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Tobacco (Nicotiana tabacum L.) is an important economic crop belonging to family Solanaceae and is widely cultivated in China (Basit 2021). From April to July in 2022, a foliar disease with symptoms similar to grey spot was extensively observed on tobacco in Guangxi Province (24°52′ N, 111°23′ E), China. Field surveys were conducted in 18 towns and the disease incidence was 0.89% to 6.95%. Symptomatic leaves displayed irregular, dark brown lesions surrounded by yellow halos and accompanied with black conidiomata in gray centers (Fig 1A-E). Symptomatic leaves were collected from 54 different tobacco plants. After surface sterilization (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on PDA and incubated at 25°C for 5 days (Fang 2007). Three single-spore isolates, GUCC BZ6-3, GUCC LJ3-4, and GUCC XH1-13 were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GUCC BZ6-3 was used for further study. The colonies on PDA were villiform, greyish (Fig 1F-G). Conidia were abundant, ovoid, with 2-6 transverse septa and 1-2 longitudinal septa 12.60 (9.43 to 14.76) × 4.30 (3.57 to 5.14) μm (n=50) (Fig 1H-S). The morphological features were consistent with Alternaria alstroemeriae E.G. Simmons & C.F. Hill (Simmons 2007; Nishikawa & Nakashima, 2013). The pathogen was confirmed to be A. alstroemeriae by amplification and sequencing of the ITS, GAPDH, LSU, TEF1, and RBP2 genes using primers ITS1/ITS4, gpd1/gpd2, LSU1Fd/LR5, EF1-728F/EF1-986R, and RPB2-5F2/fRPB2-7cR, respectively (Woudenberg 2013). The sequences of the PCR products were deposited in GenBank with accession numbers ON693856 (RBP2), ON714497 (ITS), ON694345 (GAPDH), ON931420 (TEF1) and ON714499 (LSU). BLAST searches of the obtained sequences revealed 99% (565/567 nucleotides), 99% (577/579 nucleotides), 99% (908/911 nucleotides), 99% (238/239 nucleotides), and 99% (751/753 nucleotides) homology with those of A. alstroemeriae in GenBank (MH863036, KP124154, MH874589, KP125072, and KP124765, respectively). Phylogenetic analyses of the sequence data consisted of Bayesian and Maximum likelihood analyses of the combined aligned dataset (MEGA 7.0 and PhyloSuite 1.2.2). The GUCC BZ6-3 in a well-supported cluster with A. alstroemeriae (Fig 2). The pathogen was thus identified as A. alstroemeriae based on morphological characterization and molecular analyses. The pathogenicity of GUCC BZ6-3 was tested through pot assay and carried out three times (Fang 2007). Ten healthy 30-day-old tobacco plants were inoculated by spraying a spore suspension (106 spores·ml−1) of strain GUCC BZ6-3 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The symptoms developed on all inoculated leaves but not on the control. The lesions were first visible 48 h after inoculation, and typical lesions similar to those observed on field plants appeared after 7 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments fulfilled Koch’s postulates. To our knowledge, this is the first report of grey spot disease on tobacco caused by A. alstroemeriae in China. Our findings would be of great importance for the diagnosis and control of the emerging grey spot on tobacco.

Bacillus velezensis GUAL210 was isolated from the rhizosphere of healthy pepper plants growing in high-incidence anthracnose fields in Guizhou, China. GUAL210 could be used as a potential biocontrol agent against pepper anthracnose and other soil-borne diseases. The GUAL210 genome consisted of a single circular chromosome 4,011,788 bp in length, with an average GC content of 46.41%, and did not harbor any plasmids. A total of 4,115 protein-coding genes, 27 rRNAs, 87 tRNAs, and 12 secondary metabolite biosynthesis gene clusters were identified. The products of the gene clusters included bacilysin, surfactin, bacteriocin, bacillaene, terpene, and so on, which might help host plants inhibit pathogens. The two clusters predicted to produce terpene had not typically been found in other Bacillus spp. The findings of this study will provide valuable data to explore the biocontrol mechanisms of B. velezensis strains.

Rhododendron delavayi is an evergreen shrub with large scarlet flowers that make it highly attractive as an ornamental species. The species is native to southwest China (Cai et al. 2015). From May to July in 2022, symptoms of leaf spot were observed on R. delavayi over a wide portion of the Baili Azalea Forest Area (N 27°10'-27°20', E 105°04'-106°04'), Guizhou Province, China. About 500 plants were surveyed and the incidence of leaf spot on R. delavayi leaves was 20 to 30%, significantly reducing their ornamental and economic value. The affected leaves had irregular, dark brown lesions with a clear blackish brown boundary and black conidiomata in a grayish center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GUDJ 61, GUDJ 62, GUDJ 63) that produced identical in morphology were obtained. The isolate GUDJ 61 was used for further study. Colonies on PDA grew velvety white on the upper surface and light yellow on the lower surface. Conidia were 5-celled, spindle- to ellipsoid-shaped, straight or slightly curved, 4-septate, and measured 39.0 ± 3.7 × 10.4 ± 0.79 µm (n=50). The morphological features were consistent with the description of Pestalotiopsis scoparia Maharachch., K.D. HydeCrous, (Maharachchikumbura et al. 2014). The pathogen was confirmed to be P. scoparia by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), and the partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b, and EF1-728F/EF-2, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers OP048045 (ITS), OP058111 (TUB) and OP058114 (TEF), respectively. BLAST searches of the sequences revealed 99% (549/552 nt), 99% (711/714 nt), and 82% (130/158 nt) homology with those of P. scoparia CBS 176.25T form GenBank (KM199330, KM199393, and KM199478), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUDJ 61 in a well-supported cluster with P. scoparia. The pathogen was thus identified as P. scoparia based on the morphological characterization and molecular analyses. The pathogenicity of GUDJ 61was tested through a pot assay. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying a spore suspension (106 spores mL-1) of GUDJ 61 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times (Fang 2007). After 12 days, the treated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. delavayi caused by P. scoparia in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. delavayi.

To carry out genetic diagnosis for a fetus.Chromosome G-banding and chromosomal microarray analysis (CMA) were carried out for a fetus with abnormal morphology of lateral cerebral fissure.The karyotype of the fetus was normal, but CMA showed that it has carried a 1.4 Mb deletion at 17p13.3 region, which suggested a diagnosis of Miller-Dieker syndrome (MDS).Familiarity with clinical features and proper selection of genetic testing method are crucial for the diagnosis of MDS. Attention should be paid to microdeletions and microduplications which can be missed by conventional chromosomal karyotyping.

Background C1orf61 is a specific transcriptional activator that is highly up-regulated during weeks 4–9 of human embryogenesis, the period in which most organs develop. We have previously demonstrated that C1orf61 acts as a tumor activator in human hepatocellular carcinoma (HCC) tumorigenesis and metastasis. However, the underlying molecular mechanisms of tumor initiation and progression in HCC remain obscure. Methods In this study, we demonstrated that the pattern of C1orf61 expression was closely correlated with metastasis in liver cancer cells. Gene expression profiling analysis indicated that C1orf61 regulated diverse genes related to cell growth, migration, invasion and epithelial-mesenchymal transition (EMT). Results Results showed that C1orf61 promotes hepatocellular carcinoma metastasis by inducing cellular EMT in vivo and in vitro. Moreover, C1orf61-induced cellular EMT and migration are involved in the activation of the STAT3 and Akt cascade pathways. We also found that C1orf61 was associated with HBV infection-induced cell migration in HCC. In addition, C1orf61 expression improved the efficacy of the anticancer therapy sorafenib in HCC patients. For the first time, we report a regulatory pathway by which C1orf61 promoted cancer cell metastasis and regulated the therapeutic response to sorafenib. Conclusions These findings increased our understanding of the molecular events that regulate metastasis and treatment in HCC.

The identification and development of new cytoplasmic male sterility (CMS) lines in higher plants is important for the preservation of grain security and the prevention of homogenization of hybrid rice. Molecular markers assisted selection (MAS) based on CMS-associated genes or mitochondrial-specific chimeric sequences are important for rapid and effective breeding of new CMS lines and hybrids. In our study, the distribution and allele variation of orfH79 and orf290 genes were characterized from 273 wild and cultivated rice in the AA genome species. Based on the alignment of nucleotide and amino acid sequences, four accessions with orfH79 and three accessions with orf290 were screened. Four novel CMS lines carrying orfH79 haplotypes and three novel CMS lines carrying orf290 haplotypes were then developed using multiple backcross generations with a maintainer line under MAS. The breeding process used in our study provides an efficient and feasible approach for selecting new CMS lines. CMS lines selected in our study are important for enriching rice germplasm resources and guaranteeing rice breeding programs.

Background Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus. Results Based on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets. Conclusion A rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research.

Calmodulin-like proteins (CMLs) have been shown to play key regulatory roles in calcium signaling in plants. However, few bona-fide CMLs binding proteins have been characterized in rice, a monocot model plant. Here, through large-scale screening of a yeast-two hybrid (Y2H) cDNA library with OsCML16 as a bait, six new putative interacting partners of OsCML16 were discovered and confirmed by both pairwise Y2H and bimolecular fluorescence complementation (BiFC) assays. Interestingly, the in vitro peptide-binding assays manifested that OsERD2 could bind both OsCaM1 and OsCML16 whereas other five target proteins could specifically bind OsCML16 but not OsCaM1. Furthermore, Ca2+ and TFP, a calmodulin (CaM) antagonist, were involved in the ABA-induced transcription of OsCML16 and its target genes, and they were also obviously induced by cold, drought, and salt stresses. Taken together, our new findings have provided the basis for the novel signaling pathways of OsCML16 in the abiotic stress response in rice.

Due to its unique natural conditions, the Wuling Mountain region is a key area of prevention and control of rice blast disease in China and an ideal area for investigation of its causal fungus, Magnaporthe oryzae (M. oryzae) research. A total of 237 M. oryzae isolates collected from the Wuling Mountain region in 2017–2018 were used for the study of genetic diversity and pathogenicity dynamics. A set of differential varieties (DVs), including 7 Chinese DVs, 12 Japanese DVs and 21 monogenic lines, were established, which differentiate the 237 isolates in this study into different physiological races. Cluster analysis of the combined molecular marker system (RAPD, REMAP, rep-PCR and the avirulence (Avr) gene) results showed that Baiyang and Jianshi had the greatest genetic diversity, which is consistent with the pathogenic results. The proportion of the two mating types was roughly equal each year; however, the dominance of the mating type between the two years was opposite. Furthermore, four isolates contained both mating types, which were assumed to be dual maters. Isolates with amplified bands of Avr genes (Avr-Pizt and Avr-Pita) and corresponding pathogenicity were selected for Sanger sequencing. The result showed that point mutations and small indels resulted in the loss of avirulence function, indicating that mutations are potential mechanism for the rich pathogenic and genetic diversity of M. oryzae in the Wuling Mountain region.

Background Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus. Results Based on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets. Conclusion A rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research.

Additional file 4. Primers used in this study.

Additional file 3. PCR amplification for transgenic-positive identification in calli. a Amplification of DsRed from calli of Jia 9709, Jia 2016, Zhong shuang 8, Zhong shuang 11 and Zhong you 821, respectively. P, plasmid. WT, wild type plant. Marker, DL 100 bp ladder. b Amplification of BnaA07g17400D from calli in 7633, B 351 and Shan 3B, respectively. P, plasmid. N, wild type plant. Marker, DL 2000 bp.

Additional file 1. Formation of calli to a regenerated plantlet on a hypocotyl. a and b calli that were induced from hypocotyls. c Calli that were prepared for swollen to shoots. d and e Shoot formation and cotyledon development accompanied by the appearance of a growth point.

Additional file 2. Visual screening of transgenic-positive and/or -negative tissues under a fluorescence stereomicroscope. Red fluorescence could be observed under fluorescence stereomicroscope to facilitate visual screening in tissue culture medium. The image was captured in bright field a and its corresponding dark field b, respectively. The tissues indicated by the arrow represent transgenic-positive calli.

C1orf61 is a specific transcriptional activator that is highly up-regulated during weeks 4-9 of human embryogenesis, the period in which most organs develop. We have previously demonstrated that C1orf61 acts as a tumor activator in human hepatocellular carcinoma (HCC) tumorigenesis and metastasis. However, the underlying molecular mechanisms of tumor initiation and progression in HCC remain obscure. In this study, we demonstrated that the pattern of C1orf61 expression was closely correlated with metastasis in liver cancer cells. Gene expression profiling analysis indicated that C1orf61 regulated diverse genes related to cell growth, migration, invasion and epithelial-mesenchymal transition (EMT). Results showed that C1orf61 promotes hepatocellular carcinoma metastasis by inducing cellular EMT in vivo and in vitro. Moreover, C1orf61-induced cellular EMT and migration are involved in the activation of the STAT3 and Akt cascade pathways. In addition, C1orf61 expression improved the efficacy of the anticancer therapy sorafenib in HCC patients. For the first time, we report a regulatory pathway by which C1orf61 promoted cancer cell metastasis and regulated the therapeutic response to sorafenib. These findings increased our understanding of the molecular events that regulate metastasis and treatment in HCC.

Post-translation modification through the SUMO pathway is cell cycle regulated, with specific SUMO conjugates accumulating in mitotic cells. The basis for this regulation, however, and its functional significance remain poorly understood. We present evidence that in budding yeast sumoylation during mitosis may be controlled through the SUMO deconjugating enzyme Smt4/Ulp2. We isolated the polo kinase Cdc5 as an Ulp2-interacting protein, and find a C-terminal region of Ulp2 is phosphorylated during mitosis in a Cdc5-dependent manner. cdc5 mutants display reduced levels of mitotic SUMO conjugates, suggesting Cdc5 may negatively regulate Ulp2 to promote sumoylation. Previously, we found one phenotype associated with ulp2 mutants is an inability to maintain chromatid cohesion at centromere-proximal chromosomal regions. We now show this defect is rescued by inactivating Cdc5, indicating Ulp2 maintains cohesion by counter-acting Cdc5 activity. The cohesinregulator Pds5 is a likely target of this pathway, as Cdc5 overproduction forces Pds5 dissociation from chromosomes and Pds5 overproduction restores cohesion in ulp2 mutants. Overall, these observations reveal Cdc5 is a novel regulator of the SUMO pathway and suggest the outlines of a broader circuitry in which Ulp2 and Cdc5 act in a mutually antagonistic fashion to modulate maintenance and dissolution of cohesion at centromeres.

Spindle disruption or DNA damage prevents sister chromatid separation through the activation of checkpoint pathways that inhibit anaphase entry by stabilizing the anaphase inhibitor Pds1. Mutation of CDC55 , which encodes a B regulatory subunit of protein phosphatase 2A (PP2A), results in precocious sister chromatid separation when spindle is disrupted. Here we report that decreased Pds1 levels in Δcdc55 mutants contribute to sister chromatid separation in the presence of nocodazole, a microtubule-depolymerizing drug. However, in the presence of DNA damage, Δcdc55 mutant cells separate sister chromatids without noticeable decrease of Pds1 or cohesin Mcd1/Scc1 levels. Further analysis demonstrates that Δcdc55 mutants lose cohesion along the entire chromosomes when the spindle is disrupted. In contrast, separation of sister chromatids is limited to the centromeric regions in Δcdc55 cells after DNA damage. Moreover, mutation of TPD3 , which encodes the A regulatory subunit of PP2A, also results in sister chromatid separation in DNA- or spindle-damage-arrested cells. These data suggest that PP2A regulates sister chromatid cohesion in Pds1-dependent and -independent manners.

Extracellular acidosis is involved in various pathological situations of central nervous system and the effects are largely mediated by acid sensing ion channels (ASICs). However, it remains unclear whether extracellular acidosis affects immune cells. Macrophages are immune cells that play important role in immune reactions. In this study we investigated the impact of extracellular acidosis on the function of bone marrow derived macrophages (BMMs). The results showed that extracellular acidosis upregulated the endocytosis, surface molecular expression and interleukin-10 secretion of BMMs, in which the expression of ASIC1 and ASIC3 was detected. Notably, extracellular acidosis stimulated endocytosis and upregulation of surface molecules expression in BMMs could be abolished by amiloride, a blocker of ASICs, and nonsteroid anti-inflammatory drugs. Our findings provide new insight into the role of extracellular acidosis in the regulation of immune function and suggest ASICs as new targets for the modulation of immune response.

During mitosis in budding yeast, cortically anchored dynein exerts pulling forces on cytoplasmic microtubules, moving the mitotic spindle into the mother-bud neck. Anchoring of dynein requires the cortical patch protein Num1, which is hypothesized to interact with PI(4,5)P(2) via its C-terminal pleckstrin homology (PH) domain. Here we show that the PH domain and PI(4,5)P(2) are required for the cortical localization of Num1, but are not sufficient to mediate the cortical assembly of Num1 patches. A GFP fusion to the PH domain localizes to the cortex in foci containing approximately 2 molecules, whereas patches of full-length Num1-GFP contain approximately 14 molecules. A membrane targeting sequence containing the CAAX motif from the yeast Ras2 protein can compensate for the PH domain to target Num1 to the plasma membrane as discrete patches. The CAAX-targeted Num1 exhibits overlapping but largely distinct localization from wild-type Num1. However, it is fully functional in the dynein pathway. More importantly, cortical PI(4,5)P(2) is dispensable for the localization and function of the CAAX-targeted Num1. Together, these results demonstrate that cortical assembly of Num1 into functional dynein-anchoring patches is independent of its interaction with PI(4,5)P(2).

Herpes simplex virus type 2 (HSV-2) infects the genital epithelium and can be transmitted to the central nervous system, establishing lifelong infection. Toll-like receptors (TLRs) have been reported to initiate innate immune responses to microorganisms. It has been shown that TLR2 and TLR9 are involved in recognition of different HSV strains by dendritic cells (DCs). Our previous study has shown that HSV-2 infection leads to dramatic activation of the NF-κB signaling pathway and induction of IL-6 and IFN-beta. We report here that HSV-2 activates NF-κB reporter and TLR4/MD2 augments this activation. The NF-κB reporter construct was co-transfected into human cervical epithelial (HCE) cells together with either vectors or TLR4/MD2 expression plasmids. The transfected cells were then infected with HSV-2 at 3 MOI and luciferase activity was measured and normalized to Renilla luciferase activity. We observed a relatively low, but reproducible induction (approximately 1.5-fold) of reporter activity in HCE cells with HSV-2 infection compared to that in mock-infected cells. Importantly, an augmented induction of the NF-κB reporter was seen while cells were co-transfected with TLR4/MD2 in HSV-2 infected cells. Thus, TLR4 potentially plays a role in mediating its activation. Effect of shRNA against TLR4 on this induction is under active investigation. This work was supported by grants from the National Natural Science Foundation of China (No. 30500465, 30810103052)