Zhongkang Wang

Ovarian cancer is the most frequent cause of death among gynecological cancers. In the present study, the anti‑cancer effect of liriopesides B, a steroidal saponin from Liriope spicata var. prolifera, against A2780 cells was investigated. Transwell chambers were adopted to assess its effect on cell invasion and chemotaxis abilities. Flow cytometry was used to analyze the cell cycle and apoptosis. Reverse transcription‑quantitative PCR was employed to examine gene expression levels. Western blot analysis was performed to detect protein expression levels. Liriopesides B inhibited the invasion and chemotactic movement ability of A2780 cells in a dose‑dependent manner. Furthermore, liriopesides B caused cell cycle arrest in A2780 cells at the G1 phase following incubation for 24, 48 and 72 h. Hoechst 33258 staining indicated that, following incubation for 48 h, liriopesides B induced cell apoptosis in a dose‑dependent manner. Flow cytometry verified that liriopesides B induced apoptosis in A2780 cells and induced late apoptosis in a dose‑dependent manner. Furthermore, liriopesides B significantly increased the mRNA expression levels of E‑CADHERIN, p21 and p27 and decreased the gene expression levels of BCL‑2, which was consistent with its protein expression levels. In conclusion, liriopesides B possess anti‑cancer properties, including inhibition of metastasis‑associated behaviors, cell cycle arrest and induction of apoptosis. Therefore, liriopesides B may be considered as a candidate drug against ovarian cancer.

Summary: Microsclerotia ( MS ) are pseudoparenchymatous aggregations of hyphae of fungi that can be induced in liquid culture for biocontrol applications. Previously, we determined that the high‐osmolarity glycerol ( HOG ) signalling pathway was involved in regulating MS development in the dimorphic insect pathogen Metarhizium rileyi . To further investigate the mechanisms by which the signalling pathway is regulated, we characterized the transcriptional factor MrMsn2, a homologue of the yeast C ₂ H ₂ transcriptional factor Msn2, which is predicted to function downstream of the HOG pathway in M. rileyi . Compared with wild‐type and complemented strains, disruption of MrMsn2 increased the yeast‐to‐hypha transition rate, enhanced conidiation capacity and aggravated pigmentation in M. rileyi . The ▵MrMsn2 mutants were sensitive to stress, produced morphologically abnormal clones and had significantly reduced MS formation and decreased virulence levels. Digital expression profiling revealed that genes involved in antioxidation, pigment biosynthesis and ion transport and storage were regulated by MrMsn2 during conidia and MS development. Taken together, our findings confirm that MrMsn2 controlled the yeast‐to‐hypha transition, conidia and MS formation, and virulence.

Internal oxidative stress can trigger microsclerotia (MS) formation of Metarhizium rileyi in liquid culture. Activator protein 1 (AP1) is a transcription factor and an important determinant of the response to oxidative stress. To investigate how M. rileyi responds to internal oxidative stress and how MS development is regulated, the Mrap1 gene was characterized. Mrap1 was highly expressed during periods of invasive hyphal growth and in response to changing culture conditions during MS development. Compared with the wild-type and complemented strains, ΔMrap1 mutants exhibited various defects in aerial hyphal growth, yeast-to-hypha transition, and conidia and MS formation. ΔMrap1 mutants also displayed sensitivity to oxidative stress, were morphologically abnormal, and responded differently to oxidative stress during MS development. ΔMrap1 mutants had significantly reduced conidial (74–82%) and MS (99%) yields. Insect bioassays revealed that ΔMrap1 mutants displayed reduced virulence in topical (43–76%) and injection (45–70%) bioassays. Moreover, the ability of ΔMrap1 mutants to grow out of the cuticle was reduced due to impaired conidiation on the host cadaver. Digital gene expression profiling revealed that genes involved in antioxidation, pigment biosynthesis, and ion transport were regulated by Mrap1 during MS development. Taken together, our results confirm the importance of Mrap1 in vegetative growth, conidia and MS formation, and virulence.

Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0–4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.

Microsclerotia (MS) formation was successfully induced in Metarhizium rileyi (Ascomycetes: Hypocreales) in liquid culture. To optimise the process variables of liquid fermentation, we first used a two-level fraction design to confirm the variables, including inoculum density, initial pH, shaker speed, and temperature, affecting M. rileyi MS production. Three variables were found to be important. Subsequently, a 2³ full factorial central composite design (CCD) and response surface methodology were applied to ascertain the optimal level of each variable. A second-order polynomial was determined and shaker speed and inoculum density were found to be the primary variables affecting MS yields. Finally, we realised and optimised M. rileyi MS submerged fermentation based on previous findings. A maximum MS yields (3.84 × 10⁴ MS/mL) were recorded in submerged fermentation at an initial pH of 5.5, growth temperature of 26°C, inoculum density of 10%, higher aeration rate (150 rpm in the initial 3 days and 200 rpm in the subsequent 3 days), and higher agitation rate of 800 L/h sterile air.

Microsclerotia (MS) formation was successfully induced in Metarhizium rileyi under changing liquid culture conditions. Mitogen-activated protein kinases (MAPKs) play important roles in fungal development and in coordinating many stress responses. To investigate how M. rileyi transduces growth stress and regulates MS differentiation, we characterized the roles of two MAPKs, Hog1- and Slt2-type orthologues, in M. rileyi. Compared with the wild-type strain, the deletion mutants of Mrhog1 (ΔMrhog1) and Mrslt2 (ΔMrslt2) delayed germination and vegetative growth, displayed sensitivities to various stress, and produced morphologically abnormal clones. The ΔMrhog1 and ΔMrslt2 mutants significantly reduced conidial (42–99%) and MS (96–99%) yields. A transcriptional analysis showed that the two MAPKs regulate MS development in a cooperative manner. Insect bioassays revealed that ΔMrhog1 and ΔMrslt2 had decreased virulence levels in topical (36–56%) and injection (78–93%) bioassays. Our results confirmed the roles of MrHog1 and MrSlt2 in sensing growth-related stress and in regulating MS differentiation.

Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; PX. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications.

Polarity proteins cdc24 and bem1 regulate polar growth, cytoskeleton morphogenesis, and the generation of reactive oxygen species (ROS) in filamentous fungi. In this study, the Mrcdc24 and Mrbem1 genes were cloned from Metarhizium rileyi and found to encode 1006 and 562 amino acid proteins, respectively. To determine the functions of Mrcdc24 and Mrbem1, gene-silencing mutants Mrcdc24RNAi, Mrbem1RNAi, and Mrcdc24&Nrbem1RNAi were generated by RNA silencing. The mutants presented significant phenotype changes in hyphal growth, and conidial yields, microsclerotia yields and virulence were reduced. Furthermore, the transcription levels of the MrnoxA and MrnoxR, which regulate ROS generation in the Mrcdc24RNAi and Mrbem1RNAi strains, were reduced. The transcriptional levels of MrracA and Mrcdc42 were significantly reduced in the Mrcdc24RNAi strains. Our results confirmed the crucial role of genes Mrcdc24 and Mrbem1 in hyphal growth, conidiation, microsclerotia formation, ROS generation and virulence.