Variant Isoforms Research Articles

Galactinol synthase 1 improves cucumber performance under cold stress by enhancing assimilate translocation.

Cucumber (Cucumis sativus L.) predominantly translocates raffinose family oligosaccharides (RFOs) in the phloem and accumulates RFOs in leaves. Galactinol synthase (GolS) catalyzes the critical step of RFO biosynthesis, and determining the functional diversity of multiple GolS isoforms in cucumber is of scientific significance. In this study, we found that all four isoforms of CsGolS in the cucumber genome were upregulated by different abiotic stresses. β-Glucuronidase staining and tissue separation experiments suggested that CsGolS1 is expressed in vascular tissues, whereas the other three CsGolSs are located in mesophyll cells. Further investigation indicates that CsGolS1 plays double roles in both assimilate loading and stress response in minor veins, which could increase the RFO concentration in the phloem sap and then improve assimilate transport under adverse conditions. Cold-induced minor vein-specific overexpression of CsGolS1 enhanced the assimilate translocation efficiency and accelerated the growth rates of sink leaves, fruits, and whole plants under cold stress. Finally, our results demonstrate a previously unknown response to adverse environments and provide a potential biotechnological strategy to improve the stress resistance of cucumber.

CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells.

Chaperone-mediated autophagy (CMA) is a highly specific lysosomal-dependent protein degradation pathway. A critical molecular component of CMA is the lysosome-associated membrane protein (LAMP) type 2A, which is required for substrate uptake by the lysosome. Defects in the CMA pathway have been associated with various human pathologies, including malignancies, increasing the overall interest in methods to monitor this selective autophagy process. Yet isogenic LAMP-2A knockout cancer cell models are still lacking. This is likely to depend on challenges related to that human LAMP-2 gene undergoes alternative splicing of its pre-mRNA, generating three isoform variants, LAMP-2A, LAMP-2B, and LAMP-2C. However, without assessment of the impact of LAMP-2A loss of function specifically in human cells, the involvement of CMA in human pathologies, including carcinogenesis remains speculative. Here, we describe the generation of isoform-specific CRISPR-Cas9 genomic editing of LAMP-2A in human cancer cells, without affecting the other two isoforms, allowing for experimental evaluation of LAMP-2A, thus CMA in human cancer models.

Glycogen Synthase Kinase-3 Isoform Variants and Their Inhibitory Phosphorylation in Human Testes and Spermatozoa.

To clarify (phospho-) glycogen synthase kinase-3 (GSK3) isoform variants in the germline and soma of human testes and spermatozoa. GSK3 isoform variants in normospermatogenic and Sertoli cell-only (SCO) testicular biopsies and spermatozoa were examined. In normospermatogenic testes, GSK3α and GSK3β variants 1 and 2 different in low complexity region (LCR) were expressed and their levels were decreased in SCO testes. GSK3β variant 3 was only expressed in SCO testes. GSK3β as well as GSK3α, the dominant isoforms in testes were decreased in SCO testes. In normospermatogenic testes, GSK3β were found in spermatogonia and markedly decreased in meiotic germ cells in which GSK3α was dominant. p-GSK3α/β were marginal in spermatogonia and early spermatocytes. In SCO testes, GSK3α/β immunoreactivity in seminiferous epithelia was weaker than those of normospermatogenic testes whereas p-GSK3α/β(Ser) immunoreactivity was visibly increased in Sertoli cells. GSK3α was dominant in ejaculated spermatozoa in which GSK3α and p-GSK3α(Ser) were found in the head, midpiece, and tail. In acrosome-reacted spermatozoa, GSK3α was found in the equatorial region of head, midpiece, and tail, and p-GSK3α(Ser) was only found in midpiece. During sperm capacitation, p-GSK3α(Ser) was significantly increased together with phosphotyrosine proteins and motility. In human male germ cells, GSK3 isoforms different in LCRs switch from GSK3β to GSK3α during meiotic entry, suggesting the isoform-specific roles of GSK3α and GSK3β in meiosis and stemness or proliferation of spermatogonia, respectively. In dormant Sertoli cells of SCO testes kinase activity of GSK3 might be downregulated via inhibitory phosphorylation. In spermatozoa, inhibitory phosphorylation of GSK3α might be coupled with activation of motility during capacitation.

Derivation and characterization of the induced pluripotent stem cell line CUIMCi004-A from a patient with a novel frameshift variant in exon 18a of OCRL

OCRL encodes for an inositol polyphosphate 5-phosphatase, located in the trans-Golgi network, endosomes, endocytic clathrin-coated pits, primary cilia. Mutations in OCRL causes Lowe syndrome (LS), a rare and complex disorder characterized by congenital cataracts, renal tubular dysfunction, and mental retardation. Here we generated an induced pluripotent stem cell (iPSC) line from Peripheral Blood Mononuclear Cell (PBMCs) of a 5-year-old boy with severe obesity carrying a novel pathogenic variant in the brain-expressed isoform of OCRL. The Sendai virus approach was used for reprogramming. The iPSC line CUIMCi004-A may serve as a useful resource to further investigate the tissue-specific function of OCRL.

Microarray analysis to explore the effect of CXCL12 isoforms in a pancreatic pre-tumor cell model.

CXCL12 expression was significantly lower in tumor samples than in corresponding normal samples. CXCL12 expression was significantly positively related to the infiltration levels of T cells, dendritic cells (DCs), immature DCs, cytotoxic cells, Tfh cells, mast cells, B cells, Th1 cells, natural killer (NK) cells, pDCs, neutrophils, and T helper cells (Spearman correlation coefficient > 0.5, P < 0.001) and negatively correlated with the infiltration level of NK CD56bright cells. In addition, pancreatic hTERT-HPNE cells treated with three diverse CXCL12 isoforms exhibited changes mainly in the regulation of the epithelial-mesenchymal transition activation pathway.

Distinct binding kinetics of E-, P- and L-selectins to CD44.

Molecular-level selectin-cluster of differentiation 44 (CD44) interactions are far from clear because of the complexity and diversity of CD44 glycosylation and isoforms expressed on various types of cells. By combining experimental measurements and simulation predictions, the binding kinetics of three selectin members to the recombinant CD44 were quantified and the corresponding microstructural mechanisms were explored, respectively. Experimental results showed that the E-selectin-CD44 interactions mainly mediated the firm adhesion of microbeads under shear flow with the strongest rupture force. P- and L-selectins had similar interaction strength but different association and dissociation rates by mediating stable rolling and transient adhesions of microbeads, respectively. Molecular docking and molecular dynamics (MD) simulations predicted that the binding epitopes of CD44 to selectins are all located at the side face of each selectin, although the interfaces denoted as the hinge region are between lectin and epidermal growth factor domains of E-selectin, Lectin domain side of P-selectin and epidermal growth factor domain side of L-selectin, respectively. The lowest binding free energy, the largest rupture force and the longest lifetime for E-selectin, as well as the comparable values for P- and L-selectins, demonstrated in both equilibration and steered MD simulations, supported the above experimental results. These results offer basicdata for understanding the functional differences of selectin-CD44 interactions.

CD44: A Multifunctional Mediator of Cancer Progression.

CD44, a non-kinase cell surface transmembrane glycoprotein, has been widely implicated as a cancer stem cell (CSC) marker in several cancers. Cells overexpressing CD44 possess several CSC traits, such as self-renewal and epithelial-mesenchymal transition (EMT) capability, as well as a resistance to chemo- and radiotherapy. The CD44 gene regularly undergoes alternative splicing, resulting in the standard (CD44s) and variant (CD44v) isoforms. The interaction of such isoforms with ligands, particularly hyaluronic acid (HA), osteopontin (OPN) and matrix metalloproteinases (MMPs), drive numerous cancer-associated signalling. However, there are contradictory results regarding whether high or low CD44 expression is associated with worsening clinicopathological features, such as a higher tumour histological grade, advanced tumour stage and poorer survival rates. Nonetheless, high CD44 expression significantly contributes to enhanced tumourigenic mechanisms, such as cell proliferation, metastasis, invasion, migration and stemness; hence, CD44 is an important clinical target. This review summarises current research regarding the different CD44 isoform structures and their roles and functions in supporting tumourigenesis and discusses CD44 expression regulation, CD44-signalling pathways and interactions involved in cancer development. The clinical significance and prognostic value of CD44 and the potential of CD44 as a therapeutic target in cancer are also addressed.

Altered cell and RNA isoform diversity in aging Down syndrome brains

Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.

Role of neurexin heparan sulfate in the molecular assembly of synapses - expanding the neurexin code?

Synapses are the minimal information processing units of the brain and come in many flavors across distinct circuits. The shape and properties of a synapse depend on its molecular organisation, which is thought to largely depend on interactions between cell adhesion molecules across the synaptic cleft. An established example is that of presynaptic neurexins and their interactions with structurally diverse postsynaptic ligands: the diversity of neurexin isoforms that arise from alternative promoters and alternative splicing specify synaptic properties by dictating ligand preference. The recent finding that a majority of neurexin isoforms exist as proteoglycans with a single heparan sulfate (HS) polysaccharide adds to this complexity. Sequence motifs within the HS polysaccharide may differ between neuronal cell types to contribute specificity to its interactions, thereby expanding the coding capacity of neurexin diversity. However, an expanding number of HS-binding proteins have been found capable to recruit neurexins via the HS chain, challenging the concept of a code provided by neurexin splice isoforms. Here we discuss the possible roles of the neurexin HS in light of what is known from other HS-protein interactions, and propose a model for how the neurexin HS polysaccharide may contribute to synaptic assembly. We also discuss how the neurexin HS may be regulated by co-secreted carbonic anhydrase-related and FAM19A proteins, and highlight some key issues that should be resolved to advance the field.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in a Preclinical Model By Monoclonal Anti-HPA-1a Antibody Prophylaxis: Structural and Functional Properties of Antibody Variant Isoforms

Maternal alloantibodies to paternally inherited platelet antigens can cause fetal/neonatal alloimmune thrombocytopenia (FNAIT), rendering the fetus or newborn prone to bleeding and intracranial hemorrhage (ICH) with risk of lifelong disabilities or death. In Caucasians, the vast majority of cases are due to antibodies to the Human Platelet Antigen (HPA)-1a epitope on glycoprotein (GP)IIIa. To date, there are neither any means to prevent or reduce the risk of alloimmunization and subsequent FNAIT, nor safe and efficient treatment during affected pregnancies. Thus, a prophylactic regimen by administration of monoclonal antibodies to women at risk would be highly beneficial. Knowledge about the optimal functional design of prophylactic monoclonal antibodies for antenatal or post-natal use is however still limited. We have previously isolated and characterized a fully human anti-HPA-1a monoclonal antibody, mAb26.4. In the current study we have explored the prophylactic potential of this antibody by testing a panel of different IgG1 designs, including the wild-type mAb26.4, variants with modified Fc-region N-glycans, as well as an effector silent variant. We performed analyses of properties relevant for immunosuppression: in vitro Fc-receptor binding and capacity to induce phagocytosis, in vivo half-life measurements in humanized FcRn mice and platelet clearance in a recently developed transgenic mouse strain with a recreated HPA-1a epitope on murine GPIIIa. The prophylactic capacity by antibody-mediated immune suppression in vivo, were further tested in the FNAIT pre-clinical murine model, in which BALB/c females can be immunized by transfusion of HPA-1a-expressing platelets from the transgenic mice and where subsequent breeding of pre-immunized mice with transgenic males cause thrombocytopenia in off-springs mimicking FNAIT. By intravenous administrations of mAb26.4 variants prior to platelet transfusions, the mice generated no or low anti-platelet responses compared to control mice, and normal platelet counts in pups upon subsequent breeding. Our data thus successfully demonstrates efficient immunosuppression and prevention of FNAIT by anti-HPA-1a human monoclonal variants, providing further support for potential use in humans. DisclosuresSkogen: Prophylix Pharma AS: Current holder of individual stocks in a privately-held company. Newman: Rallybio: Consultancy, Research Funding.

Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

SummaryAlternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. Global patterns of transcript diversity are similar between human and mouse cortex, although certain genes are characterized by striking differences between species. We also identify developmental changes in alternative splicing, with differential transcript usage between human fetal and adult cortex. Our data confirm the importance of alternative splicing in the cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide transcript-level data for human and mouse cortex as a resource to the scientific community.

Disrupted Calcium Homeostasis in Duchenne Muscular Dystrophy: A Common Mechanism behind Diverse Consequences.

Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the DMD gene encoding diverse isoforms of dystrophin. Loss of full-length dystrophins is both necessary and sufficient for causing degeneration and wasting of striated muscles, neuropsychological impairment, and bone deformities. Among this spectrum of defects, abnormalities of calcium homeostasis are the common dystrophic feature. Given the fundamental role of Ca2+ in all cells, this biochemical alteration might be underlying all the DMD abnormalities. However, its mechanism is not completely understood. While abnormally elevated resting cytosolic Ca2+ concentration is found in all dystrophic cells, the aberrant mechanisms leading to that outcome have cell-specific components. We probe the diverse aspects of calcium response in various affected tissues. In skeletal muscles, cardiomyocytes, and neurons, dystrophin appears to serve as a scaffold for proteins engaged in calcium homeostasis, while its interactions with actin cytoskeleton influence endoplasmic reticulum organisation and motility. However, in myoblasts, lymphocytes, endotheliocytes, and mesenchymal and myogenic cells, calcium abnormalities cannot be clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. Nevertheless, DMD gene mutations in these cells lead to significant defects and the calcium anomalies are a symptom of the early developmental phase of this pathology. As the impaired calcium homeostasis appears to underpin multiple DMD abnormalities, understanding this alteration may lead to the development of new therapies. In fact, it appears possible to mitigate the impact of the abnormal calcium homeostasis and the dystrophic phenotype in the total absence of dystrophin. This opens new treatment avenues for this incurable disease.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80kDa) translated from full-length OCRL, whereas only one protein (80kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50%enzyme activity, whereas the Lowe syndrome variants retained <20%activity. We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Suppression of CPSF6 Enhances Apoptosis Through Alternative Polyadenylation-Mediated Shortening of the VHL 3'UTR in Gastric Cancer Cells.

Alternative polyadenylation (APA) is an important RNA post-transcriptional process, which can generate diverse mRNA isoforms. Increasing evidence shows that APA is involved in cell self-renewal, development, immunity, and cancer. CPSF6 is one of the core proteins of CFIm complex and can modulate the APA process. Although it has been reported to play oncogenic roles in cancer, the underlying mechanisms remain unclear. The aim of the present study was to characterize CPSF6 in human gastric cancer (GC). We observed that CPSF6 was upregulated in GC. Knockdown of CPSF6 inhibited proliferation and enhanced apoptosis of GC cells both in vitro and in vivo. Global APA site profiling analysis revealed that knockdown of CPSF6 induced widespread 3′UTR shortening of genes in GC cells, including VHL. We also found CPSF6 negatively regulated the expression of VHL through APA and VHL short-3′UTR isoform enhanced apoptosis and inhibited cell growth in GC cells. Our data suggested that CPSF6-induced cell proliferation and inhibition of apoptosis were mediated by the preferential usage of poly(A) in VHL. Our data provide insights into the function of CPSF6 and may imply potential therapeutic targets against GC.

A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

SplicingFactory-splicing diversity analysis for transcriptome data.

MotivationAlternative splicing contributes to the diversity of RNA found in biological samples. Current tools investigating patterns of alternative splicing check for coordinated changes in the expression or relative ratio of RNA isoforms where specific isoforms are up- or down-regulated in a condition. However, the molecular process of splicing is stochastic and changes in RNA isoform diversity for a gene might arise between samples or conditions. A specific condition can be dominated by a single isoform, while multiple isoforms with similar expression levels can be present in a different condition. These changes might be the result of mutations, drug treatments or differences in the cellular or tissue environment. Here, we present a tool for the characterization and analysis of RNA isoform diversity using isoform level expression measurements.ResultsWe developed an R package called SplicingFactory, to calculate various RNA isoform diversity metrics, and compare them across conditions. Using the package, we tested the effect of RNA-seq quantification tools, quantification uncertainty, gene expression levels and isoform numbers on the isoform diversity calculation. We analyzed a set of CD34+ hematopoietic stem cells and myelodysplastic syndrome samples and found a set of genes whose isoform diversity change is associated with SF3B1 mutations.Availability and implementationThe SplicingFactory package is freely available under the GPL-3.0 license from Bioconductor for the Windows, MacOS and Linux operating systems (https://www.bioconductor.org/packages/release/bioc/html/SplicingFactory.html).Supplementary information Supplementary data are available at Bioinformatics online.

Plasma Membrane Ca2+ Pump PMCA4z Is More Active Than Splicing Variant PMCA4x.

The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms “x” and “z”. While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants “x” and “z”, maintaining constant the most frequent splice variant “b” at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant “z” of the splicing site A.

Deconvolution of expression for nascent RNA-sequencing data (DENR) highlights pre-RNA isoform diversity in human cells.

MotivationQuantification of isoform abundance has been extensively studied at the mature RNA level using RNA-seq but not at the level of precursor RNAs using nascent RNA sequencing.ResultsWe address this problem with a new computational method called Deconvolution of Expression for Nascent RNA-sequencing data (DENR), which models nascent RNA-sequencing read-counts as a mixture of user-provided isoforms. The baseline algorithm is enhanced by machine-learning predictions of active transcription start sites and an adjustment for the typical ‘shape profile’ of read-counts along a transcription unit. We show that DENR outperforms simple read-count-based methods for estimating gene and isoform abundances, and that transcription of multiple pre-RNA isoforms per gene is widespread, with frequent differences between cell types. In addition, we provide evidence that a majority of human isoform diversity derives from primary transcription rather than from post-transcriptional processes.Availability and implementationDENR and nascentRNASim are freely available at https://github.com/CshlSiepelLab/DENR (version v1.0.0) and https://github.com/CshlSiepelLab/nascentRNASim (version v0.3.0).Supplementary information Supplementary data are available at Bioinformatics online.

A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat.

Summary Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7.We utilized whole‐genome sequencing, association mapping, gene‐disrupted mutants, gain‐of‐function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions.A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light‐dependent pathway, whereas Snn7‐mediated PCD functioned in a light‐independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3.We validated a novel effector operating in an inverse‐gene‐for‐gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph–host interaction.

Aberration-corrected ultrafine analysis of miRNA reads at single-base resolution: a k-mer lattice approach.

Raw sequencing reads of miRNAs contain machine-made substitution errors, or even insertions and deletions (indels). Although the error rate can be low at 0.1%, precise rectification of these errors is critically important because isoform variation analysis at single-base resolution such as novel isomiR discovery, editing events understanding, differential expression analysis, or tissue-specific isoform identification is very sensitive to base positions and copy counts of the reads. Existing error correction methods do not work for miRNA sequencing data attributed to miRNAs’ length and per-read-coverage properties distinct from DNA or mRNA sequencing reads. We present a novel lattice structure combining kmers, (k – 1)mers and (k + 1)mers to address this problem. The method is particularly effective for the correction of indel errors. Extensive tests on datasets having known ground truth of errors demonstrate that the method is able to remove almost all of the errors, without introducing any new error, to improve the data quality from every-50-reads containing one error to every-1300-reads containing one error. Studies on experimental miRNA sequencing datasets show that the errors are often rectified at the 5′ ends and the seed regions of the reads, and that there are remarkable changes after the correction in miRNA isoform abundance, volume of singleton reads, overall entropy, isomiR families, tissue-specific miRNAs, and rare-miRNA quantities.