Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane.

Phospholipid scrambling in dying cells promotes phosphatidylserine exposure, a critical process for efferocytosis. We previously identified the Xkr family protein Xkr4 as a phospholipid-scrambling protein, but its activation mechanisms remain unknown. Here we show that Xkr4 is activated in two steps: dimer formation by caspase-mediated cleavage and structural change caused by activating factors. To identify the factors, we developed a new screening system, "revival screening," using a CRISPR sgRNA library. Applying this system, we identified the nuclear protein XRCC4 as the single candidate for the Xkr4 activator. Upon apoptotic stimuli, XRCC4, contained in the DNA repair complex, is cleaved by caspases, and its C-terminal fragment with an intrinsically disordered region is released into the cytoplasm. Protein interaction screening showed that the fragment interacts directly with the Xkr4 dimer to activate it. This study demonstrates that caspase-mediated cleavage releases a nuclear protein fragment for direct regulation of lipid dynamics on the plasma membrane.

Genetic Characterization of Three Distinct Mechanisms Supporting RNA-Driven DNA Repair and Modification Reveals Major Role of DNA Polymerase ζ.

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.

C-DNA may facilitate homologous DNA pairing.

Recombination-independent homologous pairing represents a prominent yet largely enigmatic feature of chromosome biology. As suggested by studies in the fungus Neurospora crassa, this process may be based on the direct pairing of homologous DNA molecules. Theoretical search for the DNA structures consistent with those genetic results has led to an all-atom model in which the B-DNA conformation of the paired double helices is strongly shifted toward C-DNA. Coincidentally, C-DNA also features a very shallow major groove that could permit initial homologous contacts without atom-atom clashes. The hereby conjectured role of C-DNA in homologous pairing should encourage the efforts to discover its biological functions and may also clarify the mechanism of recombination-independent recognition of DNA homology.

Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Evaluation of reverse transcription yield of RNA standards and forensic samples based on droplet digital PCR.

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 µg/µL - 0.24 ng/µL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 µL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.

Nucleic acid-based electrochemical biosensor for detection of influenza B by gold nanoparticles.

The influenza virus is a pervasive pathogen that exhibits increased prevalence during colder seasons, resulting in a significant annual occurrence of infections. Notably, pharmaceutical interventions effective against influenza A strains often exhibit limited efficacy against influenza B variants. Against this backdrop, the need for innovative approaches to accurately and swiftly differentiate and detect influenza B becomes evident. Biosensors play a pivotal role in this detection process, offering rapid, specific, and sensitive identification of the virus, facilitating timely intervention and containment efforts. Oligonucleotide sequences targeting the conserved B/Victoria/2/87 influenza virus NP region were designed. Nasopharyngeal swabs were collected from patients suspected of influenza virus infection, and viral RNA was extracted. RNA quality was assessed through one-step PCR. cDNA synthesis was performed using random hexamers, and real-time PCR quantified the influenza genome. Gold nanoparticles were immobilized on a surface to immobilize the specific DNA probe, and electrochemical hybridization was electrochemically followed. The biosensor exhibited high selectivity and effective distinction of complementary sequences from mismatches and influenza virus cDNA genome. The biosensor successfully detected the influenza B virus genome in real samples. Non-influenza samples yielded no significant hybridization signals. The comparison between the results obtained from the biosensor and real-time PCR revealed full agreement of these methods. The biosensor utilized electrochemical detection of hybridization and proved effective in detecting the influenza B virus genome with high specificity, sensitivity, and selectivity. Comparative analysis with real-time PCR underscored the accuracy and potential applicability of the biosensor in rapid and specific virus detection. This innovative approach holds promise for future diagnostic and epidemiological applications in detecting influenza B virus and other pathogens.

Full-length sequence analysis of hepatitis C virus genotype 3b strains and development of an in vivo infectious 3b cDNA clone.

IMPORTANCE: HCV genotype 3b is a difficult-to-treat subtype, associated with accelerated progression of liver disease and resistance to antivirals. Moreover, its prevalence has significantly increased among persons who inject drugs posing a serious risk of transmission in the general population. Thus, more genetic information and antiviral testing systems are required to develop novel therapeutic options for this genotype 3 subtype. We determined the complete genomic sequence and complexity of three genotype 3b isolates, which will be beneficial to study its biology and evolution. Furthermore, we developed a full-length in vivo infectious cDNA clone of genotype 3b and showed its robustness and genetic stability in human-liver chimeric mice. This is, to our knowledge the first reported infectious cDNA clone of HCV genotype 3b and will provide a valuable tool to evaluate antivirals and neutralizing antibodies in vivo, as well as in the development of infectious cell culture systems required for further research.

Process conditions affect microbial diversity and activity in a haloalkaline biodesulfurization system.

Biodesulfurization (BD) systems that treat sour gas employ mixtures of haloalkaliphilic sulfur-oxidizing bacteria to convert sulfide to elemental sulfur. In the past years, these systems have seen major technical innovations that have led to changes in microbial community composition. Different studies have identified and discussed the microbial communities in both traditional and improved systems. However, these studies do not identify metabolically active community members and merely focus on members' presence/absence. Therefore, their results cannot confirm the activity and role of certain bacteria in the BD system. To investigate the active community members, we determined the microbial communities of six different runs of a pilot-scale BD system. 16S rRNA gene-based amplicon sequencing was performed using both DNA and RNA. A comparison of the DNA- and RNA-based sequencing results identified the active microbes in the BD system. Statistical analyses indicated that not all the existing microbes were actively involved in the system and that microbial communities continuously evolved during the operation. At the end of the run, strains affiliated with Alkalilimnicola ehrlichii and Thioalkalivibrio sulfidiphilus were confirmed as the most active key bacteria in the BD system. This study determined that microbial communities were shaped predominantly by the combination of hydraulic retention time (HRT) and sulfide concentration in the anoxic reactor and, to a lesser extent, by other operational parameters.IMPORTANCEHaloalkaliphilic sulfur-oxidizing bacteria are integral to biodesulfurization (BD) systems and are responsible for converting sulfide to sulfur. To understand the cause of conversions occurring in the BD systems, knowing which bacteria are present and active in the systems is essential. So far, only a few studies have investigated the BD system's microbial composition, but none have identified the active microbial community. Here, we reveal the metabolically active community, their succession, and their influence on product formation.

A resource of single-cell gene expression profiles in a planarian Dugesia japonica.

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package "Seurat" and GSE223927, researchers can easily access and utilize this dataset.

A novel homozygous splice-site mutation of JK gene leads to Jk(a-b-) phenotype.

OBJECTIVES: This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient. BACKGROUND: Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking. MATERIALS AND METHODS: In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. RESULTS: The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. CONCLUSION: This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.

Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing.

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.

Hypermethylation and down-regulation of vitamin D receptor (VDR) as contributing factors for polycystic ovary syndrome (PCOS): a case-control study from Kashmir, North India.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevailing endocrinopathy affecting a significant population of women of reproductive age across the globe. A myriad set of complex intertwined factors ranging from etiological, genetic, and epigenetic reasons cause this disorder. Out of the different factors, vitamin D shows an imperative aspect in health and fertility of women with polycystic ovary syndrome (PCOS). The importance of vitamin D is facilitated by vitamin D receptor (VDR), a ligand-dependent transcription factor in the steroid/ thyroid hormone receptor superfamily that controls the pleiotropic biological properties of vitamin D. PURPOSE: The purpose of this study was to evaluate the role of promoter methylation of the VDR gene, a transcription factor with numerous biological utilities, with its relative expression and clinico-pathological findings and outcomes. METHODOLOGY: A total of 200 blood samples were collected, 100 from PCOS case subjects, and 100 from the normal healthy controls respectively, which were assessed by qRT-PCR for determining the expression summary. MS-PCR technique was used for analyzing the promoter methylation status of the VDR gene. Blood samples were withdrawn, respectively, for each case and the control study separately experimented for different stages for the given study, of which estimation of vitamin D was also a part. RESULTS: In this test-versus-control study, first, the promoter methylation status of VDR gene was identified which was found more prominent i.e., hyper-methylation of the VDR gene was identified in 84 cases (84%), and in the normal healthy controls, it was found (62%). The promoter methylation status of the VDR gene has remarkably shown the results with a significant difference (p value < 0.0001*). Second, the expression analysis of VDR gene was found to be strongly downregulated in majority (64%) of PCOS case samples analyzed by means fold change of 0.8743 (± 0.06466) (p value 0.0054**). This result is, therefore, indicative of VDR gene role in PCOS pathogenesis as the said gene is downregulated. Moreover, compared to the vitamin D parameter, hyper-methylation and expression analysis of the VDR promoter gene were found to correspond to some associations with PCOS. Certain case-and-control study analyses showed that patients with normal vitamin D levels showed less indicative effects of PCOS and vice versa. CONCLUSION: Our study, being exclusive from Kashmir, one of the foremost specified that VDR confirms anomalous methylation configuration in PCOS with subsequent downregulation in the gene expression i.e., there is an inverse correlation among VDR gene expression (downregulated) and methylation status (hyper-methylated) from the conclusion of our PCOS case-versus-control study.

Evidence for widespread existence of functional novel and non-canonical human transcripts.

BACKGROUND: Fraction of functional sequence in the human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches. RESULTS: In this work, we established a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool in aneuploid cells. Application of this approach to human cell lines in multiple phenotypic screens suggested the presence of many yet uncharacterized functional elements in the human genome, represented at least in part by novel exons of known and novel genes. The novel transcripts containing these exons can be massively, up to thousands-fold, induced by specific stresses, and at least some can represent bi-cistronic protein-coding mRNAs. CONCLUSIONS: Altogether, these results argue that many unannotated and non-canonical human transcripts, including those that appear as aberrant splice products, have biological relevance under specific biological conditions.

Cloning of human Type I interferon cDNAs.

In the late 1970s, crude interferon samples were found to exhibit anti-tumour activity. This discovery led to the interferon as a "magic drug" for cancer patients. Many groups, including those in Tokyo, Zürich, and San Francisco, attempted to identify human interferon cDNAs. Tadatsugu Taniguchi was the first to announce the cloning of human interferon-ß cDNA in the December 1979 issue of Proc. Jpn. Acad. Ser. B. This was followed by the cloning of human interferon-α by a Zürich group and interferon-γ by a group in Genentech in San Francisco. Recombinant interferon proteins were produced on a large scale, and interferon-α was widely used to treat C-type hepatitis patients. The biological functions of interferons were quickly elucidated with the purified recombinant interferons. The molecular mechanisms underlying virus-induced interferon gene expression were also examined using cloned chromosomal genes. The background that led to interferon gene cloning and its impact on cytokine gene hunting is described herein.

The effect of diet composition on the diversity of active gut bacteria and on the growth of Spodoptera exigua (Lepidoptera: Noctuidae).

Gut microbiota plays a functional role in nutrition among several insects. However, the situation is unclear in Lepidoptera. Field studies suggest the microbiome may not be stable and is determined by diet, while in the laboratory, Lepidoptera are routinely reared on diet containing antibiotics with unknown effects on microbial communities. Furthermore, molecular approaches for the characterization of lepidopteran microbiomes rarely describe the metabolically active gut bacteria. The aim of this study was to evaluate how diet and antibiotics affect Spodoptera exigua (Hübner) growth and the diversity and activity of the gut bacteria community. We assessed how alfalfa and wheat germ-based diets affected larval growth, in the presence and absence of streptomycin. Alfalfa diet improved larval growth, pupal mass, and survival, but antibiotic was only beneficial in the wheat germ diet. We observed diet-driven changes in the gut bacterial communities. In the active community, the alfalfa colony was dominated by Enterococcus and Rhodococcus whereas in the wheat germ colony, only Enterococcus was present. In contrast, spore-forming Bacilli species were very common members of the DNA community. In both cases, streptomycin had a selective effect on the relative abundance of the taxa present. Our study highlights the importance of characterizing both the diversity and activity of the gut microbiota community. DNA-derived communities may include environmental DNA, spores, or non-viable bacteria, while RNA-derived communities are more likely to give an accurate representation of the diversity of active members that are potentially directly involved in the metabolic processes of the host.

The Current Situation and Development Prospect of Whole-Genome Screening.

High-throughput genetic screening is useful for discovering critical genes or gene sequences that trigger specific cell functions and/or phenotypes. Loss-of-function genetic screening is mainly achieved through RNA interference (RNAi), CRISPR knock-out (CRISPRko), and CRISPR interference (CRISPRi) technologies. Gain-of-function genetic screening mainly depends on the overexpression of a cDNA library and CRISPR activation (CRISPRa). Base editing can perform both gain- and loss-of-function genetic screening. This review discusses genetic screening techniques based on Cas9 nuclease, including Cas9-mediated genome knock-out and dCas9-based gene activation and interference. We compare these methods with previous genetic screening techniques based on RNAi and cDNA library overexpression and propose future prospects and applications for CRISPR screening.

Molecular Characterization and Pathogenicity of an Infectious cDNA Clone of Youcai Mosaic Virus on Solanum nigrum.

Virus infections cause devastative economic losses for various plant species, and early diagnosis and prevention are the most effective strategies to avoid the losses. Exploring virus genomic evolution and constructing virus infectious cDNA clones is essential to achieve a deeper understanding of the interaction between host plant and virus. Therefore, this work aims to guide people to better prevent, control, and utilize the youcai mosaic virus (YoMV). Here, the YoMV was found to infect the Solanum nigrum under natural conditions. Then, an infectious cDNA clone of YoMV was successfully constructed using triple-shuttling vector-based yeast recombination. Furthermore, we established phylogenetic trees based on the complete genomic sequences, the replicase gene, movement protein gene, and coat protein gene using the corresponding deposited sequences in NCBI. Simultaneously, the evolutionary relationship of the YoMV discovered on S. nigrum to others was determined and analyzed. Moreover, the constructed cDNA infectious clone of YoMV from S. nigrum could systematically infect the Nicotiana benthamiana and S. nigrum by agrobacterium-mediated infiltration. Our investigation supplied a reverse genetic tool for YoMV study, which will also contribute to in-depth study and profound understanding of the interaction between YoMV and host plant.

Purification, Characterization, cDNA Cloning, and Bioinformatic Analysis of Zinc-Binding Protein from Magallana hongkongensis.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis.

Functional and Structural Properties of Type V Collagen from the Skin of the Shortbill Spearfish (Tetrapturus angustirostris).

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish (Tetrapturus angustirostris). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch (Perca flavescens) instead of broadbill swordfish (Xiphias gladius). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.

Screening DHHCs of S-acylated proteins using an OsDHHC cDNA library and bimolecular fluorescence complementation in rice.

S-acylation is an important lipid modification that primarily involves DHHC proteins (DHHCs) and associated S-acylated proteins. No DHHC-S-acylated protein pair has been reported so far in rice (Oryza sativa L.) and the molecular mechanisms underlying S-acylation in plants are largely unknown. We constructed an OsDHHC cDNA library for screening corresponding pairs of DHHCs and S-acylated proteins using bimolecular fluorescence complementation assays. Five DHHC-S-acylated protein pairs (OsDHHC30-OsCBL2, OsDHHC30-OsCBL3, OsDHHC18-OsNOA1, OsDHHC13-OsNAC9, and OsDHHC14-GSD1) were identified in rice. Among the pairs, OsCBL2 and OsCBL3 were S-acylated by OsDHHC30 in yeast and rice. The localization of OsCBL2 and OsCBL3 in the endomembrane depended on S-acylation mediated by OsDHHC30. Meanwhile, all four OsDHHCs screened complemented the thermosensitive phenotype of an akr1 yeast mutant, and their DHHC motifs were required for S-acyltransferase activity. Overexpression of OsDHHC30 in rice plants improved their salt and oxidative tolerance. Together, these results contribute to our understanding of the molecular mechanism underlying S-acylation in plants.