Disease resistance often comes with a penalty in growth and yield. The microRNA miR156 and its target, the transcription factor gene IDEAL PLANT ARCHITECTURE 1 (IPA1), regulate developmental processes such as tillering and panicle branching while enhancing disease resistance and abiotic stress tolerance in rice (Oryza sativa). However, how this transcription factor regulates multiple processes remains unclear. Here, we found that IPA1 physically interacts with NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (OsNPR1) in the nucleus. Under normal conditions, the OsNPR1 expression levels are low, and OsCULLIN3a (OsCUL3a), an E3 ligase responsible for OsNPR1 degradation, keeps the abundance of monomeric OsNPR1 low in the nucleus and prevents IPA1 from transcriptionally regulating defense genes. When the plant is attacked by pathogens, OsNPR1 oligomers dissociate into monomers, which translocate into the nucleus and physically interact with IPA1, facilitating its binding to promoters of downstream genes, thereby activating positive defense regulators and repressing negative defense regulators. Simultaneously, IPA1 abundance increases, and IPA1 interacts with OsNPR1 and OsCUL3a, interfering with the OsCUL3a-OsNPR1 interaction, dampening the ubiquitin-mediated degradation of OsNPR1. The stabilization of OsNPR1 by IPA1 further enhances IPA1 transcriptional activity in disease resistance. Our work demonstrates that OsNPR1 facilitates IPA1 binding to the promoters of genes related to disease resistance and that IPA1 inhibits OsCUL3a-mediated degradation of OsNPR1.

Urinary tract infections (UTI) are among the most prevalent infectious diseases, demanding rapid diagnosis to provide timely therapeutic interventions. Urinary molecules can indicate the responses of the host cells to pathogenic infections, which may serve as markers for UTI diagnosis. Sensitive and multiplex detection of urinary markers in one-pot has remained challenging. Herein, we developed an allosteric DNAzyme-based biosensor, denoted as SMART (sensitive and multiplex detection of ATP and miRNAs for UTI diagnosis), to enable rapid UTI diagnosis. Multiple sensing modules were integrated into a unimolecular DNA strand of perfect stoichiometry and excellent thermodynamic stability that collectively led to enhanced detection capability. The binding of targets to the detection module induces conformational reconfiguration of SMART to activate sequence-specific catalytic cleavage against fluorescent RNA reporters. SMART demonstrated remarkable sensitivity (ATP: ∼pM; miRNAs: ∼fM) and multiplexing capability (∼4 markers) to realize extraction-free, preamplification-free, and rapid (∼2.5 hr) detection of UTI markers. SMART-based UTI diagnosis model yielded a detection accuracy of 95.5% in a cohort of 164 patients. SMART may serve as a technical platform for detecting various markers that could be applied to the diagnosis of UTI and many other diseases in the future.

Artificial selection has greatly shaped crop agronomic traits1-3; however, the mechanistic basis of how immunity is selected remains unclear. Here we identify the Oryza sativa nucleotide-binding site and leucine-rich repeat (NLR) receptor XA48 and downstream transcription factors OsVOZ1 and OsVOZ2 (OsVOZ1/2), which confer resistance to bacterial blight. XA48 perceives the ancient pathogen effector XopG, activating effector-triggered immunity by degrading the negative regulator OsVOZ1/2. The XA48-OsVOZ1 module has undergone subspecies-specific selection: Xa48 is retained only in Oryza sativa indica and was lost in Oryza sativa japonica. By contrast, OsVOZ1 has diverged into two haplotypes-O. s. indica retains both OsVOZ1A/S alleles compatible with Xa48, whereas O. s. japonica has only OsVOZ1A. Reintroducing Xa48 into O. s. japonica severely compromises yield owing to the XA48-OsVOZ1A-mediated immune incompatibility. Stacking XA48-mediated effector-triggered immunity with XA21-mediated pattern-triggered immunity reconstitutes the broad-spectrum resistance from wild rice. Our study therefore reveals how asymmetric selection of an NLR-transcription factor module shapes disease resistance and reproductive development, providing a strategy for breeding crops by harnessing the relative immunity of wild rice.

Circulating nucleic acids are emerging disease markers whose clinical applications are hindered by the lack of rapid, sensitive, convenient, and cost-effective detection assays. Inspired by the natural replication of virus, here, we developed a biomimetic one-step, one-pot, isothermal detection assay named RAPID to realize rapid and sensitive detection of nucleic acids with minimal reliance on instruments. The core element of RAPID is an autocatalytic molecular sensor that exploits the viral replication endonuclease of duck circovirus (i.e., DCV) to transform rolling circle amplification (RCA) from linear to exponential. DCV cleaves target-induced, RCA-generated amplicons into target analogs to prime secondary RCA reactions to catalytically propagate the amplification sensor. RAPID enables rapid (∼10 min), ultrasensitive (attomolar sensitivity), and direct (RNA extraction-free) detection of microRNAs and viral RNAs that is compatible with smartphone-based fluorescence detection devices. RAPID exhibited pronounced clinical translational capability by quantitatively profiling a panel of six miRNAs to achieve accurate discrimination of prostate cancer from benign prostatic hyperplasia that is exceptionally important but challenging in clinics. Furthermore, RAPID demonstrated rapid detection of influenza A viral infections of high accuracy in point-of-care settings. Simple nucleic acid detection assays like RAPID could largely promote the development and application of liquid biopsy molecular diagnostics.

Objective·To achieve the rapid detection of Staphylococcus aureus using nanopore Cas9-targeted sequencing (nCATS) technology, and simultaneously perform staphylococcal protein A (spa) typing and staphylococcal cassette chromosome mec (SCCmec) typing.Methods·The spa gene and SCCmec gene elements were selected as two regions of interest (ROIs) for targeted sequencing. Four types of CRISPR RNAs (crRNAs) were designed to form Cas9 ribonucleoproteins (RNPs) to cleave sequences flanking the two ROIs. For each crRNA, a 42 bp synthetic target DNA was designed. Appropriate crRNAs were screened, the cleavage system was optimized, and both the cleavage reaction time and Cas9 RNP synthesis temperature were determined based on the results of Cas9 RNP cleavage efficiency testing. Genomic DNA was extracted from a methicillin-resistant Staphylococcus aureus (MRSA) strain, and the ends flanking the cleaved ROIs were dephosphorylated and dA-tailed. Sequencing adapters were ligated, and sequencing was performed using a nanopore platform. The quality scores of the sequencing data were analyzed, and the obtained nucleic acid sequences were compared with those in the mecA, spa and SCCmec databases. Based on the comparison results, the presence of Staphylococcus aureus, MRSA or not, and spa and SCCmec types were determined.Results·Two sets of crRNAs were designed. Based on grayscale analysis of electrophoresis results, the set with higher cleavage efficiency was selected for further experiments. Optimization showed that a 1∶1 ratio of Cas9 RNP to target DNA, a 15 min cleavage reaction time, and a Cas9 RNP synthesis temperature of 25 ℃ yielded a cleavage efficiency of 87.41%. nCATS sequencing quality scores ranged between 15 (Q15) and 20 (Q20), indicating an approximate sequencing accuracy of 99%. Sequence comparisons with the mecA, spa and SCCmec databases revealed that the strain's spa type was t2 and its SCCmec type was Ⅱ. These results were consistent with those obtained by PCR amplification sequencing and multiplex PCR.Conclusion·The nCATS technique enables rapid detection of Staphylococcus aureus, while simultaneously providing spa and SCCmec typing information.

Pathogens constantly attack staple crops, leading to substantial yield losses. Plant-pathogen interactions activate endogenous plant-secreted peptides, which act as immunity inducers and are promising breeding targets for enhancing crop resistance to pathogens. However, the identification and mechanisms of immunogenic peptides in staple crops remain largely unexplored. Here, we demonstrated that plant elicitor peptides (TaPeps) in wheat (Triticum aestivum), processed by a metacaspase, are competent to trigger plant immunity and contribute to resistance against Fusarium head blight (FHB). Using exogenous phytocytokine peptide screens, we identified three potential TaPeps acting as elicitors that significantly improve FHB resistance. Mechanistically, these elicitors activate innate immune signals and calcium dynamics in response to the Fusarium pathogen via wheat PEP RECEPTOR 1 (TaPEPR1). Overexpression of endogenous PRECURSOR OF PEPs (TaPROPEPs) further reduces FHB severity. Moreover, we characterized the natural form of TaPeps in planta, revealing that the wheat type-II metacaspase TaMCA-IIa cleaves TaPROPEPs at a conserved arginine residue, promoting TaPep maturation and immune activation. In Tamca-IIa mutants, the efficiency of TaPep maturation was decreased and calcium dynamics were impaired, resulting in FHB susceptibility. Conversely, overexpressing TaMCA-IIa in wheat enhanced the immune response and FHB resistance without causing pleiotropic growth penalties. Our findings highlight TaPeps as potential immune-inducing biologicals for crop protection and uncover the metacaspase–Peps–receptor module in mediating plant disease resistance.

The rising global burden of cancer underscores the urgent demand for minimally invasive precision diagnostic methods. Extracellular vesicles (EVs) are emerging cancer liquid biopsy biomarkers carrying promising molecular markers such as membrane proteins. However, conventional approaches to EV membrane protein profiling remain limited by low multiplexing capability, high sample consumption, and complex operational workflows. Herein, we report a DNA encoded multi-round profiling of extracellular vesicle membrane proteins for cancer diagnostics (DETECT) strategy that enables detection of EV membrane proteins with high sensitivity and scalability. This method leverages engineered aptamer probes to facilitate the capture and multi-round in situ detection of 9 EV surface proteins. DETECT integrates aptamer recognition with hybridization chain reaction (HCR) for signal amplification, followed by enzymatic cleavage for complete signal erasure, thereby enabling cyclic detection of multiple protein targets on the same EVs population. Clinical validation with EVs isolated from 48 serum samples of three cancer (gastric, breast, and prostate) demonstrated DETECT's capability to uncover cancer-specific membrane protein fingerprints, which achieved 100 % accuracy in differentiating cancers from noncancers and 83.3 % classification accuracy in differentiating three cancer types. DETECT represents a feasible, robust, and scalable technical platform for profiling EV surface proteins that shall hold great application potential in cancer diagnostics and beyond.

MicroRNAs (miRNAs) play important regulatory roles in biology. Direct sequencing of miRNAs in full-length can reveal comprehensive information on their sequences, abundance, and modifications, which, however, has yet to be achieved due to their extremely short length (∼22 nt). Herein, we developed Direct-miR-seq, a nanopore-based direct RNA sequencing (DRS) method that elongates miRNAs at both the 5' and 3' ends by ligating with custom nucleic acid adaptors to ensure full-length sequencing of miRNAs with high yield and accuracy. Compared to standard DRS, Direct-miR-seq enabled sequencing of the whole sequence of miRNAs, achieved a 26-fold sequencing yield, and exhibited reduced bias across miRNA species along with low sequencing error rates. We applied Direct-miR-seq to native RNA populations from cells and human serum to demonstrate its capability to selectively capture miRNAs of known sequences in complex RNA environments for revealing quantitative information in abundance and m6A modification at single-molecule and single-base resolution of ∼100 miRNA species in a single sequencing event. We envision that Direct-miR-seq may be translated toward a variety of biological and medical applications by sequencing miRNAs and other small RNAs.

Diagnosis of influenza A viral infection is crucial for preventing disease transmission and providing effective clinical treatments. There is an increasing need for convenient detection methods to enable simple yet precise identification of viral infections. Herein, a nucleic acid probe-enabled lateral flow assay (NALFA) is developed to realize visual detection and identification of influenza A viral infections (H1N1 and H3N2) of high sensitivity and specificity. Viral RNA targets are recognized by a padlock probe, which is circularized to induce rolling circle amplification (RCA). RCA products are enzymatically cleaved into short amplicons to complex with capture DNA probes for gold colloidal-induced visual lateral flow assay. NALFA achieved attomolar (aM) sensitivity for both standard viral RNAs, along with high specificity. While applying clinical samples (16 H1N1 patients, 12 healthy controls), NALFA exhibited high detection accuracy to successfully discriminate infected samples from noninfected samples. NALFA represents a potent and convenient nucleic acid detection assay that shall find its applications in fields of viral detection and beyond.

Cell membrane vesicles (CMVs) have been extensively used as delivery vehicles for a variety of cargos, which are generally prepared via membrane extrusion. Extruded CMVs are not necessarily to have the outer membrane facing outward due to the randomness of membrane wrapping. Nanoparticles have been used to serve as cores to direct the membrane orientation of CMVs; nevertheless, there is a lack of methods to efficiently sort coreless CMVs of desired orientations. Herein, we utilized a group of functional DNA probes to reveal the random distribution of membrane orientations of coreless CMVs after extrusion, producing either right-side-out vesicles (RSVs) or inside-out vesicles (ISVs). More importantly, DNA probes that protrude out from the outer membrane can serve as handles for efficiently sorting out RSVs from ISVs to produce vesicles with a dominant right-side-out orientation. We investigated three methods to enrich RSVs, including strand displacement reaction, photo cleavage (PC), and enzymatic cleavage. Among them, PC exhibits the highest enrichment efficiency (∼93%) and RSVs purity (∼85.4%), which therefore is recommended for future applications. This work revealed the mixed orientations of coreless CMVs and provided a technical platform to efficiently enrich CMVs of wanted membrane orientations that shall be useful toward a vast array of biomedical applications.

RNA-based fluorescent light-up aptamers (FLAPs) have been progressively developed as imaging probes because of their high signal-to-noise ratio. However, it remains a challenge to use these light-up aptamers due to their poor folding and stability. Leveraging DNA nanotechnology, we investigated whether a DNA origami template could improve folding and further enhance the functionality of FLAPs, namely, the corresponding fluorescence intensities. We utilized aptamer Broccoli and its cognate fluorogen DFHBI-1T as a model. When singular aptamer Broccoli was scaffolded on DNA origami, DNA brick-based nanostructures, DNA double helices, and even on structures as simple as a DNA hairpin stem, our results showed that the fluorescence intensities could be significantly enhanced. These findings show a positive correlation between the fluorogen activity of light-up aptamers and the DNA stem length, potentially mediated by the improved structural stability of the DNA stem, as determined by their simulated thermodynamic properties. Our studies provide a new method to design and enhance the fluorescence behavior of FLAPs, especially structures with a G-quadruplex-based fluorogen recognition region.

Plant proteins that belong to the nonexpressor of pathogenesis-related (NPR) gene family are paralogous receptors of the plant defense hormone salicylic acid and essential regulators of hormone-dependent plant immunity against diseases caused by various pathogens. Previous studies have established NPR1 and NPR3 as a transcriptional activator and a transcriptional repressor, respectively, of defense-gene expression to promote and inhibit broad-spectrum resistance against different strains of pathogens. However, the regulatory mechanism that underlies the opposing roles of NPR1 and NPR3 in defense-gene activation remains unclear. Here, we report that a rice transcript splicing factor, Oryza sativa RNA-binding protein 11 (OsRBP11), promotes alternative splicing of OsNPR3 to modulate the defense function of OsNPR1 in rice plants infected by Xanthomonas oryzae pathovars, which are important bacterial pathogens of rice. We discovered that 11 transcription activator-like effectors identified in representative bacterial strains activate OsRBP11 expression. The OsRBP11 protein, in turn, facilitates alternative splicing of the OsNPR3 mRNA precursor, leading to the production of truncated OsNPR3 protein variants. The OsNPR3 variants exacerbate bacterial diseases by sequestering OsNPR1 from defense-gene activation. By contrast, both artificial and natural variations in OsRBP11 prevent the alternative splicing of OsNPR3, restore the defense function of OsNPR1, and enhance rice resistance to different bacterial strains. These findings not only reveal a novel regulatory pathway exploited by bacterial pathogens to facilitate their pathogenicity and subvert plant defense but also provide a genetic basis for biotechnological strategies aimed at developing broad-spectrum resistance in crops.

Although both protein arginine methylation (PRMT) and jasmonate (JA) signaling are crucial for regulating plant development, the relationship between these processes in the control of spikelet development remains unclear. In this study, we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-offunction mutants that exhibit various abnormal spikelet structures. Interestingly, we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7. We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs, thereby promoting the ubiquitination of OsJAZ1 by the SCFOsCOI1a/OsCOI1b complex and degradation via the 26S proteasome. This process ultimately releases OsMYC2, a core transcriptional regulator in the JA signaling pathway, to activate or repress JA-responsive genes, thereby maintaining normal plant (spikelet) development. However, in the osprmt6a-1 mutant, reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/ OsCOI1b in the presence of JAs. As a result, OsJAZ1 proteins become more stable, repressing JA responses, thus causing the formation of abnormal spikelet structures. Moreover, we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner, thereby establishing a negative feedback loop to balance JA signaling. We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures. Collectively, our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

Transgenes are often spontaneously silenced, which hinders the application of genetic modifications to crop breeding. While gene silencing has been extensively studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanism of transgene silencing remains elusive in crop plants. We used rice (Oryza sativa) plants silenced for a 35S::OsGA2ox1 (Gibberellin 2-oxidase 1) transgene to isolate five elements mountain (fem) mutants showing restoration of transgene expression. In this study, we isolated multiple fem2 mutants defective in a homolog of Required to Maintain Repression 1 (RMR1) of maize (Zea mays) and CLASSY (CLSY) of Arabidopsis. In addition to failing to maintain transgene silencing, as occurs in fem3, in which mutation occurs in NUCLEAR RNA POLYMERASE E1 (OsNRPE1), the fem2 mutant failed to establish transgene silencing of 35S::OsGA2ox1. Mutation in FEM2 eliminated all RNA POLYMERASE IV (Pol-IV)-FEM1/OsRDR2 (RNA-DEPENDENT RNA POLYMERASE 2)-dependent small interfering RNAs (siRNAs), reduced DNA methylation on genome-wide scale in rice seedlings, caused pleiotropic developmental defects, and increased disease resistance. Simultaneous mutation in 2 FEM2 homologous genes, FEM2-Like 1 (FEL1) and FEL2, however, did not affect DNA methylation and rice development and disease resistance. The predominant expression of FEM2 over FEL1 and FEL2 in various tissues was likely caused by epigenetic states. Overexpression of FEL1 but not FEL2 partially rescued hypomethylation of fem2, indicating that FEL1 maintains the cryptic function. In summary, FEM2 is essential for establishing and maintaining gene silencing; moreover, FEM2 is solely required for Pol IV-FEM1 siRNA biosynthesis and de novo DNA methylation.

Cell membranes are widely recognized for their ability to effectively block the entry of harmful substances and impede the absorption of useful substances, such as drugs, by cells. Biomolecular drugs suffer from reduced stability and are vulnerable to degradation by various biological enzymes, thereby reducing their capacity to effectively target their intended site of action. This chapter investigates the impact of DNA nanostructure geometry as a drug delivery platform on cytokinesis. It discusses various design methods of DNA nanostructures, such as DNA origami, single-stranded DNA tiles, and dynamic DNA structures. The chapter provides an overview of the types and mechanisms of endocytosis, including phagocytosis, macropinocytosis, and protein-mediated internalization pathways. It summarizes the mechanisms of how different morphologies of DNA nanostructures enter the cell and their resulting cellular effects. The utilization of dynamic DNA structures has significantly increased in the biomedical field, as well as in fundamental research, due to their various applications.

Biomarkers are crucial physiological and pathological indicators in the host. Over the years, numerous detection methods have been developed for biomarkers, given their significant potential in various biological and biomedical applications. Among these, the detection system based on functionalized DNA origami has emerged as a promising approach due to its precise control over sensing modules, enabling sensitive, specific, and programmable biomarker detection. We summarize the advancements in biomarker detection using functionalized DNA origami, focusing on strategies for DNA origami functionalization, mechanisms of biomarker recognition, and applications in disease diagnosis and monitoring. These applications are organized into sections based on the type of biomarkers - nucleic acids, proteins, small molecules, and ions - and concludes with a discussion on the advantages and challenges associated with using functionalized DNA origami systems for biomarker detection.

Tumor-derived small extracellular vesicle (sEV) microRNAs (miRNAs) are emerging biomarkers for cancer diagnostics. Conventional sEV miRNA detection methods necessitate the lysis of sEVs, rendering them laborious and time-consuming and potentially leading to damage or loss of miRNAs. Membrane fusion-based in situ detection of sEV miRNAs involves the preparation of probe-loaded vesicles (e.g., liposomes or cellular vesicles), which are typically sophisticated and require specialist equipment. Membrane perforation methods employ chemical treatments that can induce severe miRNA degradation or leaks. Inspired by previous studies that loaded nucleic acids into EVs or cells using hydrophobic tethers for therapeutic applications, herein, we repurposed this strategy by conjugating a hydrophobic tether onto molecular beacons to aid their transportation into sEVs, allowing for in situ detection of miRNAs in a fusion-free and multiplexing manner. This method enables simultaneous detection of multiple miRNA species within serum-derived sEVs for the diagnosis of prostate cancer, breast cancer, and gastric cancer with an accuracy of 83.3%, 81.8%, and 100%, respectively, in a cohort of 66 individuals, indicating that it holds a high application potential in clinical diagnostics.

How to realize fM-level detection of viral DNA without target amplification is a challenge. Traditionally, preamplification of low-abundant DNA targets is a prerequisite, which not only increases the risk of infectious material leakage, enlarges the chance of false positive detection, but also prolongs the overall detection time. Herein, we developed a combinatory CRISPR-Cas12a detection system from three aspects to enhance its limit of detection (LOD) to the femtomolar level which is three orders of magnitude lower than a conventional protocol. Specifically, the detection sensitivity of CRISPR-Cas12a system was enhanced by utilizing multiplex crRNAs for simultaneous recognition of multiple distinct sites of the same viral DNA target, by employing a novel molecular reporter with G-triplex structure that exhibits a significantly enhanced cleaving tendency by Cas12a, by exploring the optimal molecular coexistence reaction environment for maintaining enzyme activity which was the key for sensing. It is worth noting that we have for the first time discovered an environment where sensitive G-triplex reporter forms at a low K+ concentration without damaging CRISPR-Cas12a activity. Using this system, we demonstrated ultrasensitive detection of plasmids containing monkeypox (Mpox) viral DNA sequences in a mimicking physiological scenario. More importantly, we realized sensitive and specific detection and classification of human papillomavirus (HPV) subtypes from clinical samples. Moreover, this design successfully circumvented intricate procedures and the signal readout was not contingent upon the use of unconventional equipment, suggesting its great promise of expanding into a portable, field-deployable test kit for rapid, sensitive, and specific detection of various pathogens.