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.

Intracellular pathogens including Staphylococcus aureus contribute to the non-healing phenotype of chronic wounds. Lactobacilli, well known as beneficial bacteria, are also reported to modulate the immune system, yet their role in cutaneous immunity remains largely unknown. We explored the therapeutic potential of bacteria-free postbiotics, bioactive lysates of lactobacilli, to reduce intracellular S. aureus colonization and promote healing. Fourteen postbiotics derived from various lactobacilli species were screened, and Latilactobacillus curvatus BGMK2-41 was selected for further analysis based on the most efficient ability to reduce intracellular infection by S. aureus diabetic foot ulcer clinical isolate and S. aureus USA300. Treatment of both infected keratinocytes in vitro and infected human skin ex vivo with BGMK2-41 postbiotic cleared S. aureus. Keratinocytes treated in vitro with BGMK2-41 upregulated expression of antimicrobial response genes, of which DEFB4, ANG, and RNASE7 were also found upregulated in treated ex vivo human skin together with CAMP exclusively upregulated ex vivo. Furthermore, BGMK2-41 postbiotic treatment has a multifaceted impact on the wound healing process. Treatment of keratinocytes stimulated cell migration and the expression of tight junction proteins, while in ex vivo human skin BGMK2-41 increased expression of anti-inflammatory cytokine IL-10, promoted re-epithelialization, and restored the epidermal barrier via upregulation of tight junction proteins. Together, this provides a potential therapeutic approach for persistent intracellular S. aureus infections.

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.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects the motoneurons. More than 40 genes are related with ALS, and amyloidogenic proteins like SOD1 and/or TDP-43 mutants are directly involved in the onset of ALS through the formation of polymorphic fibrillogenic aggregates. However, efficacious therapeutic approaches are still lacking. Notably, heterozygous missense mutations affecting the gene coding for RNase 5, an enzyme also called angiogenin (ANG), were found to favor ALS onset. This is also true for the less-studied but angiogenic RNase 4. This review reports the substrate targets and illustrates the neuroprotective role of native ANG in the neo-vascularization of motoneurons. Then, it discusses the molecular determinants of many pathogenic ANG mutants, which almost always cause loss of function related to ALS, resulting in failures in angiogenesis and motoneuron protection. In addition, ANG mutations are sometimes combined with variants of other factors, thereby potentiating ALS effects. However, the activity of the native ANG enzyme should be finely balanced, and not excessive, to avoid possible harmful effects. Considering the interplay of these angiogenic RNases in many cellular processes, this review aims to stimulate further investigations to better elucidate the consequences of mutations in ANG and/or RNase 4 genes, in order to achieve early diagnosis and, possibly, successful therapies against ALS.

The unfolded protein response can switch from a pro-survival to a maladaptive, pro-apoptotic mode. During ER stress, IRE1α sensors dimerize, become phosphorylated, and activate XBP1 splicing, increasing folding capacity in the ER protein factory. The steps that turn on the IRE1α endonuclease activity against endogenous mRNAs during maladaptive ER stress are still unknown. Here, we show that although necessary, IRE1α dimerization is not sufficient to trigger phosphorylation. Random and/or guided collisions among IRE1α dimers are needed to elicit cross-phosphorylation and endonuclease activities. Thus, reaching a critical concentration of IRE1α dimers in the ER membrane is a key event. Formation of stable IRE1α clusters is not necessary for RNase activity. However, clustering could modulate the potency of the response, promoting interactions between dimers and decreasing the accessibility of phosphorylated IRE1α to phosphatases. The stepwise activation of IRE1α molecules and their low concentration at the steady state prevent excessive responses, unleashing full-blown IRE1 activity only upon intense stress conditions.

How do biological networks evolve and expand? We study these questions in the context of the plant collaborative-non-self recognition self-incompatibility system. Self-incompatibility evolved to avoid self-fertilization among hermaphroditic plants. It relies on specific molecular recognition between highly diverse proteins of two families: female and male determinants, such that the combination of genes an individual possesses determines its mating partners. Though highly polymorphic, previous models struggled to pinpoint the evolutionary trajectories by which new specificities evolved. Here, we construct a novel theoretical framework, that crucially affords interaction promiscuity and multiple distinct partners per protein, as is seen in empirical findings disregarded by previous models. We demonstrate spontaneous self-organization of the population into distinct \"classes\" with full between-class compatibility and a dynamic long-term balance between class emergence and decay. Our work highlights the importance of molecular recognition promiscuity to network evolvability. Promiscuity was found in additional systems suggesting that our framework could be more broadly applicable.

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, whereas both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Furthermore, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

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.