbon chain lengths (C2-C16). Thus, the GPD method could be used for the de novo design of industrial enzymes and protein drugs. The code was released at https://github.com/decodermu/GPD.

Truncated FRMD7 proteins in congenital Nystagmus: novel frameshift mutations and proteasomal pathway implications.

Idiopathic congenital nystagmus (ICN) manifests as involuntary and periodic eye movements. To identify the genetic defect associated with X-linked ICN, Whole Exome Sequencing (WES) was conducted in two affected families. We identified two frameshift mutations in FRMD7, c.1492dupT/p.(Y498Lfs*15) and c.1616delG/p.(R539Kfs*2). Plasmids harboring the mutated genes and qPCR analysis revealed mRNA stability, evading degradation via the NMD pathway, and corroborated truncated protein production via Western-blot analysis. Notably, both truncated proteins were degraded through the proteasomal (ubiquitination) pathway, suggesting potential therapeutic avenues targeting this pathway for similar mutations. Moreover, we conducted a comprehensive analysis, summarizing 140 mutations within the FRMD7 gene. Our findings highlight the FERM and FA structural domains as mutation-prone regions. Interestingly, exons 9 and 12 are the most mutated regions, but 90% (28/31) mutations in exon 9 are missense while 84% (21/25) mutations in exon 12 are frameshift. A predominant occurrence of shift code mutations was observed in exons 11 and 12, possibly associated with the localization of premature termination codons (PTCs), leading to the generation of deleterious truncated proteins. Additionally, our conjecture suggests that the loss of FRMD7 protein function might not solely drive pathology; rather, the emergence of aberrant protein function could be pivotal in nystagmus etiology. We propose a dependence of FRMD7 protein normal function primarily on its anterior domain. Future investigations are warranted to validate this hypothesis.

Dissecting the features of TGA gene family in Saccharum and the functions of ScTGA1 under biotic stresses.

Sugarcane is an important sugar and energy crop and smut disease caused by Sporisorium scitamineum is a major fungal disease which can seriously reduce the yield and quality of sugarcane. In plants, TGACG motif binding (TGA) transcription factors are involved in the regulation of salicylic acid (SA) and methyl jasmonate (MeJA) signaling pathways, as well as in response to various biotic and abiotic stresses. However, no TGA-related transcription factor has been reported in Saccharum. In the present study, 44 SsTGA genes were identified from Saccharum spontaneum, and were assorted into three clades (I, II, III). Cis-regulatory elements (CREs) analysis revealed that SsTGA genes may be involved in hormone and stress response. RNA-seq data and RT-qPCR analysis indicated that SsTGAs were constitutively expressed in different tissues and induced by S. scitamineum stress. In addition, a ScTGA1 gene (GenBank accession number ON416997) was cloned from the sugarcane cultivar ROC22, which was homologous to SsTGA1e in S. spontaneum and encoded a nucleus protein. It was constitutively expressed in sugarcane tissues and up-regulated by SA, MeJA and S. scitamineum stresses. Furthermore, transient overexpression of ScTGA1 in Nicotiana benthamiana could enhance its resistance to the infection of Ralstonia solanacearum and Fusarium solani var. coeruleum, by regulating the expression of immune genes related to hypersensitive response (HR), ethylene (ET), SA and jasmonic acid (JA) pathways. This study should contribute to our understanding on the evolution and function of the SsTGA gene family in Saccharum, and provide a basis for the functional identification of ScTGA1 under biotic stresses.

Genome-wide characterization of sugarcane catalase gene family identifies a ScCAT1 gene associated disease resistance.

In plants, catalase (CAT) mainly scavenges H2O2 from reactive oxygen species (ROS) and regulates the growth and development. So far, genome-wide identification of CAT gene family in Saccharum has not yet been reported. Here, 16 SsCAT genes were identified based on a Saccharum spontaneum genome. They were clustered into three subfamilies, with closer genes sharing similar structures. Most SsCAT proteins contained three conserved amino acids, one active catalytic site, one heme-ligand signature, and three peroxisomal targeting signal 1 (PTS1) sequences. The cis-regulatory element prediction revealed that SsCAT genes were involved in growth and development, and in response to various hormones and stresses. RNA-Seq databases showed that SsCAT genes were differentially expressed in Saccharum tissues and under cold, drought, and Sporisorium scitamineum stresses. The ScCAT1 gene transcript (GenBank accession number KF664183) and relevant CAT activity were up-regulated under S. scitamineum stress. Overexpression of ScCAT1 gene in Nicotiana benthamiana could enhance its resistance to pathogen infection through scavenging of excessive toxic ROS and up-regulated expressions of genes related to hypersensitive response (HR), ROS and salicylic acid (SA) pathways. This study provides comprehensive information for the CAT gene family and sets up a basis for its function identification in sugarcane.

Transcriptomics Integrated with Metabolomics Unveil Carotenoids Accumulation and Correlated Gene Regulation in White and Yellow-Fleshed Turnip (Brassica rapa ssp. rapa).

Turnip (Brassica rapa ssp. rapa) is considered to be a highly nutritious and health-promoting vegetable crop, whose flesh color can be divided into yellow and white. It is widely accepted that yellow-fleshed turnips have higher nutritional value. However, reports about flesh color formation is lacking. Here, the white-fleshed inbred line, W21, and yellow-fleshed inbred line, W25, were profiled from the swollen root of the turnip at three developmental periods to elucidate the yellow color formation. Transcriptomics integrated with metabolomics analysis showed that the PSY gene was the key gene affecting the carotenoids formation in W25. The coding sequence of BrrPSY-W25 was 1278 bp and that of BrrPSY-W21 was 1275 bp, and BrrPSY was more highly expressed in swollen roots in W25 than in W21. Transient transgenic tobacco leaf over-expressing BrrPSY-W and BrrPSY-Y showed higher transcript levels and carotenoids contents. Results revealed that yellow turnip formation is due to high expression of the PSY gene rather than mutations in the PSY gene, indicating that a post-transcriptional regulatory mechanism may affect carotenoids formation. Results obtained in this study will be helpful for explaining the carotenoids accumulation of turnips.

Characterization of Annexin gene family and functional analysis of RsANN1a involved in heat tolerance in radish (Raphanus sativus L.).

Plant annexins are a kind of conserved Ca2+-dependent phospholipid-binding proteins which are involved in plant growth, development and stress tolerance. Radish is an economically important annual or biennial root vegetable crop worldwide. However, the genome-wide characterization of annexin (RsANN) gene family remain largely unexplored in radish. In this study, a comprehensive identification of annexin gene family was performed at the whole genome level in radish. In total, ten RsANN genes were identified, and these putative RsANN proteins shared typical characteristics of the annexin family proteins. Phylogenetic analysis showed that the RsANNs together with annexin from Arabidopsis and rice were clustered into five groups with shared similar motif patterns. Chromosomal localization showed that these ten RsANN genes were distributed on six chromosomes (R3-R8) of radish. Several cis-elements involved in abiotic stress response were identified in the promoter regions of RsANN genes. Expression profile analysis indicated that the RsANN genes exhibited tissue-specific patterns at different growth stages and tissues. The Real-time quantitative PCR (RT-qPCR) revealed that the expression of most RsANN genes was induced under various abiotic stresses including heat, drought, salinity, oxidization and ABA stress. In addition, stress assays showed that overexpression of RsANN1a improved plant's growth and heat tolerance, while artificial microRNAs (amiRNA)-mediated knockdown of RsANN1a caused dramatically decreased survival ratio of Arabidopsis plants. These findings not only demonstrate that RsANN1a might play a critical role in the heat stress response of radish, but also facilitate clarifying the molecular mechanism of RsANN genes in regulating the biological process governing plant growth and development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01056-5.

Exploring the function of genetic variants in the non-coding genomic regions: approaches for identifying human regulatory variants affecting gene expression.

Understanding the genetic basis of human traits/diseases and the underlying mechanisms of how these traits/diseases are affected by genetic variations is critical for public health. Current genome-wide functional genomics data uncovered a large number of functional elements in the noncoding regions of human genome, providing new opportunities to study regulatory variants (RVs). RVs play important roles in transcription factor bindings, chromatin states and epigenetic modifications. Here, we systematically review an array of methods currently used to map RVs as well as the computational approaches in annotating and interpreting their regulatory effects, with emphasis on regulatory single-nucleotide polymorphism. We also briefly introduce experimental methods to validate these functional RVs.

A detailed representation of electrostatic energy in prediction of sequence and pH dependence of protein stability.

A molecular mechanics model, previously validated in applications to structure prediction, is shown to reproduce experiment in predictions of protein ionization state, and in predictions of sequence and pH dependence of protein stability. Over a large dataset, 1876 values of ΔΔG of folding, the RMSD is 1.34 kcal/mol. Using an alternative measure of accuracy, either the sign of the calculated ΔΔG agrees with experiment or the absolute value of the deviation is less than 1.0 kcal/mol, 1660 of 1876 data points (88.5%) pass the condition. Relative to models used previously in computer-aided protein design, the concept, we propose, most responsible for the performance of our model, and for the extensibility to non-neutral values of pH, is the treatment of electrostatic energy. The electronic structure of the protein is modeled using distributed atomic multipoles. The structured liquid state of the solvent is modeled using a dielectric continuum. A modification to the energetics of the reaction field, induced by the protein in the dielectric continuum, attempts to account for preformed multipoles of solvent water molecules and ions. An adjustable weight (with optimal value.141) applied to the total vacuum energy accounts implicitly for electronic polarization. A threshold distance, beyond which pairwise atomic interactions are neglected, is not used. In searches through subspaces of sequences and conformations, efficiency remains acceptable for useful applications.