Chinese Medicine for Treatment of COVID-19: A Review of Potential Pharmacological Components and Mechanisms.

Coronavirus disease 2019 (COVID-19) is an acute infectious respiratory disease that has been prevalent since December 2019. Chinese medicine (CM) has demonstrated its unique advantages in the fight against COVID-19 in the areas of disease prevention, improvement of clinical symptoms, and control of disease progression. This review summarized the relevant material components of CM in the treatment of COVID-19 by searching the relevant literature and reports on CM in the treatment of COVID-19 and combining with the physiological and pathological characteristics of the novel coronavirus. On the basis of sorting out experimental methods in vivo and in vitro, the mechanism of herb action was further clarified in terms of inhibiting virus invasion and replication and improving related complications. The aim of the article is to explore the strengths and characteristics of CM in the treatment of COVID-19, and to provide a basis for the research and scientific, standardized treatment of COVID-19 with CM.

Two-Dimensional Metal-Organic Frameworks/Epoxy Composite Coatings with Superior O2/H2O Resistance for Anticorrosion Applications.

Corrosion protection technology plays a crucial role in preserving infrastructure, ensuring safety and reliability, and promoting long-term sustainability. In this study, we combined experiments and various analyses to investigate the mechanism of corrosion occurring on the epoxy-based anticorrosive coating containing the additive of two-dimensional (2D) and water-stable zirconium-based metal-organic frameworks (Zr-MOFs). By using benzoic acid as the modulator for the growth of the MOF, a 2D MOF constructed from hexazirconium clusters and BTB linkers (BTB = 1,3,5-tri(4-carboxyphenyl)benzene) with coordinated benzoate (BA-ZrBTB) can be synthesized. By coating the BA-ZrBTB/epoxy composite film (BA-ZrBTB/EP) on the surface of cold-rolled steel (CRS), we found the lowest coating roughness (RMS) of BA-ZrBTB/EP is 2.83 nm with the highest water contact angle as 99.8°, which represents the hydrophobic coating surface. Notably, the corrosion rate of the BA-ZrBTB/EP coating is 2.28 × 10-3 mpy, which is 4 orders of magnitude lower than that of the CRS substrate. Moreover, the energy barrier for oxygen diffusion through BA-ZrBTB/EP coating is larger than that for epoxy coating (EP), indicating improved oxygen resistance for adding 2D Zr-MOFs as the additive. These results underscore the high efficiency and potential of BA-ZrBTB as a highly promising agent for corrosion prevention in various commercial applications. Furthermore, this study represents the first instance of applying 2D Zr-MOF materials in anticorrosion applications, opening up new possibilities for advanced corrosion-resistant coatings.

Significance and Possible Biological Mechanism for CLDN8 Downregulation in Kidney Renal Clear Cell Carcinoma Tissues.

Background: The clinical role of claudin 8 (CLDN8) in kidney renal clear cell carcinoma (KIRC) remains unclarified. Herein, the expression level and potential molecular mechanisms of CLDN8 underlying KIRC were determined. Methods: High-throughput datasets of KIRC were collected from GEO, ArrayExpress, SRA, and TCGA databases to determine the mRNA expression level of the CLDN8. In-house tissue microarrays and immunochemistry were performed to examine CLDN8 protein expression. A summary receiver operating characteristic curve (SROC) and standardized mean difference (SMD) forest plot were generated using Stata v16.0. Single-cell analysis was conducted to further prove the expression level of CLDN8. A clustered regularly interspaced short palindromic repeats knockout screen analysis was executed to assess the growth impact of CLDN8. Functional enrichment analysis was conducted using the Metascape database. Additionally, single-sample gene set enrichment analysis was implied to explore immune cell infiltration in KIRC. Results: A total of 17 mRNA datasets comprising 1,060 KIRC samples and 452 non-cancerous control samples were included in this study. Additionally, 105 KIRC and 16 non-KIRC tissues were analyzed using in-house immunohistochemistry. The combined SMD was -5.25 (95% confidence interval (CI): -6.13 to -4.37), and CLDN8 downregulation yielded an SROC area under the curve (AUC) close to 1.00 (95% CI: 0.99 - 1.00). CLDN8 downregulation was also confirmed at the single-cell level. Knocking out CLDN8 stimulated KIRC cell proliferation. Lower CLDN8 expression was correlated with worse overall survival of KIRC patients (hazard ratio of CLDN8 downregulation = 1.69, 95% CI: 1.2 - 2.4). Functional pathways associated with CLDN8 co-expressed genes were centered on carbon metabolism obstruction, with key hub genes ACADM, ACO2, NDUFS1, PDHB, SDHD, SUCLA2, SUCLG1, and SUCLG2. Conclusions: CLDN8 is downregulated in KIRC and is considered a potential tumor suppressor. CLDN8 deficiency may promote the initiation and progression of KIRC, potentially in conjunction with metabolic dysfunction.

Immobilization of europium and terbium ions with tunable ratios on a dispersible two-dimensional metal-organic framework for ratiometric photoluminescence detection of D2O.

A two-dimensional zirconium-based metal-organic framework (2D Zr-MOF), ZrBTB (BTB = 1,3,5-tri(4-carboxyphenyl)benzene), is used as a platform to simultaneously immobilize terbium ions and europium ions with tunable ratios on its hexa-zirconium nodes by a post-synthetic modification. The crystallinity, morphology, porosity and photoluminescence (PL) properties of the obtained 2D Zr-MOFs with various europium-to-terbium ratios are investigated. With the energy transfer from the excited BTB linker to the installed terbium ions and the energy transfer from terbium ions to europium ions, a low loading of immobilized europium ions and a high loading of surrounding terbium ions in the 2D Zr-MOF result in the optimal PL emission intensities of europium; this phenomenon is not observable for the physical mixture of both terbium-installed ZrBTB and europium-installed ZrBTB. The role of installed terbium ions as efficient mediators for the energy transfer from the excited BTB linker to the installed europium ion is confirmed by quantifying PL quantum yields. As a demonstration, these materials with modulable PL characteristics are applied for the ratiometric detection of D2O in water, with the use of the stable emission from the BTB linker as the reference. With the strong emission of immobilized europium ions and the good dispersity in aqueous solutions, the optimal bimetal-installed ZrBTB, Eu-Tb-ZrBTB(1 : 10), can achieve the sensing performance outperforming those of the terbium-installed ZrBTB, europium-installed ZrBTB and the physical mixture of both.

G-quadruplex-guided cisplatin triggers multiple pathways in targeted chemotherapy and immunotherapy.

G-quadruplexes (G4s) are atypical nucleic acid structures involved in basic human biological processes and are regulated by small molecules. To date, pyridostatin and its derivatives [e.g., PyPDS (4-(2-aminoethoxy)-N 2,N 6-bis(4-(2-(pyrrolidin-1-yl) ethoxy) quinolin-2-yl) pyridine-2,6-dicarboxamide)] are the most widely used G4-binding small molecules and considered to have the best G4 specificity, which provides a new option for the development of cisplatin-binding DNA. By combining PyPDS with cisplatin and its analogs, we synthesize three platinum complexes, named PyPDSplatins. We found that cisplatin with PyPDS (CP) exhibits stronger specificity for covalent binding to G4 domains even in the presence of large amounts of dsDNA compared with PyPDS either extracellularly or intracellularly. Multiomics analysis reveals that CP can effectively regulate G4 functions, directly damage G4 structures, activate multiple antitumor signaling pathways, including the typical cGAS-STING pathway and AIM2-ASC pathway, trigger a strong immune response and lead to potent antitumor effects. These findings reflect that cisplatin-conjugated specific G4 targeting groups have antitumor mechanisms different from those of classic cisplatin and provide new strategies for the antitumor immunity of metals.

Discovery of Dual MER/AXL Kinase Inhibitors as Bifunctional Small Molecules for Inhibiting Tumor Growth and Enhancing Tumor Immune Microenvironment.

A series of bifunctional compounds have been discovered for their dual functionality as MER/AXL inhibitors and immune modulators. The furanopyrimidine scaffold, renowned for its suitability in kinase inhibitor discovery, offers at least three distinct pharmacophore access points. Insights from molecular modeling studies guided hit-to-lead optimization, which revealed that the 1,3-diketone side chain hybridized with furanopyrimidine scaffold that respectively combined amino-type substituent and 1H-pyrazol-4-yl substituent on the top and bottom of the aryl regions to produce 22 and 33, exhibiting potent antitumor activities in various syngeneic and xenograft models. More importantly, 33 demonstrated remarkable immune-modulating activity by upregulating the expression of total T-cells, cytotoxic CD8+ T-cells, and helper CD4+ T-cells in the spleen. These findings underscored the bifunctional capabilities of 33 (BPR5K230) with excellent oral bioavailability (F = 54.6%), inhibiting both MER and AXL while modulating the tumor microenvironment and highlighting its diverse applicability for further studies to advance its therapeutic potential.

Two-dimensional metal-organic framework for post-synthetic immobilization of graphene quantum dots for photoluminescent sensing.

Immobilization of graphene quantum dots (GQDs) on a solid support is crucial to prevent GQDs from aggregation in the form of solid powder and facilitate the separation and recycling of GQDs after use. Herein, spatially dispersed GQDs are post-synthetically coordinated within a two-dimensional (2D) and water-stable zirconium-based metal-organic framework (MOF). Unlike pristine GQDs, the obtained GQDs immobilized on 2D MOF sheets show photoluminescence in both suspension and dry powder. Chemical and photoluminescent stabilities of MOF-immobilized GQDs in water are investigated, and the use of immobilized GQDs in the photoluminescent detection of copper ions is demonstrated. Findings here shed the light on the use of 2D MOFs as a platform to further immobilize GQDs with various sizes and distinct chemical functionalities for a range of applications.

Ultrasmall Single-Chain Nanoparticles Derived from Amphiphilic Alternating Copolymers.

The collapse or folding of an individual polymer chain into a nanoscale particle gives rise to single-chain nanoparticles (SCNPs), which share a soft nature with biological protein particles. The precise control of their properties, including morphology, internal structure, size, and deformability, are a long-standing and challenging pursuit. Herein, a new strategy based on amphiphilic alternating copolymers for producing SCNPs with ultrasmall size and uniform structure is presented. SCNPs are obtained by folding the designed alternating copolymer in N,N-dimethylformamide (DMF) and fixing it through a photocatalyzed cycloaddition reaction of anthracene units. Molecular dynamics simulation confirms the solvophilic outer corona and solvophobic inner core structure of SCNPs. Furthermore, by adjusting the length of PEG units, precise control over the mean size of SCNPs is achieved within the range of 2.8 to 3.9 nm. These findings highlight a new synthetic strategy that enables enhanced control over morphology and internal structure while achieving ultrasmall and uniform size for SCNPs.

Detection of pTDP-43 via routine muscle biopsy: A promising diagnostic biomarker for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, pathologically characterized by TDP-43 aggregates. Recent evidence has been indicated that phosphorylated TDP-43 (pTDP-43) is present not only in motor neurons but also in muscle tissues. However, it is unclear whether testing pTDP-43 aggregation in muscle tissue would assist in the diagnosis of ALS. We propose three key questions: (i) Is aggregation of pTDP-43 detectable in routine biopsied muscles? (ii) Can detection of pTDP-43 aggregation discriminate between ALS and non-ALS patients? (iii) Can pTDP-43 aggregation be observed in the early stages of ALS? We conducted a diagnostic study comprising 2 groups: an ALS group in which 18 cases underwent muscle biopsy screened from a registered ALS cohort consisting of 802 patients and a non-ALS control group, in which we randomly selected 54 muscle samples from a biospecimen bank of 684 patients. Among the 18 ALS patients, 3 patients carried pathological GGGGCC repeats in the C9ORF72 gene, 2 patients carried SOD1 mutations, and 7 patients were at an early stage with only one body region clinically affected. The pTDP-43 accumulation could be detected in routine biopsied muscles, including biceps brachii, deltoid, tibialis anterior, and quadriceps. Abnormal aggregation of pTDP-43 was present in 94.4% of ALS patients (17/18) compared to 29.6% of non-ALS controls (16/54; p < 0.001). The pTDP-43 aggregates were mainly close to the sarcolemma. Using a semi-quantified pTDP-43 aggregates score, we applied a cut-off value of 3 as a diagnostic biomarker, resulting in a sensitivity of 94.4% and a specificity of 83.3%. Moreover, we observed that accumulation of pTDP-43 occurred in muscle tissues prior to clinical symptoms and electromyographic lesions. Our study provides proof-of-concept for the detection of pTDP-43 accumulation via routine muscle biopsy which may serve as a novel biomarker for diagnosis of ALS.

First telomere-to-telomere gapless assembly of the rice blast fungus Pyricularia oryzae.

Rice blast caused by Pyricularia oryzae (syn., Magnaporthe oryzae) was one of the most destructive diseases of rice throughout the world. Genome assembly was fundamental to genetic variation identification and critically impacted the understanding of its ability to overcome host resistance. Here, we report a gapless genome assembly of rice blast fungus P. oryzae strain P131 using PacBio, Illumina and high throughput chromatin conformation capture (Hi-C) sequencing data. This assembly contained seven complete chromosomes (43,237,743 bp) and a circular mitochondrial genome (34,866 bp). Approximately 14.31% of this assembly carried repeat sequences, significantly greater than its previous assembled version. This assembly had a 99.9% complement in BUSCO evaluation. A total of 14,982 genes protein-coding genes were predicted. In summary, we assembled the first telomere-to-telomere gapless genome of P. oryzae, which would be a valuable genome resource for future research on the genome evolution and host adaptation.

DGS1 improves rice disease resistance by elevating pathogen-associated molecular pattern-triggered immunity.

Rice yield and disease resistance are two crucial factors in determining the suitability of a gene for agricultural breeding. Decreased grain size1 (DGS1), encoding an RING-type E3 ligase, has been found to have a positive effect on rice yield by regulating rice grain number and 1000-grain weight. However, the role of DGS1 in rice blast resistance is still unknown. In this study, we report that DGS1 enhances disease resistance by improving PTI responses, including stronger ROS burst and MAPK activation, and also increased expression of defense-related genes. Furthermore, DGS1 works in conjunction with ubiquitin conjugating enzyme OsUBC45 as an E2-E3 pair to facilitate the ubiquitin-dependent degradation of OsGSK3 and OsPIP2;1, thereby influencing rice yield and immunity, respectively. Therefore, the DGS1-OsUBC45 module has the potential in facilitating rice agricultural breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00137-9.

Molecular Phylogenetics and the Evolution of Morphological Complexity in Aytoniaceae (Marchantiophyta).

Aytoniaceae are one of the largest families of complex thalloid liverworts (Marchantiopsida), consisting of about 70 species, with most species being distributed in temperate areas. However, the phylogeny and evolution of the morphological character of Aytoniaceae are still poorly understood. Here, we employed two chloroplast loci, specifically, rbcL and trnL-F, along with a 26S nuclear ribosomal sequence to reconstruct the phylogeny and track the morphological evolution of Aytoniaceae. Our results reveal that Aytoniaceae are monophyletic, and five monophyletic clades were recovered (i.e., Asterellopsis-Cryptomitrium, Calasterella, Mannia, Reboulia-Plagiochasma, and Asterella). Asterella was divided into five clades (i.e., Asterella lindenbergiana, subg. Saccatae, subg. Phragmoblepharis, subg. Wallichianae, and subg. Asterella), except for Asterella palmeri, which is the sister of Asterellopsis grollei. Bayesian molecular clock dating indicates that the five primary clades within Aytoniaceae underwent divergence events in the Cretaceous period. Asterellopsis differentiated during the early Upper Cretaceous (c. 84.2 Ma), and Calasterella originated from the late Lower Cretaceous (c. 143.0 Ma). The ancestral Aytoniaceae plant is reconstructed as the absence of a pseudoperianth, lacking equatorial apertures, and having both male and female reproductive organs on the main thallus. At present, Asterellopsis consists of two species known in Asia and America with the new transfer of Asterella palmeri to Asterellopsis. A new subgenus, Asterella subg. Lindenbergianae, is proposed.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases.

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8-dihydroxynaphthalene to generate the spiroketal linkage and 2,3-epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C-5 hydroxy group, widely found in SBNs. PalC catalyzes 1-keto reduction, the reverse 1-dehydrogenation, and 2,3-epoxide reduction. Moreover, an FAD-dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1-dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.

Synergistic Cation-π Interactions and PEDOT-Based Protective Double-Layer for High Performance Zinc Anode.

Ensuring effective and controlled zinc ion transportation is crucial for functionality of the solid electrolyte interphase (SEI) and overall performance in zinc-based battery systems. Herein the first-ever demonstration of incorporate cation-π interactions are provided in the SEI to effectively facilitate uniform zinc ion flux. The artificial SEI design involves the immobilization of 4-amino-p-terphenyl (TPA), a strong amphiphilic cation-π interaction donor, as a monolayer onto a conductive poly(3,4-ethylenedioxythiophene) (PEDOT) matrix, which enable the establishment of a robust network of cation-π interactions. Through a carefully-designed interfacial polymerization process, a high-quality, large-area, robust is achieved, thin polymeric TPA/PEDOT (TP) film for the use of artificial SEI. Consequently, this interphase exhibits exceptional cycling stability with low overpotential and enables high reversibility of Zn plating/stripping. Symmetrical cells with TP/Zn electrodes can be cycled for more than 3200 hours at 1 mA cm-2 and 1 mAh cm-2 . And the asymmetric cells can cycle 3000 cycles stably with a high Coulomb efficiency of 99.78%. Also, under the extreme conditions of lean electrolyte and low N/P ratio, the battery with TP protective layer can still achieve ultra-stable cycle.

Exploring the interplay of liquid crystal orientation and spherical elastic shell deformation in spatial confinement.

The application of liquid crystal technology typically relies on the precise control of molecular orientation at a surface or interface. This control can be achieved through a combination of morphological and chemical methods. Consequently, variations in constrained boundary flexibility can result in a diverse range of phase behaviors. In this study, we delve into the self-assembly of liquid crystals within elastic spatial confinement by using the Gay-Berne model with the aid of molecular dynamics simulations. Our findings reveal that a spherical elastic shell promotes a more regular and orderly alignment of liquid crystals compared to a hard shell. Moreover, during the cooling process, the hard-shell confined system undergoes an isotropic-smectic phase transition. In contrast, the phase behavior within the spherical elastic shell closely mirrors the isotropic-nematic-smectic phase transition observed in bulk systems. This indicates that the orientational arrangement of liquid crystals and the deformations induced by a flexible interface engage in a competitive interplay during the self-assembly process. Importantly, we found that phase behavior could be manipulated by altering the flexibility of the confined boundaries. This insight offers a fresh perspective for the design of innovative materials, particularly in the realm of liquid crystal/polymer composites.

The synthetic NLR RGA5HMA5 requires multiple interfaces within and outside the integrated domain for effector recognition.

Some plant sensor nucleotide-binding leucine-rich repeat (NLR) receptors detect pathogen effectors through their integrated domains (IDs). Rice RGA5 sensor NLR recognizes its corresponding effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae through direct binding to its heavy metal-associated (HMA) ID to trigger the RGA4 helper NLR-dependent resistance in rice. Here, we report a mutant of RGA5 named RGA5HMA5 that confers complete resistance in transgenic rice plants to the M. oryzae strains expressing the noncorresponding effector AVR-PikD. RGA5HMA5 carries three engineered interfaces, two of which lie in the HMA ID and the other in the C-terminal Lys-rich stretch tailing the ID. However, RGA5 variants having one or two of the three interfaces, including replacing all the Lys residues with Glu residues in the Lys-rich stretch, failed to activate RGA4-dependent cell death of rice protoplasts. Altogether, this work demonstrates that sensor NLRs require a concerted action of multiple surfaces within and outside the IDs to both recognize effectors and activate helper NLR-mediated resistance, and has implications in structure-guided designing of sensor NLRs.

Nakazawaea tricholomae f.a., sp. nov., a Novel Ascomycetous Yeast Species Isolated from Two Mushroom Species in China.

Two yeast strains designated as 20-27-1 and 20-28 were isolated from the fruiting bodies of Tricholoma gambosum and Marasmius maximus, respectively, which were collected in Wudaogou, Weichang county, Chengde area, Hebei Province, China. The multi-locus analysis of the sequences of the rDNA ITS, D1/D2 LSU, and SSU regions, together with partial sequences of two protein-coding genes RPB1 and TEF1 indicates that the two strains are closely related to Nakazawaea ernobii and Nakazawaea holstii, showing the similarity values of 99.3-98.7%, 97.2-97.1%, 91.9-92.5%, and 84.6% in D1/D2 LSU, ITS, TEF1, and RPB1, respectively. Physiologically, the two strains are different from N. ernobii and N. holstii in the assimilation of melibiose, inulin, and DL-lactic acid. Both the phenotypic and phylogenetic analyses indicate that those two strains represent a novel species in the genus Nakazawaea, for which the name Nakazawaea tricholomae f.a., sp. nov. (Fungal Names: FN 571492) is proposed.

Divergence of trafficking and polarization mechanisms for PIN auxin transporters during land plant evolution.

The phytohormone auxin, and its directional transport through tissues, plays a fundamental role in the development of higher plants. This polar auxin transport predominantly relies on PIN-FORMED (PIN) auxin exporters. Hence, PIN polarization is crucial for development, but its evolution during the rise of morphological complexity in land plants remains unclear. Here, we performed a cross-species investigation by observing the trafficking and localization of endogenous and exogenous PINs in two bryophytes, Physcomitrium patens and Marchantia polymorpha, and in the flowering plant Arabidopsis thaliana. We confirmed that the GFP fusion did not compromise the auxin export function of all examined PINs by using a radioactive auxin export assay and by observing the phenotypic changes in transgenic bryophytes. Endogenous PINs polarize to filamentous apices, while exogenous Arabidopsis PINs distribute symmetrically on the membrane in both bryophytes. In the Arabidopsis root epidermis, bryophytic PINs have no defined polarity. Pharmacological interference revealed a strong cytoskeletal dependence of bryophytic but not Arabidopsis PIN polarization. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues of individual species. These results collectively reveal the divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and the co-evolution of PIN sequence-based and cell-based polarity mechanisms.

The GRAS protein OsDLA involves in brassinosteroid signalling and positively regulates blast resistance by forming a module with GSK2 and OsWRKY53 in rice.

Brassinosteroids (BRs) play a crucial role in shaping the architecture of rice (Oryza sativa) plants. However, the regulatory mechanism of BR signalling in rice immunity remains largely unexplored. Here we identify a rice mutant dla, which exhibits decreased leaf angles and is insensitive to 24-epiBL (a highly active synthetic BR), resembling the BR-deficient phenotype. The dla mutation caused by a T-DNA insertion in the OsDLA gene leads to downregulation of the causative gene. The OsDLA knockout plants display reduced leaf angles and less sensitivity to 24-epiBL. In addition, both dla mutant and OsDLA knockout plants are more susceptible to rice blast compared to the wild type. OsDLA is a GRAS transcription factor and interacts with the BR signalling core negative regulator, GSK2. GSK2 phosphorylates OsDLA for degradation via the 26S proteasome. The GSK2 RNAi line exhibits enhanced rice blast resistance, while the overexpression lines thereof show susceptibility to rice blast. Furthermore, we show that OsDLA interacts with and stabilizes the WRKY transcription factor OsWRKY53, which has been demonstrated to positively regulate BR signalling and blast resistance. OsWRKY53 directly binds the promoter of PBZ1 and activates its expression, and this activation can be enhanced by OsDLA. Together, our findings unravel a novel mechanism whereby the GSK2-OsDLA-OsWRKY53 module coordinates blast resistance and plant architecture via BR signalling in rice.

A gln-tRNA-based CRISPR/Cas9 knockout system enables the functional characterization of genes in the genetically recalcitrant brassica anthracnose fungus Colletotrichum higginsianum.

Colletotrichum higginsianum causes anthracnose disease in brassicas. The availability of the C. higginsianum genome has paved the way for the genome-wide exploration of genes associated with virulence/pathogenicity. However, delimiting the biological functions of these genes remains an arduous task due to the recalcitrance of C. higginsianum to genetic manipulations. Here, we report a CRISPR/Cas9-based system that can knock out the genes in C. higginsianum with a staggering 100% homologous recombination frequency (HRF). The system comprises two vectors: pCas9-Ch_tRp-sgRNA, in which a C. higginsianum glutaminyl-tRNA drives the expression of sgRNA, and pCE-Zero-HPT carrying a donor DNA cassette containing the marker gene HPT flanked by homology arms. Upon co-transformation of the C. higginsianum protoplasts, pCas9-Ch_tRp-sgRNA causes a DNA double-strand break in the targeted gene, followed by homology-directed replacement of the gene with HPT by pCE-Zero-HPT, thereby generating loss-of-function mutants. Using the system, we generated the knockout mutants of two effector candidates (ChBas3 and OBR06881) with a 100% HRF. Interestingly, the ΔChBas3 and ΔOBR06881 mutants did not seem to affect the C. higginsianum infection of Arabidopsis thaliana. Altogether, the CRISPR/Cas9 system developed in the study enables the targeted deletion of genes, including effectors, in C. higginsianum, thus determining their biological functions.