Biocontrol activity of an endophytic Alternaria alternata Aa-Lcht against apple Valsa canker.

Apple Valsa canker (AVC), caused by Valsa mali, is the most serious branch disease for apples in East Asia. Biocontrol constitutes a desirable alternative strategy to alleviate the problems of orchard environment pollution and pathogen resistance risk. It is particularly important to explore efficient biocontrol microorganism resources to develop new biocontrol technologies and products. In this study, an endophytic fungus, which results in the specific inhibition of the growth of V. mali, was isolated from the twig tissue of Malus micromalus with a good tolerance to AVC. The fungus was identified as Alternaria alternata, based on morphological observations and phylogenetic analysis, and was named Aa-Lcht. Aa-Lcht showed a strong preventive effect against AVC, as determined with an in vitro twig evaluation method. When V. mali was inhibited by Aa-Lcht, according to morphological and cytological observations, the hyphae was deformed and it had more branches, a degradation in protoplasm, breakages in cell walls, and then finally died completely due to mycelium cells. Transcriptome analysis indicated that Aa-Lcht could suppress the growth of V. mali by inhibiting the activity of various hydrolases, destroying carbohydrate metabolic processes, and damaging the pathogen membrane system. It was further demonstrated that Aa-Lcht could colonize apple twig tissues without damaging the tissue's integrity. More importantly, Aa-Lcht could also stimulate the up-regulated expression of defense-related genes in apples together with the accumulation of reactive oxygen species and callose deposition in apple leaf cells. Summarizing the above, one endophytic biocontrol resource was isolated, and it can colonize apple twig tissue and play a biocontrol role through both pathogen inhibition and resistance inducement.

Survey of Heterodera glycines population abundance and virulence phenotypes during 2021-2022 in Heilongjiang Province.

Soybean cyst nematode (SCN), Heterodera glycines, poses a significant threat to global soybean production. Heilongjiang, the largest soybean-producing province in China, contributes over 40% to the country's total yield. This province has much longer history of SCN infestation. To assess the current situation in Heilongjiang, we conducted a survey to determine the SCN population density and virulence phenotypes during 2021-2022 and compared the data with a previous study in 2015. A total of 377 soil samples from 48 counties representing eleven major soybean-planting regions were collected. The prevalence of SCN increased from 55.4% in 2015 to 59% in the current survey. The population densities ranged from 80 to 26,700 eggs and juveniles per 100 cm3 of soil. Virulence phenotypes were evaluated for 60 representative SCN populations using the HG type test, revealing nine different HG types. The most common virulence phenotypes were HG types 7 and 0, accounting for 56.7% and 20% of all SCN populations, respectively. The prevalence of populations with a reproductive index (FI) greater than 10% on PI548316 increased from 64.5% in 2015 to 71.7%. However, the FI on the commonly used resistance sources PI 548402 (Peking) and PI 437654 remained low at 3.3%. These findings highlight the increasing prevalence and changing virulence phenotypes of SCN in Heilongjiang. They also emphasize the importance of rotating soybean varieties with different resistance sources and urgently identifying new sources of resistance to combat SCN.

Part-Aware Point Cloud Completion through Multi-Modal Part Segmentation.

Point cloud completion aims to generate high-resolution point clouds using incomplete point clouds as input and is the foundational task for many 3D visual applications. However, most existing methods suffer from issues related to rough localized structures. In this paper, we attribute these problems to the lack of attention to local details in the global optimization methods used for the task. Thus, we propose a new model, called PA-NET, to guide the network to pay more attention to local structures. Specifically, we first use textual embedding to assist in training a robust point assignment network, enabling the transformation of global optimization into the co-optimization of local and global aspects. Then, we design a novel plug-in module using the assignment network and introduce a new loss function to guide the network's attention towards local structures. Numerous experiments were conducted, and the quantitative results demonstrate that our method achieves novel performance on different datasets. Additionally, the visualization results show that our method efficiently resolves the issue of poor local structures in the generated point cloud.

Bile acids regulate the epithelial Na+ channel in native tissues through direct binding at multiple sites.

Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the ß and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. KEY POINTS: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel. We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability. Photoaffinity labelling experiments showed specific binding to the ß and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes. Taurocholic acid regulation of ENaC was voltage-dependent, providing evidence for binding to pore-forming helices. Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems.

A fungal microRNA-like RNA subverts host immunity and facilitates pathogen infection by silencing two host receptor-like kinase genes.

Small RNAs (sRNAs) play important roles in various biological processes by silencing their corresponding target genes in most eukaryotes. However, cross-kingdom regulation mediated by fungal microRNA-like RNAs (milRNAs) in plant-pathogen interactions is still largely unknown. Using molecular, genetic, histological, and biochemical approaches, we found that the apple tree Valsa canker pathogen Valsa mali milRNA Vm-milR1 could suppress the host immunity by silencing two host receptor-like kinase genes, MdRLKT1 and MdRLKT2. Vm-milR1 was highly induced during V. mali infection. Deletion of Vm-milR1 precursor abolished the generation of Vm-milR1 and reduced the virulence of V. mali. Inoculation of Vm-milR1 deletion mutants induced the host defence responses, including reactive oxygen species (ROS) accumulation, callose deposition, and high expression of defence-related genes. Furthermore, Vm-milR1 was confirmed to be able to suppress the expression of MdRLKT1 and MdRLKT2 in a sequence-specific manner. Moreover, overexpression of either MdRLKT1 or MdRLKT2 enhanced apple resistance to V. mali by activating the host defence responses. Furthermore, knockdown of MdRLKT1 or MdRLKT2 compromised the host resistance to V. mali. Our study revealed that V. mali was equipped with Vm-milR1 as an sRNA effector to silence host receptor-like kinase genes, suppress the host defence responses, and facilitate pathogen infection.

In vivo and in vitro antifungal activities of five alkaloid compounds isolated from Picrasma quassioides (D. Don) Benn against plant pathogenic fungi.

Highly active and novel antifungal compounds are continuously researched from natural products for pesticide development. Picrasma quassioides (D. Don) Benn, a species of Simaroubaceae, is used in traditional Chinese medicine to treat colds and upper respiratory infections. In this study, the active ingredients of P. quassioides and their antifungal activities against plant pathogenic fungi are investigated to explore the practical application of the plant in the agricultural field. The results showed that the extracts of P. quassioides exhibited highly significant preventive and curative effects on apple valsa canker (AVC) with a reduction of lesion diameter were 80.28% and 83.63%, respectively, and can improve the resistance of apple trees to a pathogen. Five antifungal compounds, namely, canthin-6-one (T1), nigakinone (T2), 4,5-dimethoxycanthin-6-one (T3), 1-methoxycarbonyl-ß-carboline (T4), and 1-methoxycarbonyl-3-methoxyl-ß-carboline (T5), are isolated from P. quassioides using the bioassay-guided method. This is the first report of 1-methoxycarbonyl-3-methoxyl-ß-carboline as a natural product. Canthin-6-one shows strong in vitro inhibitory activity against 11 species of plant pathogenic fungi, and their EC50 values range from 1.49 to 8.80 mg/L. The control efficacy of canthin-6-one at 2000 mg/L are 87.88% and 94.37% against AVC and 80.10% and 84.73% against apple anthracnose (C. gloeosporioides), respectively. Additionally, V. mali is observed after treatment with cannin-6-one, although microscopic. This is the first study on the control of the secondary metabolites of P. quassioides against plant fungal diseases. The results show that P. quassioides is a potential resource for the development of botanical fungicides.

Ethyl Phloretate and Ethyl p-Coumarate: Two Phytotoxins from Valsa mali and Their Pathogenic Activities.

Valsa mali, the causal agent of apple Valsa canker, produces several phytotoxic metabolites to promote infection. Bioassay and 1H nuclear magnetic resonance (NMR)-guided isolation from the culture filtrate of V. mali strain 03-8 led to the identification of seven compounds including three unreported ones, ethyl phloretate (1), ethyl p-coumarate (2), and 1-p-hydroxybenzoyl glycerol (3). Compounds 1 and 2 produced significant phytotoxicity, with average lesion areas of 6.22 and 3.74 mm2, along with 2.96 and 3.47 mm2 at 1 mg/ml on mature and tissue-cultured apple leaves, respectively, whereas compound 3 did not cause any symptoms on host plants. The necrotic lesion area of compounds 1 and 2 on tobacco leaves was 52.65 and 48.28 mm2, respectively, compared with the negative control (0.46 mm2) at 1 mg/ml. At the same concentration, compounds 1 and 2 showed no significant influence on the germination rate of lettuce seeds while significantly decreasing the root length of lettuce seedlings to 6.74 and 4.67 mm, respectively, compared with that treated with sterile distilled water (22.01 mm). The discovery indicated that compounds 1 and 2 could be considered as non-host-specific toxins. Furthermore, compounds 1 and 2 could cause cell shrinkage, organelle damage, plasmolysis, and eventually ruptured protoplasmic membranes with cell death for their phytotoxicity in the host plants under optical microscopy and transmission electron microscopy. The results shed light on the mechanism for toxins 1 and 2 in V. mali-infected plants at the macroscopic and cellular levels.

Adaptive regulation of virulence genes by microRNA-like RNAs in Valsa mali.

MicroRNAs play important roles in the regulation of gene expression in plants and animals. However, little information is known about the action mechanism and function of fungal microRNA-like RNAs (milRNAs). In this study, combining deep sequencing, molecular and histological assays, milRNAs and their targets in the phytopathogenic fungus Valsa mali were isolated and identified. A critical milRNA, Vm-milR16, was identified to adaptively regulate the expression of virulence genes. Fourteen isolated milRNAs showed high expression abundance. Based on the assessment of a pathogenicity function of these milRNAs, Vm-milR16 was found to be a critical milRNA in V. mali by regulating sucrose non-fermenting 1 (VmSNF1), 4,5-DOPA dioxygenase extradiol (VmDODA), and a hypothetical protein (VmHy1). During V. mali infection, Vm-milR16 is downregulated, while its targets are upregulated. Overexpression of Vm-milR16, but not mutated Vm-milR16, significantly reduces the expression of targets and virulence of V. mali. Furthermore, deletion of VmSNF1, VmDODA and VmHy1 significantly reduce virulence of V. mali. All three targets seem to be essential for oxidative stress response and VmSNF1 is required for expression of pectinase genes during V. mali-host interaction. Our results demonstrate Vm-milRNAs contributing to the infection of V. mali on apple trees by adaptively regulating virulence genes.

Investigation on the Antifungal Ingredients of Saccharothrix Yanglingensis Hhs.015, an Antagonistic Endophytic Actinomycete Isolated from Cucumber Plant.

Abstract: Apple tree canker infected by Valsa mali var. mali is a serious and widely distributed disease in China. Saccharothrix yanglingensis Hhs.015 is an endophytic actinomycete isolated from cucumber roots, and it has been proven that this strain is a promising biocontrol agent on apple tree canker in previous studies. The aim of this study was to elucidate the active ingredients in its metabolites. Two pentaene macrolides, WH01 and WH02, were isolated from strain Hhs.015, and their structures were elucidated based on the extensive spectroscopic analysis. WH01 and WH02 were identified as fungichromin and 1'-deoxyfungichromin, among which WH02 is a novel compound. These two compounds showed strong in vitro and in vivo antifungal activity against V. mali. By comparison of the structures of hyphae cells treated by pure compound and fermentation broth, it has been proven that pentaene macrolides are the main active ingredients in the metabolites of strain Hhs.015. This is the first report on the antifungal activity of fungichromin and its analogs on V. mali, and the 28-member pentaene macrolides were also firstly isolated from the genus of Saccharothrix.

Two transcription factors, AcREM14 and AcC3H1, enhance the resistance of kiwifruit Actinidiachinensis var. chinensis to Pseudomonas syringae pv. actinidiae.

Kiwifruit bacterial canker is a global disease caused by Pseudomonas syringae pv. actinidiae (Psa), which poses a major threat to kiwifruit production worldwide. Despite the economic importance of Actinidia chinensis var. chinensis, only a few resistant varieties have been identified to date. In this study, we screened 44 kiwifruit F1 hybrid lines derived from a cross between two A. chinensis var. chinensis lines and identified two offspring with distinct resistance to Psa: resistant offspring RH12 and susceptible offspring SH14. To identify traits associated with resistance, we performed a comparative transcriptomic analysis of these two lines. We identified several highly differentially expressed genes (DEGs) associated with flavonoid synthesis, pathogen interactions, and hormone signaling pathways, which play essential roles in disease resistance. Additionally, using weighted gene co-expression network analysis, we identified six core transcription factors. Moreover, qRT-PCR results demonstrated the high expression of AcC3H1 and AcREM14 in Psa-induced highly resistant hybrid lines. Ultimately, Overexpression of AcC3H1 and AcREM14 in kiwifruit enhanced disease resistance, and this was associated with upregulation of enzymatic activity and gene expression in the salicylic acid (SA) signaling pathway. Our study elucidates a molecular mechanism underlying disease resistance in kiwifruit and contributes to the advancement of research on kiwifruit breeding.

Interfacial engineering of CoP/CoS2 heterostructure for efficiently electrocatalytic pH-universal hydrogen production.

The design and development of high-performance, low-cost catalysts with long-term durability are crucial for hydrogen generation from water electrolysis. Interfacial engineering is an appealing strategy to boost the catalytic performance of electrode materials toward hydrogen evolution reaction (HER). Herein, we report a simple phosphidation followed by sulfidation treatment to construct heterogeneous cobalt phosphide-cobalt sulfide nanowire arrays on carbon cloth (CoP/CoS2/CC). When evaluated as catalysts toward the HER, the resultant CoP/CoS2/CC exhibits efficient pH-universal hydrogen production due to the heterostructure, synergistic contribution of CoP and CoS2, and conductive substrate. To attain a current density of 10 mA cm-2, overpotentials of only 111.2, 58.1, and 182.9 mV for CoP/CoS2/CC are required under alkaline, acidic, and neutral conditions, respectively. In particular, the as-prepared CoP/CoS2/CC shows markedly improved HER electroactivity in 1.0 M KOH, even outperforming commercial Pt-C/CC at a current density of >50 mA cm-2. In addition, the self-assembled CoP/CoS2||NiFe layered double hydroxide electrolyzer demonstrates efficient catalytic performance and long-time stability, excelling the benchmark Pt-C||IrO2. These findings indicate an effective pathway for the fabrication of high-performance heterogeneous electrocatalysts for hydrogen production in the future.

Advances in Cardiac Organoids.

Cardiovascular diseases rank as the leading cause of death worldwide and are a major contributor to disability, posing a significant threat to human health. Organoids offer a partial simulation of the structure and function of the tissue of origin. It is a promising model that can supplement the disadvantages of two-dimensional culture and animal models. Due to the complexity of heart development, the research of cardiac organoids is still maturing. The advancement of technology has helped address certain challenges, but it has also unveiled new issues and complexities. This paper summarizes the application of organoids technology in the cardiovascular field, the common construction methods of cardiac organoids, and the latest progress of cardiac organoids in the fields of disease model construction, cardiac development research, drug research, and regenerative medicine. The future development and challenges of cardiac organoids are also addressed.

Chemoproteomic Profiling Reveals the Mechanism of Bile Acid Tolerance in Bacteria.

Bile acids (BAs) are a class of endogenous metabolites with important functions. As amphipathic molecules, BAs have strong antibacterial effects, preventing overgrowth of the gut microbiota and defending the invasion of pathogens. However, some disease-causing pathogens can survive the BA stress and knowledge is limited about how they develop BA tolerance. In this work, we applied a quantitative chemoproteomic strategy to profile BA-interacting proteins in bacteria, aiming to discover the sensing pathway of BAs. Using a clickable and photo-affinity BA probe with quantitative mass spectrometry, we identified a list of histidine kinases (HKs) of the two-component systems (TCS) in bacteria as the novel binding targets of BA. Genetic screening revealed that knocking out one specific HK, EnvZ, renders bacteria with significant sensitivity to BA. Further biochemical and genetic experiments demonstrated that BA binds to a specific pocket in EnvZ and activates a downstream signaling pathway to help efflux of BA from bacteria, resulting in BA tolerance. Collectively, our data revealed that EnvZ is a novel sensor of BA in bacteria and its associated TCS signaling pathway plays a critical role in mediating bacterial BA tolerance, which opens new opportunities to combat BA-tolerating pathogens.

Role of the Type VI Secretion System in the Pathogenicity of Pseudomonas syringae pv. actinidiae, the Causative Agent of Kiwifruit Bacterial Canker.

The type VI secretion system (T6SS), a macromolecular machine, plays an important role in the pathogenicity of many Gram-negative bacteria. However, the role of T6SS in the pathogenicity of Pseudomonas syringae pv. actinidiae (Psa), the pathogen of kiwifruit bacterial canker, is yet to be studied. Here, we found a T6SS gene cluster consisting of 13 core genes (A-J) in the genome of Psa M228 based on a genome-wide analysis. To determine whether the T6SS gene cluster affects the pathogenicity of Psa M228, T6SS and its 13 core gene deletion mutants were constructed and their pathogenicity was determined. The deletion mutants showed different degrees of reduction in pathogenicity compared with the wild-type strain M228; in tssM and tssJ mutants, pathogenicity was significantly reduced by 78.7 and 71.3%, respectively. The pathogenicity results were also confirmed by electron microscopy. To further confirm that the reduction in pathogenicity is related to the function of T6SS, we selected the T6SS gene cluster, comprising tssM and tssJ, for further analyses. Western blot results revealed that tssM and tssJ were necessary for hemolytic co-regulatory protein secretion, indicating that they encode a functional T6SS. Further, we explored the mechanism by which T6SS affects the pathogenicity of Psa M228. The ability of bacterial competition, biofilm formation, hydrogen peroxide tolerance, and proteolytic activity were all weakened in the deletion mutants M228ΔT6SS, M228ΔtssM, and M228ΔtssJ. All these properties of the two gene complementation mutants were restored to the same levels as those of the wild-type strain, M228. Quantitative real-time results showed that during the interaction between the deletion mutant M228ΔT6SS and the host, expression levels of T3SS transcriptional regulatory gene hrpR, structural genes hrpZ, hrcC, hopP1, and effector genes hopH1 and hopM1 were down-regulated at different levels. Taken together, our data provide evidence for the first time that the T6SS plays an important role in the pathogenicity of Psa, probably via effects on bacterial competition, biofilm formation, and environmental adaptability. Moreover, a complicated relationship exists between T6SS and T3SS.

FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis.

The Diels-Alder reaction is one of the most powerful and widely used methods in synthetic chemistry for the stereospecific construction of carbon-carbon bonds. Despite the importance of Diels-Alder reactions in the biosynthesis of numerous secondary metabolites, no naturally occurring stand-alone Diels-Alderase has been demonstrated to catalyse intermolecular Diels-Alder transformations. Here we report a flavin adenine dinucleotide-dependent enzyme, Morus alba Diels-Alderase (MaDA), from Morus cell cultures, that catalyses an intermolecular [4+2] cycloaddition to produce the natural isoprenylated flavonoid chalcomoracin with a high efficiency and enantioselectivity. Density functional theory calculations and preliminary measurements of the kinetic isotope effects establish a concerted but asynchronous pericyclic pathway. Structure-guided mutagenesis and docking studies demonstrate the interactions of MaDA with the diene and dienophile to catalyse the [4+2] cycloaddition. MaDA exhibits a substrate promiscuity towards both dienes and dienophiles, which enables the expedient syntheses of structurally diverse natural products. We also report a biosynthetic intermediate probe (BIP)-based target identification strategy used to discover MaDA.