Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

The eTM-miR858-MYB62-like module regulates anthocyanin biosynthesis under low-nitrogen conditions in Malus spectabilis.

The anthocyanin content increases in Malus spectabilis leaves under low-nitrogen conditions. Noncoding RNAs are indicated to play key regulatory roles in anthocyanin biosynthesis. However, the functional roles of noncoding RNAs in anthocyanin biosynthesis under low-nitrogen conditions remain elusive. In this study, miR858 was screened as a key regulator of anthocyanin biosynthesis under low-nitrogen conditions through whole-transcriptome sequencing. Then, we used miR858 as an entry point to explore the regulatory network of lncRNA-miRNA-mRNA by dual-luciferase reporter assays and GUS histochemical staining assays, as well as to identify the mechanism of this regulatory network in anthocyanin biosynthesis by both transient and stable transformation experiments in Malus. MiR858 overexpression increased total anthocyanin content. MiR858 acted by negatively regulating its target gene, MsMYB62-like, under the low-nitrogen condition. MsMYB62-like inhibited the expression of MsF3'H, thereby negatively regulating anthocyanin biosynthesis. In addition, eTM858-1 and eTM858-2 were identified as endogenous target mimics of miR858 that bind to miR858 to prevent cleavage of MsMYB62-like and thereby negatively regulate anthocyanin biosynthesis. The results clarify the mechanism through which the eTM-miR858-MYB62-like module regulates anthocyanin biosynthesis in Malus under low-nitrogen conditions.

First report of Leaf Blight of Liquidambar formosana Hance caused by Alternaria tenuissima in China.

Liquidambar formosana Hance is widely planted in urban landscapes in China owing to its ornamental red leaves. In June 2020, a distinctive leaf spot disease was observed on L. formosana in Nanjing Forestry University, Jiangsu Province of China (32°4'49"N, 118°48'56"E). Approximately 61% (14 out of 23) of the trees displayed leaf spots. The diseased symptoms included irregularly distributed spots that showed black or dark brown, and occasionally with pale green halo. Two representative trees were selected for sampling and five leaves with typical symptoms were selected randomly for isolation. The tissues from the margin of the lesions (0.2 cm × 0.2 cm) were cut and disinfected in 1% sodium hypochlorite for 90 s, rinsed in sterile water twice for 30 s, and dried with sterile paper. Then, 20 tissues were incubated on 2% potato dextrose agar (PDA) supplemented with 100 mg/L Ampicillin Sodium and incubated in the dark at 25℃ for 4 days. Seventeen single-spore fungi were isolated from lesion tissues as described by Woudenberg et al. (2013). The colony morphology of 17 isolates was extremely similar, so 3 isolates (NFUA01, NFUA02, and NFUA03) were selected randomly for further study. Colonies on PDA were circular, gray, and slightly raised loose cotton mycelium, while the reverse side was olive green in the center with white margins. Conidiophores were brown, simple or branched, and produced numerous conidia in short chains. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 7.1 to 32.5 × 3.3 to 13.3 µm (n=50). The morphological observations were consistent with the description of the genus Alternaria sp. (Woudenberg et al. 2013). Six gene fragments, including SSU, LSU, ITS, GAPDH, RPB2 and EF-1 region, were amplified and sequenced. The primers of six nuclear loci were used by NS1 / NS4((White et al. 1990), LSU1Fd (Crous et al. 2009)/ LR5 (Vilgalys & Hester 1990), V9G (De Hoog & Gerrits van den Ende 1998)/ ITS4 (White et al. 1990), gpd1 / gpd2 (Berbee et al. 1999), RPB2-5F2 / fRPB2-7cR (Liu et al. 1999), and EF1-728F / EF1-986R (Carbone & Kohn 1999). The sequences were submitted in GenBank (SSU, ON237470 to ON237472; LSU, ON237464 to ON237466; ITS, ON197354 to ON197356; GAPDH, ON237476 to ON237478; RPB2, ON237467 to ON237469; EF-1, ON237473 to ON237475). BLAST result showed that SSU, LSU, ITS, GAPDH, RPB2, and EF-1 sequences of NFUA01, NFUA02, and NFUA03 were identical to A. tenuissima at a high level (>99%, Table 1). A maximum likelihood and Bayesian posterior probability analysis were performed by IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (Guindon et al. 2010; Ronquist et al. 2012). The representative strains which selected for Phylogenetic analyses were chosen from the strains which mentioned by Woudenberg et al (2013) and obtained the sequences from NCBI. The concatenated sequences placed NFUA01, NFUA02 and NFUA03 in the clade of Alternaria tenuissima with a high confidence level (ML/BI= 100/1). A pathogenicity assay was done using isolate NFUA01 on 3-year-old L. formosana seedlings. L. formosana leaves were wounded by a sterilized needle (0.5-mm-diam), and inoculated with spore suspension (106 conidia/mL), and L. formosana leaves inoculated with sterile water were used as the control. Each treatment had 5 leaves, and incubated at 25℃ under high moisture conditions. The experiments were conducted three times. Seven days after inoculation, leaves inoculated with spore suspension showed brown leaf blights resembling the original disease symptoms, whereas the control remained healthy. The fungus was reisolated from the lesions and was confirmed as A. tenuissima based on morphologically characteristics and ITS sequence analysis. To our knowledge, this is the first report of A. tenuissima associated with leaf blight on L. formosana. The finding provides clear pathogen information for further evaluation of the disease control strategies.

WITHDRAWN: Modulation of adipocyte function by the TGF-ß family.

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A Review of Temperature Effects on Membrane Filtration.

Membrane technology plays a vital role in drinking water and wastewater treatments. Among a number of factors affecting membrane performance, temperature is one of the dominant factors determining membrane performance. In this review, the impact of temperature on membrane structure, fouling, chemical cleaning, and membrane performance is reviewed and discussed with a particular focus on cold temperature effects. The findings from the literature suggest that cold temperatures have detrimental impacts on membrane structure, fouling, and chemical cleaning, and thus could negatively affect membrane filtration operations and performance, while warm and hot temperatures might expand membrane pores, increase membrane flux, improve membrane chemical cleaning efficiency, and interfere with biological processes in membrane bioreactors. The research gaps, challenges, and directions of temperature effects are identified and discussed indepth. Future studies focusing on the impact of temperature on membrane processes used in water and wastewater treatment and the development of methods that could reduce the adverse effect of temperature on membrane operations are needed.

Species of the Colletotrichum spp., the Causal Agents of Leaf Spot on European Hornbeam (Carpinus betulus).

European hornbeam (Carpinus betulus L.) is widely planted in landscaping. In October 2021 and August 2022, leaf spot was observed on C. betulus in Xuzhou, Jiangsu Province, China. To identify the causal agent of anthracnose disease on C. betulus, 23 isolates were obtained from the symptomatic leaves. Based on ITS sequences and colony morphology, these isolates were divided into four Colletotrichum groups. Koch's postulates of four Colletotrichum species showed similar symptoms observed in the field. Combining the morphological characteristics and multi-gene phylogenetic analysis of the concatenated sequences of the internal transcribed spacer (ITS) gene, Apn2-Mat1-2 intergenic spacer (ApMat) gene, the calmodulin (CAL) gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene, Glutamine synthetase (GS) gene, and beta-tubulin 2 (TUB2) genes, the four Colletotrichum groups were identified as C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense. This study is the first report of four Colletotrichum species causing leaf spot on European hornbeam in China, and it provides clear pathogen information for the further evaluation of the disease control strategies.

Cyclocarya paliurus Reprograms the Flavonoid Biosynthesis Pathway Against Colletotrichum fructicola.

Cyclocarya paliurus is an endemic Chinese tree species with considerable medicinal, timber, and horticultural value. The anthracnose disease of C. paliurus is caused by the fungal pathogen Colletotrichum fructicola, which results in great losses in yield and quality. Here, resistance evaluation of six cultivars of C. paliurus exhibited varying degrees of resistance to C. fructicola infection, where Wufeng was the most resistant and Jinggangshan was the most susceptive. Physiological measurements and histochemical staining assays showed that the Wufeng cultivar exhibits intense reactive oxygen species accumulation and defense capabilities. A multiomics approach using RNA sequencing and metabolome analyses showed that resistance in C. paliurus (Wufeng) is related to early induction of reprogramming of the flavonoid biosynthesis pathway. In vitro antifungal assays revealed that the flavonoid extracts from resistant cultivars strongly inhibited C. fructicola hyphal growth than susceptible cultivars. Relative gene expression analysis further demonstrated the pivotal antifungal role of C. paliurus flavonoids in targeting Colletotrichum appressorium formation. Together, these results represent a novel resistance mechanism of C. paliurus against anthracnose through the reprogramming of flavonoids, which will lay a foundation for breeding anthracnose-resistant varieties and the application of flavonoid extraction of C. paliurus as a natural antifungal treatment.

MrMYB44-Like Negatively Regulates Anthocyanin Biosynthesis and Causes Spring Leaf Color of Malus 'Radiant' to Fade From Red to Green.

The "Spring-red-leaf" crabapple cultivar has young red leaves and mature green leaves. However, the mechanism of anthocyanin biosynthesis in crabapple leaves in spring remains unknown. In this study, Illumina RNA sequencing (RNA-Seq) was performed on Malus 'Radiant' leaf tissues in different stages of development. Twenty-two genes in the anthocyanin biosynthesis pathway and 44 MYB transcription factors (TFs) were significantly enriched among differentially expressed genes (DEGs). Three R2R3-MYB TFs in subgroup 22 of the MYB TF family, MrMYB44-like1, MrMYB44-like2, and MrMYB44-like3, were highly expressed in green leaves according to RNA-Seq and quantitative real-time quantitative PCR results. Their expression levels were negatively correlated with anthocyanin content. In transient assays, overexpression of MrMYB44-like1, MrMYB44-like2, or MrMYB44-like3 inhibited anthocyanin accumulation and reduced pigment in leaf disks of M. 'Radiant' and fruit peels of M. domestica 'Fuji.' When the conserved region of the three MrMYB44-likes was silenced, the anthocyanin biosynthesis pathway was activated and pigments increased in both tissues. Moreover, bimolecular fluorescence complementation assays showed MrMYB44-likes interacted with MrWRKY6 to form protein complexes that regulated anthocyanin biosynthesis.

Modulation of the cellular content of metabolites in adipocytes by insulin.

Although the insulin-mediated cell signaling pathway has been extensively examined, changes in the cellular content of metabolites currently remain unclear. We herein examined metabolite contents in 3T3-L1 adipocytes treated with insulin using a metabolomic analysis. Fifty-four compounds were detected, and the contents of metabolites from the citric acid cycle increased in response to the insulin treatment for 4 h, which was sensitive to U0126 and LY294002, inhibitors for mitogen-activated protein kinase kinase-1 and phosphoinositide 3-kinase, respectively. The cellular contents of fumaric acid and malic acid were increased more by insulin than those of citric acid and succinic acid. Time-course changes in metabolites from the citric acid cycle exhibited oscillations with a 2-h cycle. A metabolic pathway analysis also indicated that insulin affected the metabolism of alanine, aspartate and glutamate, as well as that of arginine and proline. The contents of free amino acids were slightly decreased by the insulin treatment, while the co-treatment with U0126 and LY294002 abrogated these insulin-mediated decreases. The present study revealed the unexpected accumulation of citric acid cycle metabolites in adipocytes by insulin. Our results indicate the usefulness of metabolomic analyses for obtaining a more comprehensive understanding of the regulation of metabolic pathways in cell-culture systems.

Effects of vitamin a status on expression of ucp1 and brown/beige adipocyte-related genes in white adipose tissues of beef cattle.

We previously reported the presence of brown/beige adipocytes in the white fat depots of mature cattle. The present study examined the effects of dietary vitamin A on the expression of brown/beige adipocyte-related genes in the white fat depots of fattening cattle. No significant differences were observed in the expression of Ucp1 between vitamin A-deficient cattle and control cattle. However, the expression of the other brown/beige adipocyte-related genes was slightly higher in the mesenteric fat depots of vitamin A-deficient cattle. The present results suggest that a vitamin A deficiency does not markedly affect the expression of Ucp1 in white fat depots, but imply that it may stimulate the emergence of beige adipocytes in the mesenteric fat depots of fattening cattle.