Significance Plasmodesmata connect the cytoplasm of neighboring plant cells across cell walls. In response to various signals, they open and close to connect and isolate cells. We have found that the plasmodesmal plasma membrane hosts a unique immune-signaling cascade—different from that in the surrounding plasma membrane—which leads to plasmodesmata closure and cell isolation upon chitin perception. This response is mediated by a specific receptor complex, which in turn activates an NADPH oxidase via a specific regulatory module. This work characterizes how a cell can produce a localized and specific response in a discrete membrane domain, identifying that there is microdomain specificity in immune signaling to a single elicitor and that cell-to-cell connections are independently controlled.

Significance It is highly controversial whether secondary reactive oxygen species generated by dysfunctional mitochondria are able to diffuse across the cytoplasm to the nucleus and cause subsequent nuclear changes. We have developed a targeted chemoptogenetic technology to induce mitochondrial dysfunction by generating short-lived highly reactive singlet oxygen exclusively in the mitochondria, with precise spatiotemporal control by light stimulation. Through careful analysis of the events involving mitochondrial dysfunction and subsequent nuclear oxidative stress that resulted in specific telomere damage, we delineated the mechanism of mitochondria–telomere axis of cellular damage. Our findings revealed a fundamental mechanism underlying the pathophysiological role of mitochondrial singlet oxygen, with important ramifications for understanding the role of mitochondrial signaling in aging and cancer among other human diseases.

Significance We report a long-sought& in vivo& physiological role for& α-synuclein (α-syn) in dopamine signaling. The results indicate that& α-syn is critical for activity-dependent dopamine plasticity, and that short repeated burst activity produces rapid presynaptic facilitation, while prolonged burst activity slowly depresses evoked dopamine release. We propose that the rapid facilitation is due to an enhanced fusion of synaptic vesicles at active zones during exocytosis while the depression is due to synaptic exhaustion. These results identify a& dynamic role of& α-syn, and are critical for defining& molecular mechanisms and therapeutic targets for various neurological disorders, where the firing properties of neurons are severely altered.

This work elucidates the mechanisms of horizontal genome transfer and uncovers a new pathway of intercellular transport.

ObjectiveNon-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which incorporates arachidonic acid into phosphatidylinositol (PI), is associated with the entire spectrum of NAFLD. In this study, we investigated the mechanism underlying this association in mice and cultured human hepatocytes.DesignWe generated the hepatocyte-specific Lpiat1 knockout mice to investigate the function of Lpiat1 in vivo. We also depleted LPIAT1 in cultured human hepatic cells using CRISPR-Cas9 systems or siRNA. The effect of LPIAT1-depletion on liver fibrosis was examined in mice fed high fat diet and in liver spheroids. Lipid species were measured using liquid chromatography-electrospray ionisation mass spectrometry. Lipid metabolism was analysed using radiolabeled glycerol or fatty acids.ResultsThe hepatocyte-specific Lpiat1 knockout mice developed hepatic steatosis spontaneously, and hepatic fibrosis on high fat diet feeding. Depletion of LPIAT1 in cultured hepatic cells and in spheroids caused triglyceride accumulation and collagen deposition. The increase in hepatocyte fat content was due to a higher triglyceride synthesis fueled by a non-canonical pathway. Indeed, reduction in the PI acyl chain remodelling caused a high PI turnover, by stimulating at the same time PI synthesis and breakdown. The degradation of PI was mediated by a phospholipase C, which produces diacylglycerol, a precursor of triglyceride.ConclusionWe found a novel pathway fueling triglyceride synthesis in hepatocytes, by a direct metabolic flow of PI into triglycerides. Our findings provide an insight into the pathogenesis and therapeutics of NAFLD.

Autism spectrum disorder (ASD) is a developmental disorder characterized by social behavior deficit in childhood without satisfactory medical intervention. Transcutaneous electrical acupoint stimulation (TEAS) is a noninvasive technique derived from acupuncture and has been shown to have similar therapeutic effects in many diseases. Valproic acid- (VPA-) induced ASD is a known model of ASD in rats. The therapeutic efficacy of TEAS was evaluated in the VPA model of ASD in the present study. The offspring of a VPA-treated rat received TEAS in the early life stage followed by a series of examinations conducted in their adolescence. The results show that following TEAS treatment in early life, the social and cognitive ability in adolescence of the offspring of a VPA rat were significantly improved. In addition, the abnormal pain threshold was significantly corrected. Additional studies demonstrated that the dendritic spine density of the primary sensory cortex was decreased with Golgi staining. Results of the transcriptomic study showed that expression of some transcription factors such as the neurotrophic factor were downregulated in the hypothalamus of the VPA model of ASD. The reduced gene expression was reversed following TEAS. These results suggest that TEAS in the early life stage may mitigate disorders of social and recognition ability and normalize the pain threshold of the ASD rat model. The mechanism involved may be related to improvement of synaptic plasticity.

Astragalus membranaceus root has been widely used for repigmentation treatment in vitiligo, but its mechanism is poorly understood. We sought to investigate the effect of astragaloside IV (AS-IV), a main active extract of the Astragalus membranaceus root, on melanin synthesis in normal human epidermal melanocytes (NHEMs) and to elucidate its underlying mechanisms. Melanin content, tyrosinase activity, qPCR, western blot, and immunofluorescence were employed. Specific inhibitors and small interfering RNA were used to investigate the possible pathway. AS-IV stimulated melanin synthesis and upregulated the expression of melanogenesis-related genes in a concentration-dependent manner in NHEMs. AS-IV could activate the aryl hydrocarbon receptor (AhR), and AS-IV-induced melanogenesis was inhibited in si-AhR-transfected NHEMs. In addition, we showed that AS-IV enhanced the phosphorylation of AKT and GSK-3β and nuclear translocation of β-catenin. AS-IV-induced MITF expression upregulation and melanin synthesis were decreased in the presence of β-catenin inhibitor FH353. Furthermore, AhR antagonist CH223191 inhibited the activation of AKT/GSK-3β/β-catenin signaling, whereas the expression of CYP1A1 (marker of AhR activation) was not affected by the AKT inhibitor in AS-IV-exposed NHEMs. Our findings show that AS-IV induces melanogenesis through AhR-dependent AKT/GSK-3β/β-catenin pathway activation and could be beneficial in the therapy for depigmented skin disorders.

Spontaneous intracerebral hemorrhage (ICH) commonly causes secondary hippocampal damage and delayed cognitive impairments, but the mechanisms remain elusive. Here, we sought to identify the molecular mechanisms underlying these hemorrhagic outcomes in a rat autologous blood model of ICH. First, a significant increase in phosphatase and tensin homolog (PTEN) expression was observed in nonhemorrhagic ipsilateral hippocampus. However, systemic administration of PTEN inhibitor BPV or hippocampal injection of PTEN siRNA could prevent hippocampal neuronal injury and cognitive dysfunctions after ICH. Furthermore, we also found that ICH robustly triggered autophagic neuronal death in the ipsilateral hippocampus, but which were strongly reduced by PTEN knockdown. Notably, suppression of autophagy effectively attenuated poststroke hippocampal inflammation, neuronal damage, and cognitive decline, suggesting the beneficial effects of PTEN deletion was associated with autophagy inactivation. Specifically, PTEN antagonized the PI3K/AKT signaling and downstream effector FoxO3a phosphorylation and subsequently enhanced nuclear translocation of FoxO3a to drive proautophagy gene program, but these changes were diminished upon PTEN inhibition. More importantly, lentivirus-mediated FoxO3a overexpression apparently abrogated the antiauotphagy effect of PTEN deletion via enhancing autophagy-related gene (ATG) transcription. Collectively, these results suggest that knockdown of PTEN alleviated progressive hippocampal injury and cognitive deficits by suppression of autophagy induction involving the AKT/FoxO3a/ATG axis after ICH. Thus, this study provides a novel and promising therapeutic target for the treatment of hemorrhagic stroke.

Abstract M cell targeting is one of the critical issues to develop efficient mucosal vaccine design. In this study, peptide ligands with high affinity to porcine TLR2, which is highly expressed in M cells and play an important role in mucosal immune responses in pigs, were identified through the cell-based phage display technique combined with high-throughput sequencing. A random phage-peptide library was applied to the porcine TLR2 overexpressing cell line and total 85, 557 unique peptide sequences were identified from approximately 9.0 × 107 reads after three rounds of both subtractive and non-subtractive biopanning via high-throughput sequencing. Among the unique sequences, three candidate peptide sequences, NAGHLSQ, VPSKPGL, and RANLDGQ, were selected based on their abundance in the third round of biopanning. Consequently, NAGHLSQ showed the highest affinity exclusively to porcine TLR2 compared with other candidates and its binding mechanism was inferred to be directly associated with ligand binding site of the TLR2 through the in vitro competitive analysis. The peptide identified in this research could be used in development of effective porcine mucosal vaccine as an M cell targeting moiety to enhance the transport of antigens into the Peyer's patch via oral route.

Abstract Background:Epidermal growth factor receptor (EGFR) activation may play an important role in blood spinal cord barrier (BSCB) disruption and secondary injury after SCI as it is significantly upregulated in the astrocytes (AS) and microvascular endothelial cells (MEC), which are the main component of cells in BSCB. EGFR inhibition alleviates the disruption of BSCB and improves the functional recovery in rats following spinal cord injury (SCI). However, the biological mechanisms underlying EGFR activation mediating secondary damage after SCI remain unclear. Methods:An in vitro model of Oxygen and glucose deprivation/reoxygenation (OGD/R) induced BSCB damage and in vivo rat SCI model was employed to define the role of EGFR activation and its induced inflammatory injury during this pathological process in AS and MEC. AS and MEC were exposed to EGFR or p38 MAPK up-regulation in the presence and absence of EGFR inhibitor, p38 MAPK inhibitor, NF-κB inhibitor, and/or appropriate shRNA. RT-PCR, ELISA and western blotting were used for mRNA and protein expression analyses of TNF-α, iNOS, COX-2 and IL-1β. Immunohistochemical staining and confocal microscopy were used to demonstrate cellular EGFR activation and to investigate the expression of tight junction (TJ) protein (ZO-1 and occludin). Measurement of transendothelial electrical resistance (TEER) and transendothelial FITC-dextran permeability were used to measure permeability of BSCB in vitro, while Evans blue dye extravasation test and evaluation spinal cord edema were used to detect permeability of BSCB in vivo.Results:The expression of pEGFR was significantly increased in BSCB component cells (AS and MEC) after SCI and BSCB damage model. EGFR activation induced inflammation injury by upregulating the expression of TNF-α, iNOS, COX-2, and IL-1β and BSCB disruption with loss of TJ protein by downregulating the expression of ZO-1 and occludin in BSCB damage model and SCI rats. Moreover, EGFR or p38 activation leads to NF-κB nuclear translocation in primary AS and MEC after OGD/R. The EGFR inhibitor as well as shRNA against EGFR markedly attenuated pro-inflammatory factor excessive producing and loss of TJ protein, and activation of the EGFR/p38/NF-κB pathway. While, EGFR overexpression significantly increased the expression of TNF-α, iNOS, COX-2, and IL-1β and decrease the expression of ZO-1 and occludin, inducing activation of the EGFR/p38/NF-κB pathway in both AS and MEC. Conclusion:This study strongly suggests that EGFR activation in BSCB component cells after SCI and BSCB damage model mediates both upregulation of pro-inflammation expression and downregulation of TJ protein downregulation via the EGFR/p38/NF-κB pathway. These findings contribute to a better understanding of the biological mechanisms underlying BSCB disruption and secondary injury following SCI mediating by EGFR activation.

Abstract Background Umbilical cord mesenchymal stem cells (HUCMSCs)-based therapies were previously predicated in cartilage regeneration due to the chondrogenic potential of MSCs. However, chondrogenic differentiation of HUMSCs is limited by administration of growth factors like TGF-β that may cause cartilage hypertrophy. It has been reported the exosomes could modulate phenotypic expression of stem cells. However, the role of human chondrogenic derived exosomes (C-EXO) in chondrogenic differentiation of HUCMSCs has not been reported. Results In this study, we successfully isolated chondrocyte-derived exosomes (C-EXO) from human multi-finger cartilage and found that C-EXO efficiently promoted the proliferation and chondrogenic differentiation of HUCMSCs, evidenced by highly expressed aggrecan (ACAN), COL2A and SOX-9. Also, the expression of the fibrotic marker, COL1A and hypertrophic marker, COL10, was significantly lower than that induced by TGF-β. In vivo, stimulation of C-EXO accelerated HUCMSCs-mediated cartilage repair in rabbit models. Furthermore, C-EXO led to increasing autophagosomes during the process of chondrogenic differentiation, indicating that C-EXO promoted cartilage regeneration might be through the activation of autophagy. Conclusions This study suggests that C-EXO has an essential role in fostering chondrogenic differentiation and proliferation of HUCMSCs, which may be a stable supply for articular cartilage repair.

Abstract BackgroundWidely used in recent years, mesenchymal stem cells (MSCs) expressing enhanced green fluorescent protein (eGFP) can be tracked during migration to injury sites, while also supporting tri-lineage differentiation. However, the relationship between the expression of green fluorescence and the magnitude of osteogenic differentiation is not clearly defined. Despite increasing use of eGFP-MSCs derived from the transgenic pigs and non-viral eGFP plasmid transfected MSCs in recent years, it remains unclear which cells are suitable for tracking during osteogenic differentiation, and whether the transfected plasmid alters osteogenic potential.MethodsWe compared the expression of green fluorescence and the magnitude of osteogenic differentiation between eGFP MSCs from a transgenic pig (group 1) and non-virally transfected eGFP-MSCs using transIT®-2020 (group 2). Non-transfected MSCs were used as control group (group 3). We also use a scaffold to compare the osteogenic induction environments created by 2-D monolayer cultures and 3-D cultures, respectivelyResultsIn the monolayer culture, flow cytometry from day 7 to day 28 showed that the percentage of green fluorescent cells in groups 1 and 2 were 99.6% and 59.7% of total cell counts, respectively. Quantification showed that eGFP expression peaked on day 7, decreased after day 14, and plateaued to day 28 in group 1 and group 2. Significant aggregation of eGFP over bone-like nodules was appreciated in group 1. In 3-D culture, eGFP expression increased from day 7 to day 28 in both groups, and was higher in group 1 than in group 2 at each time point. Osteogenic profiles and immunohistochemistry showed more significant osteogenic activity in group 1 and group 3 than in group 2. ConclusionsThe expression of eGFP in the test groups did not significantly change after osteogenic induction. However, quantification data was different in monolayer and 3-D cultures due to spatial limitations, differing extracellular environments, and heterogeneous cell morphology and methods of division. Osteogenic profiles and immunohistochemistry data confirmed that osteogenic potential did not change in transgenic pig-derived MSCs. However osteogenic potential decreased in pig MSCs (pig MSCs) treated with the transfection reagent, likely from related toxicity.

Abstract Background Antimicrobial photodynamic therapy (APDT) is a promising alternative to traditional antibiotics for bacterial infections, which inactivates a broad spectrum of bacteria. However, it has some disadvantages including poor water solubility and easy aggregation of hydrophobic photosensitizers (PS), and poor tissue penetration and cytotoxicity when using UV as the light source, leading to undesired photodynamic therapy efficacy.Results In this study, we develop a novel water-soluble natural PS (sorbicillinoids) obtained by microbial fermentation using recombinant filamentous fungus Trichoderma reesei (T. reesei). Sorbicillinoids could effectively generate singlet oxygen (1O2) under ultraviolet (UV) light irradiation, and ultimately display photoinactivation activity on Gram-positive bacteria, but not Gram-negative ones. Staphylococcus aureus (S. aureus) treated with sorbicillinoids and UV light displayed high levels of intracellular reactive oxygen species (ROS), notable DNA photocleavage, and compromised cell semi-permeability without overt cell membrane disruption. Moreover, the dark toxicity, phototoxicity or hemolysis activity of sorbicillinoids is negligible, showing its excellent biocompatibility.Conclusion Sorbicillinoids obtained from T. reesei display photoinactivation activity on Gram-positive bacteria using nontoxic dose of UV light irradiation and have an excellent biocompatibility Therefore, sorbicillinoids, a type of secondary metabolite from fungus, has a promising future as a new PS for APDT.

Abstract Background: Resisting cell death is one of the hallmarks of cancer. Necroptosis is a form of non-caspase dependent necrotic cell death mediated by receptor-interacting protein kinase-1/3 (RIP1/3), which represents another mode of programmed cell death besides apoptosis. Growing evidence supports that RIP3 has emerged as a critical regulator of necroptosis and can be activated by several stimuli to trigger necroptotic cell death in a RIP1-independent manner. RIP3 also acts as an energy metabolism regulator associated with switching cell death from apoptosis to necroptosis. Natural products provide a unique source for the discovery of innovative leading compounds and drugs, which exhibits promising anticancer activities through inducing cell death and enhancing chemotherapeutic sensitivity. Trichothecin (TCN) is a sesquiterpenoid originating from an endophytic fungus of the herbal plant Maytenus hookeri Loes and shows potent anti-tumor bioactivity. However, the underlying mechanism is not fully understood.Methods: Cell permeability assay and transmission electron microscopy were applied to identify the death pattern induced by TCN in apoptotic-resistant cancer cells. We used Seahorse extracellular flux analyzer to examine the cellular oxygen consumption rate (OCR) and flow cytometry to detect mitochondrial reactive oxygen species (ROS) content. Xenograft animal experiment was performed to assess the effect of TCN synergized with cisplatin to enhance chemotherapeutic sensitivity of tumor cells. Results: Our current findings revealed that RIP3 mediated TCN-induced necroptosis through activating mitochondria energy metabolism and ROS production in apoptotic-resistant cancer cells. RIP3 might be involved in sensitizing tumor cells to chemotherapy induced by TCN. Conclusions: Activating RIP3 to induce necroptosis through reprogramming mitochondrial energy metabolism and ROS production contributes to the anti-tumor activity of TCN. Moreover, TCN could be exploited for therapeutic gain through up-regulating RIP3 to sensitize cancer chemotherapy.

Abstract Despite continuous active development of fluorescent probes for metal-ions, their molecular design for ratiometric detection is limited owing to a narrow choice of available sensing mechanisms. We present herein a dual-emission sensing platform for metal ions based on contact interaction between a coordinated metal ion and the aromatic ring of a fluorophore (i.e., arene–metal-ion contact). Our structure-based ligand design provided a new probe possessing BPTN as the metal ion binding unit, which was flexibly concatenated to a tricyclic fluorophore. This molecular architecture allowed us to fluorescently sense various metal ions such as Zn(II), Cu(II), Cd(II), Ag(I), and Hg(II) with the red-shifted emissions. This probe design was applicable to a series of tricyclic fluorophores, enabling ratiometric detection of the metal ions across the blue to near-infrared wavelength region. X-ray crystallography and theoretical computational calculation indicated that the coordinated metal ion has van der Waals contact with the fluorophore, which perturbs its electronic structure and ring conformation to induce the emission red-shift. A set of the arene–metal-ion contact probes was used for the differential sensing of eight metal ions in a one-pot single titration via PCA analysis. Furthermore, the probe was applicable to the ratio imaging of metal ions under live-cell conditions.

Abstract Background. Injuries to the peripheral nerve system are common conditions, with broad spectrum of symptoms depending on the impaired nerves and severity of damage. Although peripheral nervous system retains a remarkable ability for regeneration, it is estimated that less than ten percent of patients fully recover function after nerve injury and the available treatments remain suboptimal. Here, we identify a role for the obestatin/GPR39 system in the regulation of the Schwann cell plasticity as well as in the preservation of neuromuscular synapses in the course of nerve repair. Methods. Utilizing a compression model of sciatic nerve injury, axonotmesis, we assessed the obestatin-related regenerative response in the peripheral nerve system. The role of the obestatin/GPR39 system was further evaluated on immortalized rat Schwann cells, IFRS1, and the model of neuronal differentiation, PC12 cells. The interactions between SCs and neurons was evaluated using a co-culture system that combine the SC cell line IFRS1 and the NGF-primed PC12. Results. Obestatin signaling directs proliferation and migration of Schwann cells that sustain axonal regrowth and later remyelinate regenerated axons. We provide evidence supporting the preservation of skeletal muscle by the maintenance of neuromuscular synapses through the axonal regulation of calpain-calpastatin proteolytic system. This encompasses the control of skeletal muscle homeostasis by regulation of the ubiquitin proteasome system and the autophagy machinery. Conclusions. These results provide important insights into how the obestatin/GPR39 system promotes nerve repair through integration of multiple molecular cues of neuron-Schwann cells crosstalk aimed to promote axon growth and guide axons back to their targets.

Abstract Background The Mediator complex transduces information from the DNA-bound transcription factors to the RNA polymerase II transcriptional machinery. Research on plant Mediator subunits was mainly performed in Arabidopsis, while very few of them have been functionally characterized in rice. Results Here the rice Mediator subunit 16, OsMed16, was studied. OsMed16 encoded a putative protein of 1301 amino acids, which is longer than the reported version. It was expressed in various rice organs, and localized in nucleus. Knockout of OsMed16 caused rice seedling lethality. Its overexpression led to rice growth retardation, low yield, and spontaneous cell death in leaf blade and leaf sheath. RNA sequencing suggested that overexpression of OsMed16 altered the expression of a large number of genes. Among them, the up-regulation of some defense-related genes was verified. Conclusions Our results demonstrated that OsMed16 can regulate the expression of a wealth of genes, and alterations in its expression have profound impact on plant growth, development and defense response in rice.

Abstract Neurite outgrowth is important in neuronal circuit formation and functions, and for regeneration of neuronal networks following trauma and disease in the brain. Thus, identification and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function and for the development of treatment of neurological disorders. In this study, we found that lysophosphatidylethanolamine (LPE), one of the lysophospholipids, influences neurite outgrowth in cultured cortical neurons. Extracellular application of either of the structurally different LPE spices, palmitoyl LPE (16:0 LPE) and stearoyl LPE (18:0 LPE) dramatically increased the areas of axon and dendrite without affecting the neuronal viability. Subsequent analysis revealed that both LPEs increased the length of neurite in a dose-dependent manner. Interestingly, inhibition of phospholipase C, one of the effectors for G-protein-coupled receptor-mediated signaling pathways, inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of protein kinase C (PKC) inhibitors on neurite outgrowth were also different. Inhibitor against PKCα, β, δ, ε, η, and θ inhibited both 16:0 LPE- and 18:0 LPE-induced neurite outgrowth. In contrast, an inhibitor against PKCα, β, γ, δ, and ζ inhibited the 18:0 LPE effect but not the 16:0 LPE effect. We also found that both 16:0 LPE and 18:0 LPE activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2. There was no substantial difference in the amount of phosphorylated MAPK/ERK1/2 between 16:0 LPE and 18:0 LPE-treated cultures. MAPK inhibitor completely inhibited 18:0 LPE-induced neurite outgrowth and partially inhibited 16:0 LPE-induced neurite outgrowth. Thus, the effect of the MAPK inhibitor differed between the 16:0 LPE- and 18:0 LPE-treated cultures. Collectively, the results suggest that the structurally different LPE species, 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through the distinct signaling cascades in cultured cortical neurons.

Abstract Background: Photoreceptor death and neurodegeneration is the leading cause of irreversible vision loss. The inflammatory response of microglia plays an important role in the process of neurodegeneration. In this study, we examined the involvement of myosin 1f as a key regulator of immune cell activation via the AKT and MAPK pathways in microglia. Methods: We chose retinal detachment as the model of photoreceptor degeneration. Immunofluorescence and Western Blot was performed to confirm the expression and location of myosin 1f in detached retina. The RD mouse model was induced in WT and myosin 1f-/- mice and confirmed by HE and TUNEL staining. The expression of inflammatory cytokine and downstream pathways was assessed via qPCR and WB.Results: Myosin 1f was upregulated after retinal detachment, and it was specifically expressed in microglia. Deficiency of myosin 1f protected against cell death by inhibiting microglia activation. The elimination of microglia can abolish the protective effect of myosin 1f deficiency. After stimulation by LPS, microglia with myosin 1f deficiency showed downregulation of the MAPK and AKT pathways.Conclusions: Myosin 1f plays a crucial role in microglia-induced neuro-inflammation after retinal injury and photoreceptor degeneration by regulating 2 classic pathways, MAPK and AKT, and thereby decreasing the expression of inflammatory cytokines. Myosin 1f can be inhibited to prevent a decline in visual acuity after photoreceptor degeneration.

Abstract DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. Here we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by the creation of transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in tobacco to target the endogenous phytoene desaturase (pds) and actin depolymerizing factor (adf) genes. Genome-edited plants were produced with an efficiency of 5.6–8.7%. We also evaluated the effects of adf mutations in T2 mutant plants under drought and salinity stress, showing that adf acts as a key regulator of osmotic stress tolerance in plants.