Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a major disease of banana plants worldwide. Effector proteins play critical roles in banana-Foc TR4 interaction. Our previous studies highlighted a ribonuclease protein belonging to the T2 family (named as FocRnt2) in the Foc TR4 secretome, which was predicted to be an effector. However, its biological function in Foc TR4 infection is still unclear. Herein, we observed significant expression of FocRnt2 during the early stage of fungal infection in planta. A yeast signal sequence trap assay showed that FocRnt2 contained a functional signal peptide for secretion. FocRnt2 possessed ribonuclease activity that could degrade the banana total RNA in vitro. Subcellular localization showed that FocRnt2 was localized in the nucleus and cytoplasm of Nicotiana benthamiana leaves. Transient expression of FocRnt2 suppressed the expression of salicylic acid- and jasmonic acid-signalling marker genes, reactive oxygen species accumulation, and BAX-mediated cell death in N. benthamiana. FocRnt2 deletion limited fungal penetration, reduced fusaric acid biosynthesis in Foc TR4, and attenuated fungal virulence against banana plants, but had little effect on Foc TR4 growth and sensitivity to various stresses. Furthermore, FocRnt2 deletion mutants induced higher expression of the defence-related genes in banana plants. These results suggest that FocRnt2 plays an important role in full virulence of Foc TR4, further improving our understanding of effector-mediated Foc TR4 pathogenesis.

Antimicrobial proteins contribute to host-microbiota interactions and are associated with inflammatory bowel disease (IBD), but our understanding on antimicrobial protein diversity and functions remains incomplete. Ribonuclease 4 (Rnase4) is a potential antimicrobial protein with no known function in the intestines. Here we find that RNASE4 is expressed in intestinal epithelial cells (IEC) including Paneth and goblet cells, and is detectable in human and mouse stool. Results from Rnase4-deficient mice and recombinant protein suggest that Rnase4 kills Parasutterella to modulate intestinal microbiome, thereby enhancing indoleamine-2,3-dioxygenase 1 (IDO1) expression and subsequently kynurenic and xanthurenic acid production in IECs to reduce colitis susceptibility. Furthermore, deceased RNASE4 levels are observed in the intestinal tissues and stool from patients with IBD, correlating with increased stool Parasutterella. Our results thus implicate Rnase4 as an intestinal antimicrobial protein regulating gut microbiota and metabolite homeostasis, and as a potential diagnostic biomarker and therapeutic target for IBD.

In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.

Phytopathogens secrete numerous molecules into the environment to establish a microbial niche and facilitate host infection. The phytopathogenic fungus Colletotrichum fructicola, which causes pear anthracnose, can colonize different plant tissues like leaves and fruits, which are occupied by a diversity of microbes. We speculate that this fungus produces antimicrobial effectors to outcompete host-associated competitive microorganisms. Herein, we identified two secreted ribonucleases, CfRibo1 and CfRibo2, from the C. fructicola secretome. The two ribonucleases both possess ribonuclease activity and showed cytotoxicity in Nicotianan benthamiana without triggering immunity in an enzymatic activity-dependent manner. CfRibo1 and CfRibo2 recombinant proteins exhibited toxicity against Escherichia coli, Saccharomyces cerevisiae, and, importantly, the phyllosphere microorganisms isolated from the pear host. Among these isolated microbial strains, Bacillus altitudinis is a pathogenic bacterium causing pear soft rot. Strikingly, CfRibo1 and CfRibo2 were found to directly antagonize B. altitudinis to facilitate C. fructicola infection. More importantly, CfRibo1 and CfRibo2 functioned as essential virulence factors of C. fructicola in the presence of host-associated microorganisms. Further analysis revealed these two ribonucleases are widely distributed in fungi and are undergoing purifying selection. Our results provide the first evidence of antimicrobial effectors in Colletotrichum fungi and extend the functional diversity of fungal ribonucleases in plant-pest-environment interactions.
OBJECTIVE: Colletotrichum fructicola is emerging as a devastating pathogenic fungus causing anthracnose in various crops in agriculture, and understanding how this fungus establishes successful infection is of great significance for anthracnose disease management. Fungi are known to produce secreted effectors as weapons to promote virulence. Considerable progress has been made in elucidating how effectors manipulate plant immunity; however, their importance in modulating environmental microbes is frequently neglected. The present study identified two secreted ribonucleases, CfRibo1 and CfRibo2, as antimicrobial effectors of C. fructicola. These two proteins both possess toxicity to pear phyllosphere microorganisms, and they efficiently antagonize competitive microbes to facilitate the infection of pear hosts. This study represents the first evidence of antimicrobial effectors in Colletotrichum fungi, and we consider that CfRibo1 and CfRibo2 could be targeted for anthracnose disease management in diverse crops in the future.

As self-incompatibility is a major issue in pummelo breeding and production, its mechanism in citrus was analyzed to improve breeding efficiency and reduce production costs. Rutaceae belongs to S-RNase type of gametophytic self-incompatibility. While the function of S-RNase/SLF and the mechanism of self-incompatibility have been studied extensively, the transcriptional regulation of S-RNase has been less studied. We performed transcriptome sequencing with the styles of \'Shatian\' pummelo on the day of anthesis and 1-5 days before anthesis, and found that the transcript level of S-RNase gradually decreased with flower development. By analyzing differentially expressed genes and correlation with the expression trend of S-RNase, we identified a candidate gene, CgHSFB1, and utilized biochemical experiments such as yeast one-hybrid assay, electrophoretic mobility shift assay and dual-luciferase assay, as well as transient transformation of citrus calli and Citrus microcarpa and demonstrated that CgHSFB1 could directly bind to the S1-RNase promoter and repress the expression of S1-RNase, which is involved in the pummelo self-incompatibility response. In contrast, CgHSFB1 did not bind to the promoter of S2-RNase, and there was specificity in the regulation of S-RNase.

Nucleic acids are major structures detected by the innate immune system. Although intracellular single-stranded DNA (ssDNA) accumulates during pathogen infection or disease, it remains unclear whether and how intracellular ssDNA stimulates the innate immune system. Here, we report that intracellular ssDNA triggers cytokine expression and cell death in a CGT motif-dependent manner. We identified Schlafen 11 (SLFN11) as an ssDNA-activated RNase, which is essential for the innate immune responses induced by intracellular ssDNA and adeno-associated virus infection. We found that SLFN11 directly binds ssDNA containing CGT motifs through its carboxyl-terminal domain, translocates to the cytoplasm upon ssDNA recognition, and triggers innate immune responses through its amino-terminal ribonuclease activity that cleaves transfer RNA (tRNA). Mice deficient in Slfn9, a mouse homolog of SLFN11, exhibited resistance to CGT ssDNA-induced inflammation, acute hepatitis, and septic shock. This study identifies CGT ssDNA and SLFN11/9 as a class of immunostimulatory nucleic acids and pattern recognition receptors, respectively, and conceptually couples DNA immune sensing to controlled RNase activation and tRNA cleavage.

Ribonuclease targeting chimera (RIBOTAC) represents an emerging strategy for targeted therapy. However, RIBOTAC that is selectively activated by bio-orthogonal or cell-specific triggers has not been explored. We developed a strategy of inducible RIBOTAC (iRIBOTAC) that enables on-demand degradation of G-quadruplex (G4) RNAs for precision cancer therapy. iRIBOTAC is designed by coupling an RNA G4 binder with a caged ribonuclease recruiter, which can be decaged by a bio-orthogonal reaction, tumor-specific enzyme, or metabolite. A bivalent G4 binder is engineered by conjugating a near-infrared (NIR) fluorescence G4 ligand to a noncompetitive G4 ligand, conferring fluorescence activation on binding G4s with synergistically enhanced affinity. iRIBOTAC is demonstrated to greatly knockdown G4 RNAs upon activation under bio-orthogonal or cell-specific stimulus, with dysregulation of gene expressions involving cell killing, channel regulator activity, and metabolism as revealed by RNA sequencing. This strategy also shows a crucial effect on cell fate with remarkable biochemical hallmarks of apoptosis. Mice model studies demonstrate that iRIBOTAC allows selective imaging and growth suppression of tumors with bio-orthogonal and tumor-specific controls, highlighting G4 RNA targeting and inducible silencing as a valuable RIBOTAC paradigm for cancer therapy.

Rapid and visual detection of SARS-CoV-2 variants is vital for timely assessment of variant transmission in resource-limited settings. Here, a closed-tube, two-stage, mixed-dye-based isothermal amplification method with ribonuclease-cleavable enhanced probes (REP), termed REP-TMAP, for dual-visualization detection of SARS-CoV-2 variants including JN.1, BA.2, BA.4/5, and Delta is introduced. The first stage of REP-TMAP is reverse transcription recombinase polymerase amplification and the second stage is dual-visualization detection synergistically mediated by the REP and the mixed dyes of cresol red and hydroxy naphthol blue. In REP-TMAP reaction, the color change under ambient light indicates SARS-CoV-2 infection, while the fluorescence change under blue light excitation specifies variant type. On detecting transcribed RNA of SARS-CoV-2 spike gene, this assay is rapid (within 40 min), highly sensitive (10-200 copies per reaction), and highly specific (identification of single-base mutations). Furthermore, the assay has been clinically validated to accurately detect JN.1, BA.2, and BA.4/5 variants from 102 human oropharyngeal swabs. The proposed assay therefore holds great potentials to provide a rapid, dual-visualization, sensitive, specific, point-of-care detection of SARS-CoV-2 variants and beyond.

RNA turnover plays critical roles in the regulation of gene expression and allows cells to respond rapidly to environmental changes. In bacteria, the mechanisms of RNA turnover have been extensively studied in the models Escherichia coli and Bacillus subtilis, but not much is known in other bacteria. Cyanobacteria are a diverse group of photosynthetic organisms that have great potential for the sustainable production of valuable products using CO2 and solar energy. A better understanding of the regulation of RNA decay is important for both basic and applied studies of cyanobacteria. Genomic analysis shows that cyanobacteria have more than 10 ribonucleases and related proteins in common with E. coli and B. subtilis, and only a limited number of them have been experimentally investigated. In this review, we summarize the current knowledge about these RNA-turnover-related proteins in cyanobacteria. Although many of them are biochemically similar to their counterparts in E. coli and B. subtilis, they appear to have distinct cellular functions, suggesting a different mechanism of RNA turnover regulation in cyanobacteria. The identification of new players involved in the regulation of RNA turnover and the elucidation of their biological functions are among the future challenges in this field.

In response to the urgent need for potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics, this study introduces an innovative nucleoside tailoring strategy leveraging ribonuclease targeting chimeras. By seamlessly integrating ribonuclease L recruiters into nucleosides, we address RNA recognition challenges and effectively inhibit severe acute respiratory syndrome coronavirus 2 replication in human cells. Notably, nucleosides tailored at the ribose 2\'-position outperform those modified at the nucleobase. Our in vivo validation using hamster models further bolsters the promise of this nucleoside tailoring approach, positioning it as a valuable asset in the development of innovative antiviral drugs.