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Abstract BackgroundAlternative splicing of precursor mRNAs serves as a crucial mechanism to enhance gene expression plasticity for organismal adaptation. However, the precise regulation and function of alternative splicing in plant immune gene regulation remain elusive. ResultsHere, by deploying in-depth transcriptome profiling with deep genome coverage coupled with differential expression, differential alternative splicing, and differential transcript usage analysis, we reveal profound and dynamic changes in alternative splicing following treatment with microbial pattern flg22 peptides inArabidopsis. Our findings highlight RNA polymerase II C-terminal domain phosphatase-like 3 (CPL3) as a key regulator of alternative splicing, preferentially influencing the splicing patterns of defense genes rather than their expression levels. CPL3 mediates the production of a flg22-induced alternative splicing variant, diacylglycerol kinase 5α (DGK5α), which differs from the canonical DGK5β in its interaction with the upstream kinase BIK1 and subsequent phosphorylation, resulting in reduced flg22-triggered production of phosphatidic acid and reactive oxygen species. Furthermore, our functional analysis suggests that DGK5β, but not DGK5α, contributes to plant resistance against virulent and avirulent bacterial infections. ConclusionsThese findings underscore the role of CPL3 in modulating alternative splicing dynamics of defense genes and DGK5 isoform-mediated phosphatidic acid homeostasis, shedding light on the intricate mechanisms underlying plant immune gene regulation. 

Abstract Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using ‘off-the-shelf’ products, such as allogeneic CAR natural killer T (^AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into^AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced^AlloCAR-NKT cells with high yield and purity. We generated^AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of^AlloCAR-NKT cells support their potential for clinical translation. 

Abstract Allogeneic Vγ9Vδ2 (Vδ2) T cells have emerged as attractive candidates for developing cancer therapy due to their established safety in allogeneic contexts and inherent tumor-fighting capabilities. Nonetheless, the limited clinical success of Vδ2 T cell-based treatments may be attributed to donor variability, short-lived persistence, and tumor immune evasion. To address these constraints, we engineer Vδ2 T cells with enhanced attributes. By employing CD16 as a donor selection biomarker, we harness Vδ2 T cells characterized by heightened cytotoxicity and potent antibody-dependent cell-mediated cytotoxicity (ADCC) functionality. RNA sequencing analysis supports the augmented effector potential of Vδ2 T cells derived from CD16 high (CD16^Hi) donors. Substantial enhancements are further achieved through CAR and IL-15 engineering methodologies. Preclinical investigations in two ovarian cancer models substantiate the effectiveness and safety of engineered CD16^HiVδ2 T cells. These cells target tumors through multiple mechanisms, exhibit sustained in vivo persistence, and do not elicit graft-versus-host disease. These findings underscore the promise of engineered CD16^HiVδ2 T cells as a viable therapeutic option for cancer treatment. 

Like many cereal crops, barley is also negatively affected by drought stress. However, due to its simple genome as well as enhanced stress resilient nature compared to rice and wheat, barley has been considered as a model to decipher drought tolerance in cereals. In the present study, transcriptomic and hormonal profiles along with several biochemical features were compared between drought-tolerant (Otis) and drought-sensitive (Baronesse) barley genotypes subjected to drought to identify molecular and biochemical differences between the genotypes. The drought-induced decrease in the leaf relative water content, net photosynthesis, and biomass accumulation was relatively low in Otis compared to Baronesse. The hormonal profiles did not reveal significant differences for majority of the compounds other than the GA20 and the cis-zeatin-o-glucoside (c-ZOG), whose levels were greatly increased in Otis compared to Baronesse under drought. The major differences that emerged from the transcriptome analysis are; (1), the overall number of differentially expressed genes was relatively low in drought-tolerant Otis compared to drought-sensitive Baronesse; (2), a wax biosynthesis gene (CER1), and NAC transcription factors were specifically induced in Otis but not in Baronesse; (3), the degree of upregulation of betaine aldehyde dehydrogenase and a homeobox transcription factor (genes with proven roles in imparting drought tolerance), was greater in Otis compared to Baronesse; (4) the extent of downregulation of gene expression profiles for proteins of the reaction center photosystem II (PSII) (D1 and D2) was low in Otis compared to Baronesse; and, (5), alternative splicing (AS) was also found to differ between the genotypes under drought. Taken together, the overall transcriptional responses were low in drought-tolerant Otis but the genes that could confer drought tolerance were either specifically induced or greatly upregulated in the tolerant genotype and these differences could be important for drought tolerance in barley. 

Abstract Background Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. Results We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. Conclusions AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species. 

SUMMARY Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA‐seq data, and enables detailed investigations of transcriptional and post‐transcriptional regulation. Using novel computational methods and a combination of PacBio Iso‐seq and Illumina short‐read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2‐row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high‐resolution reverse transcriptase‐polymerase chain reaction and 5'‐RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high‐quality resource for advanced transcriptomic analyses, including both transcriptional and post‐transcriptional regulation, with exceptional resolution and precision.