Ubiquitin Gene Research Articles

A Genome-Wide Alternative Splicing Landscape Specifically Associated with Durable Rice Blast Resistance

The rice blast, caused by the hemibiotrophic plant pathogen Magnaporthe oryzae, is a devastating disease that threatens rice crop production worldwide. The molecular interactions that underlie the rice-M. oryzae interaction have received much attention. However, genome-wide research focusing on alternative splicing (AS) has not been well-studied in rice—M. oryzae interactions. AS in plants leads to diverse proteomes without an expansion in gene numbers to regulate cellular processes during abiotic or biotic stress. The Pi21 gene negatively regulates rice resistance to M. oryzae infection, and thus the Pi21-RNAi silenced transgenic line (#241) exhibits partial but durable resistance. We compared the AS landscape in #241 and “Nipponbare” (Nip) during interacting with M. oryzae Guy11, and the alternative 3′ splice-site (A3SS) is the most common AS type. GO enrichment analysis of #241-specific differentially alternatively spliced genes (DASGs) revealed that WRKY transcription factors (TFs), bHLH TFs, F-box protein with leucine rich repeats, AAA-type ATPase, and protein kinase were enriched in the GO terms “response to jasmonate acid (JA)” and “ethylene (ET)” at 24 h post-inoculation (hpi). At 48 hpi, one #241-specific DASG, ubiquitin gene (Os08g0295000), was predicted to be involved in endoplasmic reticulum (ER) stress. In silico analysis combined with PCR amplification confirmed that multiple isoforms are produced by Os08g0295000 and a skipped exon (SE) event results in isoform switching during interaction with M. oryzae. Protein–protein interaction (PPI) network analysis predicted that Os08g0295000-encoding proteins may interact with SNARE protein Q9LGF8 (Uniprot ID) to cooperatively regulate rice’s response to M. oryzae. This study uncovered the AS profile of rice in response to M. oryzae, which will help to explore the linkage between AS and durable rice resistance.

Yeast polyubiquitin unit regulates synaptonemal complex formation and recombination during meiosis.

Ubiquitin is highly conserved in most eukaryotes and involved in diverse physiological processes, including cell division, protein quality control, and protein degradation mediated by the ubiquitin-proteasome system after heat shock, glucose-starvation, and oxidative stress. However, the role of the ubiquitin gene UBI4, which contains five consecutive head-to-tail ubiquitin repeats, in meiosis has not been investigated. In this study, we show that the Saccharomyces cerevisiae polyubiquitin precursor gene, UBI4, is required to promote synaptonemal complex (SC) formation and suppress excess double-strand break formation. Moreover, the proportion of Zip1 polycomplexes, which indicate abnormal SC formation, in cells with a mutation in UBI4 (i.e., ubi4Δ cells) is higher than that of wild-type cells, implying that the UBI4 plays an important role in the early meiotic prophase I. Interestingly, although ubi4Δ cells rarely form full-length SCs in the pachytene stage of prophase I, the Zip3 foci are still seen, as in wild-type cells. Moreover, ubi4Δ cells proficiently form crossover and noncrossover products with a slight delay compared to wild-type cells, suggesting that UBI4 is dispensable in SC-coupled recombination. Our findings demonstrate that UBI4 exhibits dual functions that are associated with both positive and negative roles in SC formation and recombination during meiosis.

Fluorescein-labeled ThUBD probe for super-sensitive visualization of polyubiquitination signal in situ cells

Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the ubiquitin and ubiquitin chains. The preferences of UBDs for ubiquitin chains of specific length and linkage are central to their functions. We demonstrated that an artificial tandem hybrid UBD (ThUBD) exhibits an unbiased high affinity to all ubiquitin chains and is a promising tool for global ubiquitination profiling research. In this study, we labeled fluorescein on the four cysteine residues in the N-terminal glutathione S-transferase (GST) tag of ThUBD, generating a fluorescein-labeled ThUBD (ThUBD-Flu) probe for direct polyubiquitination signal imaging and visualization. Compared to the canonical ubiquitin antibody method, the ThUBD-Flu is hyper-sensitive and accurate to detect ubiquitination signal. More importantly, the ThUBD-Flu probe provided, for the first time, a widely applicable, super-sensitive and unbiased technique for in situ detection of intracellular polyubiquitination signal through immunofluorescence staining, which was only achievable with recombinant fluorescence tag fused ubiquitin gene previously. We propose that ThUBD-Flu, combined with evolving microscopy technology, could serve as prototypes to track and trace cellular polyubiquitination signal in vivo.

The Clinical Prediction Value of the Ubiquitination Model Reflecting the Immune Traits in LUAD.

BackgroundIncreasing evidence shows that the ubiquitin–proteasome system has a crucial impact on lung adenocarcinoma. However, reliable prognostic signatures based on ubiquitination and immune traits have not yet been established.MethodsBioinformatics was performed to analyze the characteristic of ubiquitination in lung adenocarcinoma. Principal component analysis was employed to identify the difference between lung adenocarcinoma and adjacent tissue. The ubiquitin prognostic risk model was constructed by multivariate Cox regression and least absolute shrinkage and selection operator regression based on the public database The Cancer Genome Atlas, with evaluation of the time-dependent receiver operating characteristic curve. A variety of algorithms was used to analyze the immune traits of model stratification. Meanwhile, the drug response sensitivity for subgroups was predicted by the “pRRophetic” package based on the database of the Cancer Genome Project.ResultsThe expression of ubiquitin genes was different in the tumor and in the adjacent tissue. The ubiquitin model was superior to the clinical indexes, and four validation datasets verified the prognostic effect. Additionally, the stratification of the model reflected distinct immune landscapes and mutation traits. The low-risk group was infiltrating plenty of immune cells and highly expressed major histocompatibility complex and immune genes, which illustrated that these patients could benefit from immune treatment. The high-risk group showed higher mutation and tumor mutation burden. Integrating the tumor mutation burden and the immune score revealed the patient’s discrepancy between survival and drug response. Finally, we discovered that the drug targeting ubiquitin and proteasome would be a beneficial prospective treatment for lung adenocarcinoma.ConclusionThe ubiquitin trait could reflect the prognosis of lung adenocarcinoma, and it might shed light on the development of novel ubiquitin biomarkers and targeted therapy for lung adenocarcinoma.

Caenorhabditis elegans Parkin: Regulators of its abundance and role in autophagy-lysosomal dynamics

Background: Parkin, which when mutated leads to early-onset Parkinson’s disease, acts as an E3 ubiquitin ligase. How Parkin is regulated for selective protein and organelle targeting is not well understood. Here, we used protein interactor and genetic screens in Caenorhabditis elegans (C. elegans) to identify new regulators of Parkin abundance and showed their impact on autophagy-lysosomal dynamics and alpha-Synuclein processing. Methods: We generated a transgene encoding mCherry-tagged C. elegans Parkin – Parkinson’s Disease Related 1 (PDR-1). We performed protein interactor screen using Co-immunoprecipitation followed by mass spectrometry analysis to identify putative interacting partners of PDR-1. Ribonucleic acid interference (RNAi) screen and an unbiased mutagenesis screen were used to identify genes regulating PDR-1 abundance. Confocal microscopy was used for the identification of the subcellular localization of PDR-1 and alpha-Synuclein processing. Results: We show that the mCherry::pdr-1 transgene rescues the mitochondrial phenotype of pdr-1 mutants and that the expressed PDR-1 reporter is localized in the cytosol with enriched compartmentalization in the autophagy-lysosomal system. We determined that the transgenic overexpression of the PDR-1 reporter, due to inactivated small interfering RNA (siRNA) generation pathway, disrupts autophagy-lysosomal dynamics. From the RNAi screen of putative PDR-1 interactors we found that the inactivated Adenine Nucleotide Translocator ant-1.1/hANT, or hybrid ubiquitin genes ubq-2/hUBA52 and ubl-1/hRPS27A encoding a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively, induced PDR-1 abundance and affected lysosomal dynamics. In addition, we demonstrate that the abundant PDR-1 plays a role in alpha-Synuclein processing. Conclusions: These data show that the abundant C. elegans Parkin ortholog affects the autophagy-lysosomal system together with alpha-Synuclein processing which can help in understanding the pathology in Parkin-related diseases.

Validation and Invalidation of SARS-CoV-2 Papain-like Protease Inhibitors.

SARS-CoV-2 encodes two viral cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), both of which are validated antiviral drug targets. PLpro is involved in the cleavage of viral polyproteins as well as immune modulation by removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PLpro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PLpro inhibitors through high-throughput screenings. In this study, we aim to validate/invalidate the reported PLpro inhibitors using a combination of PLpro target-specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and cell-based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of PLpro in the TSA binding assay, which might explain their weak enzymatic inhibition in the FRET assay. In addition, none of the compounds showed cellular PLpro inhibition as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for potent and specific SARS-CoV-2 PLpro inhibitors.

An Optimised TRIzol-based Protocol for the Improvement of RNA Extraction Yield of Tomato Stem

One of the most common methods for purifying RNA is using TRIzol reagent because of its simplicity and economic feasibility. However, the drawback of this method is frequently the low quality of extracted RNA due to contaminants from the residue of phenol and guanidinium thiocyanate from the reagents. This study aimed to evaluate the improvement in the quality and concentration of RNA after the optimisation treatment. One-month-old tomato (Solanum lycopersicum) stem was used in this research. TRIzol or acid guanidinium thiocyanate-phenol-chloroform-based method was given optimisation treatments of the initial sample amount, twice chloroform extraction, overnight precipitation at low temperature, and three times final washing with ethanol. The results showed no significant improvement (p > 0.05) in the purity ratio A260/A280. At the same time, there was a significant improvement (p < 0.05) in RNA yield and purity ratio A260/A230. The quality of RNA was verified using agarose-formaldehyde electrophoresis gel. Eight of nine samples (89%) from the optimised group had better RNA integrity characterised by sharp bands for 28S and 18S rRNA. Furthermore, a representative sample from the optimised group was successfully synthesised into complementary DNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with primers of the ubiquitin (UBI3) gene. To sum up, optimised TRIzol-based protocol provides meaningful insight to produce RNA with better quality and suitability for downstream applications.

Lettuce-based production of an oral vaccine against porcine edema disease for the seed lot system.

Plant-made oral vaccines can be a cost-effective method to control infectious diseases of humans and farm animals. Pig edema is a bacterial disease caused by enterohemorrhagic Escherichia coli producing the toxin Shiga toxin 2e (Stx2e). In our previous report, we chose the non-toxic B subunit of Stx2e (Stx2eB) as a vaccine antigen, and Stx2eB was expressed in lettuce (Lactuca sativa L., cv. Green wave). We found that a double repeated Stx2eB (2×Stx2eB) accumulates to higher levels than a single Stx2eB. In this study, we analyzed progeny plants introduced with 2×Stx2eB in which the gene was expressed under the control of conventional cauliflower mosaic virus 35S RNA (CaMV 35S) promoter, and found that the lettuce underwent transgene silencing and bore few seeds. We resolved these problems by using a transgene cassette which harbored a transcriptional promoter derived from the lettuce ubiquitin gene and a longer version of HSPT. The lettuce harboring this expression construct will be valuable in establishing the seed lot system on the basis that thousands of seeds can be obtained from one plant body and the resulting progeny plants accumulate 2×Stx2eB at high levels without the transgene silencing.

Structural insights into the interaction of papain-like protease 2 from the alphacoronavirus porcine epidemic diarrhea virus and ubiquitin.

Porcine epidemic diarrhea is a devastating porcine disease that is caused by the alphacoronavirus porcine epidemic diarrhea virus (PEDV). Like other members of the Coronaviridae family, PEDV encodes a multifunctional papain-like protease 2 (PLP2) that has the ability to process the coronavirus viral polyprotein to aid in RNA replication and antagonize the host innate immune response through cleavage of the regulatory proteins ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15) (deubiquitination and deISGylation, respectively). Because Betacoronavirus PLPs have been well characterized, it was sought to determine how PLP2 from the alphacoronavirus PEDV differentiates itself from its related counterparts. PEDV PLP2 was first biochemically characterized, and a 3.1 Å resolution crystal structure of PEDV PLP2 bound to Ub was then solved, providing insight into how Alphacoronavirus PLPs bind to their preferred substrate, Ub. It was found that PEDV PLP2 is a deubiquitinase and readily processes a variety of di-Ub linkages, in comparison with its Betacoronavirus counterparts, which have a narrower range of di-Ub activity but process both Ub and ISG15.

SARS-CoV-2 Papain-Like Protease Potential Inhibitors-In Silico Quantitative Assessment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes the papain-like protease (PLpro). The protein not only plays an essential role in viral replication but also cleaves ubiquitin and ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from host proteins, making it an important target for developing new antiviral drugs. In this study, we searched for novel, noncovalent potential PLpro inhibitors by employing a multistep in silico screening of a 15 million compound library. The selectivity of the best-scored compounds was evaluated by checking their binding affinity to the human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which, as a deubiquitylating enzyme, exhibits structural and functional similarities to the PLpro. As a result, we identified 387 potential, selective PLpro inhibitors, from which we retrieved the 20 best compounds according to their IC50 values toward PLpro estimated by a multiple linear regression model. The selected candidates display potential activity against the protein with IC50 values in the nanomolar range from approximately 159 to 505 nM and mostly adopt a similar binding mode to the known, noncovalent SARS-CoV-2 PLpro inhibitors. We further propose the six most promising compounds for future in vitro evaluation. The results for the top potential PLpro inhibitors are deposited in the database prepared to facilitate research on anti-SARS-CoV-2 drugs.

Flipping the substrate preference of Hazara virus ovarian tumour domain protease through structure-based mutagenesis.

Nairoviruses are arthropod-borne viruses with a nearly global geographical distribution. Several are known causative agents of human disease, including Crimean-Congo hemorrhagic fever virus (CCHFV), which has a case fatality rate that can exceed 30%. Nairoviruses encode an ovarian tumour domain protease (OTU) that can suppress the innate immune response by reversing post-translational modifications by ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15). As a result, the OTU has been identified as a potential target for the development of CCHFV therapeutics. Despite sharing the same general fold, nairoviral OTUs show structural and enzymatic diversity. The CCHFV OTU, for example, possesses activity towards both Ub and ISG15, while the Hazara virus (HAZV) OTU interacts exclusively with Ub. Virology studies focused on the OTU have mostly been restricted to CCHFV, which requires BSL-4 containment facilities. Although HAZV has been proposed as a BSL-2 alternative, differences in the engagement of substrates by CCHFV and HAZV OTUs may present complicating factors when trying to model one using the other. To understand the molecular underpinnings of the differences in activity, a 2.78 Å resolution crystal structure of HAZV OTU bound to Ub was solved. Using structure-guided site-directed mutagenesis, HAZV OTUs were engineered with altered or eliminated deubiquitinase activity, including one with an exclusive activity for ISG15. Additionally, analysis of the structure yielded insights into the difference in inhibition observed between CCHFV and HAZV OTUs with a Ub-based inhibitor. These new insights present opportunities to utilize HAZV as a model system to better understand the role of the OTU in the context of infection.

Identification of responsive genes and analysis of genes with bacterial-inducible cis-regulatory elements in the promoter regions in Oryza sativa L.

Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most critical diseases in rice. In order to study rice responsive genes to bacterial stress, microarray data were retrieved from GEO dataset. To identify the responsive genes to biotic stress (bacteria) bioinformatic tools were employed and the data presented in the forms of heatmap, gene ontology, gene network, and cis-element prediction were used. Almost all responsive genes were down-regulated at around 3 h time point and up-regulated 24 h time point in response to bacterial stress in rice varieties (Oryza sativa subs. japonica ‘IR64’, ‘IRBB5’, ‘IRBB7’ and ‘Y73’). Gene ontology showed that genes are involved in different biological processes including translation and cellular protein metabolic processes. Network analysis showed that genes expressed in response to pathogen infection (Xoo) included protein translation, eukaryotic initiation factors (eIFs), ribosomal proteins, protein ubiquitin, and MAPK genes. The genes expressed in response to bacterial stress can enable plant balance between synthesis and degradation of proteins which in turn allows plants for further growth and development. TATA-box and CAAT box had the highest number of cis elements involved in bacterial stress. These genes can provide novel insights into regulatory mechanisms in biotic stress responses in rice. Identification of bacterial stress response/tolerance genes of rice can assist the molecular breeding of new rice varieties tolerant to bacterial stress.

Cloning, sequencing and transcriptional analysis of ubiquitin gene from Alexandrium catenella under different nutrient conditions

Cloning, sequencing and transcriptional analysis of ubiquitin gene from Alexandrium catenella under different nutrient conditions

The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker β-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.

Upregulation of ubiquitinated proteins and their degradation pathway in muscle atrophy induced by cisplatin in mice

We previously demonstrated that cisplatin administration in mice induces muscle atrophy and an increase in the expression of two muscle-specific ubiquitin E3 ligase genes, muscle ring finger protein 1 (MuRF1), and atrophy gene-1 (atrogin-1), in skeletal muscle. Ubiquitination serves as a degradation signal in both the ubiquitin-proteasome and selective autophagy pathways. In the present study, we investigated changes in the expression of ubiquitin and ubiquitinated proteins and their degradation pathways. Ubiquitin and ubiquitinated protein levels were increased by cisplatin compared with those in the vehicle and dietary restriction (DR) groups. To quantify the levels of ubiquitin and ubiquitinated proteins, we conducted a dot blot assay using an anti-ubiquitin antibody. The expression of ubiquitin was also significantly increased by cisplatin compared with that in the vehicle and DR groups. Since the ubiquitin proteins were upregulated by cisplatin, we measured the mRNA levels of the ubiquitin genes: Ubb, Ubc, Rps27a, and Uba52. All these four genes were increased by cisplatin administration compared with those in both the vehicle-treated and DR groups in quadriceps muscle tissue. The anti-ubiquitin antibody-sensitive bands increased when C2C12 myotubes were treated with cisplatin. Furthermore, MG-132 (26 s proteasome inhibitor), but not bafilomycin A1 (autophagy inhibitor), caused a further increase in expression. In conclusion, ubiquitin and ubiquitinated proteins are upregulated in cisplatin-induced muscle atrophy. Cisplatin-induced ubiquitinated proteins are degraded by the 26 s proteasome pathway.

A novel polyubiquitin chain linkage formed by viral Ubiquitin is resistant to host deubiquitinating enzymes.

The Baculoviridae family of viruses encode a viral Ubiquitin (vUb) gene. Though the vUb is homologous to the host eukaryotic Ubiquitin (Ub), its preservation in the viral genome indicates unique functions that are not compensated by the host Ub. We report the structural, biophysical, and biochemical properties of the vUb from Autographa californica multiple nucleo-polyhedrosis virus (AcMNPV). The packing of central helix α1 to the beta-sheet β1-β5 is different between vUb and Ub. Consequently, its stability is lower compared with Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin-binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54), and the deubiquitinating enzymes are ineffective against the K54-linked polyubiquitin chains. We propose that the modification of host/viral proteins with the K54-linked chains is an effective way selected by the virus to protect the vUb signal from host DeUbiquitinases.

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean.

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

Identification of suitable reference genes for normalization of real-time quantitative PCR data in pecan (Carya illinoinensis)

This study proposed the combination of PR26S and PP1 as a good choice for RT-qPCR normalization in pecan under abiotic stress, developing kernels, grafting, and various tissues. Reference gene selection is an essential pre-requisite to generate reliable results in real-time quantitative polymerase chain reaction (RT-qPCR) analysis. However, studies regarding systematic validation of suitable reference genes in pecan are still lacking. In this study, 17 candidate reference genes were selected and evaluated for their expression stabilities in pecan under various experimental conditions, including various tissues, developing kernels, grafting, and two plant tissues (leaves and roots) subjected to three abiotic stresses (salt, drought, and Zn deficiency). The stability of the candidate genes was assessed by geNorm, NormFinder, and BestKeeper, and their outputs were integrated to obtain a final comprehensive rank of stability based on the geometric mean. The results indicated that samples under different experimental conditions possessed their own best reference genes, and using two reference genes for RT-qPCR normalization was recommended for the tested experiments. Overall, the combination of 26S protease regulatory subunit 7A (PR26S) and serine/threonine-protein phosphatase-1 (PP1) was recognized as a good choice for RT-qPCR normalization in pecan across all the treatments. More importantly, the widely used alpha-tubulin (α-TUB), ubiquitin (UBQ), and actin (ACT) genes were not the best suitable reference genes in most of our experiments. Our results will be helpful for future gene-expression studies in pecan.

Characterization and Noncovalent Inhibition of the Deubiquitinase and deISGylase Activity of SARS-CoV-2 Papain-Like Protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19, is a novel human betacoronavirus that is rapidly spreading worldwide. The outbreak currently includes over 3.7 million cases and 260,000 fatalities. As a betacoronavirus, SARS-CoV-2 encodes for a papain-like protease (PLpro) that is likely responsible for cleavage of the coronavirus (CoV) viral polypeptide. The PLpro is also responsible for suppression of host innate immune responses by virtue of its ability to reverse host ubiquitination and ISGylation events. Here, the biochemical activity of SARS-CoV-2 PLpro against ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) substrates is evaluated, revealing that the protease has a marked reduction in its ability to process K48 linked Ub substrates compared to its counterpart in SARS-CoV. Additionally, its substrate activity more closely mirrors that of the PLpro from the Middle East respiratory syndrome coronavirus and prefers ISG15s from certain species including humans. Additionally, naphthalene based PLpro inhibitors are shown to be effective at halting SARS-CoV-2 PLpro activity as well as SARS-CoV-2 replication.

De novo Transcriptome Reveals Gene Changes in the Development of the Endosperm Chalazal Haustorium in Taxillus chinensis (DC.) Danser.

Loranthus (Taxillus chinensis) is a facultative, hemiparasite and stem parasitic plant that attacks other plants for living. Transcriptome sequencing and bioinformatics analysis were applied in this study to identify the gene expression profiles of fresh seeds (CK), baby (FB), and adult haustoria tissues (FD). We assembled 160,571 loranthus genes, of which 64,926, 35,417, and 47,249 were aligned to NR, GO, and KEGG pathway databases, respectively. We identified 14,295, 15,921, and 16,402 genes in CK, FB, and FD, respectively. We next identified 5,480 differentially expressed genes (DEGs) in the process, of which 258, 174, 81, and 94 were encoding ribosomal proteins (RP), transcription factors (TF), ubiquitin, and disease resistance proteins, respectively. Some DEGs were identified to be upregulated along with the haustoria development (e.g., 68 RP and 26 ubiquitin genes). Notably, 36 RP DEGs peak at FB; 10 ER, 5 WRKY, 6 bHLH, and 4 MYB TF genes upregulated only in FD. Further, we identified 4 out of 32 microRNA genes dysregulated in the loranthus haustoria development. This is the first haustoria transcriptome of loranthus, and our findings will improve our understanding of the molecular mechanism of haustoria.