Decolorization and detoxication of malachite green by engineered Saccharomyces cerevisiae expressing novel thermostable laccase from Trametes trogii.

Malachite Green (MG) is a widely used industrial dye that is hazardous to health. Herein, the decolourisation and detoxification of MG were achieved using the engineered Saccharomyces cerevisiae expressing novel thermostable laccase lcc1 from Trametes trogii. The engineered strain RCL produced a high laccase activity of 121.83 U L-1. Lcc1 was stable at temperatures ranging from 20 ℃ to 60 ℃ and showed a high tolerance to organic solvents. Moreover, Lcc1 could decolorize different kinds of dyes (azo, anthraquinone and triphenylmethane), among which, the decolorization ability of MG is the highest, reaching 95.10 %, and the decolorization rate of other triphenylmethane dyes also over 50 %. The RCL decolorized about 95 % of 50 mg L-1 of MG dye in 10 h at 30 ℃. The MG degradation products were analyzed. The industrial application potential of the RCL was evaluated by treating industrial wastewater and the decolourisation rates were over 90 %.

Biosynthetic mechanism of the yellow pigments in the marine bacterium Pseudoalteromonas sp. strain T1lg65.

The Pseudoalteromonas genus marine bacteria have attracted increasing interest because of their abilities to produce bioactive metabolites. The pigmented Pseudoalteromonas group encodes more secondary metabolite biosynthetic gene clusters (BGCs) than the non-pigmented group. Here, we report a yellow pigmented bacterium Pseudoalteromonas sp. strain T1lg65, which was isolated from a mangrove forest sediment. We showed that the yellow pigments of T1lg65 belong to the group of lipopeptide alterochromides. Further genetic analyses of the alterochromide BGC revealed that the yellow pigments are biosynthesized by aryl-polyene synthases and nonribosomal peptide synthases. Within the gene cluster, altA encodes a tyrosine ammonia acid lyase, which catalyzes synthesis of the precursor 4-hydroxycinnamic acid (4-HCA) from tyrosine in the alterochromide biosynthetic pathway. In addition, altN, encoding a putative flavin-dependent halogenase, was proven to be responsible for the bromination of alterochromides based on gene deletion, molecular docking, and site mutagenesis analyses. In summary, the biosynthetic pathway, precursor synthesis, and bromination mechanism of the lipopeptide alterochromides were studied in-depth. Our results expand the knowledge on biosynthesis of Pseudoalteromonas pigments and could promote the development of active pigments in the future.IMPORTANCEThe marine bacteria Pseudoalteromonas spp. are important biological resources because they are producers of bioactive natural products, including antibiotics, pigments, enzymes, and antimicrobial peptides. One group of the microbial pigments, alterochromides, holds a great value for their novel lipopeptide structures and antimicrobial activities. Previous studies were limited to the structural characterization of alterochromides and genome mining for the alterochromide biosynthesis. This work focused on the biosynthetic mechanism for alterochromide production, especially revealing functions of two key genes within the gene cluster for the alterochromide biosynthesis. On the one hand, our study provides a target for metabolic engineering of the alterochromide biosynthesis; on the other hand, the 4-HCA synthase AltA and brominase AltN show potential in the biocatalyst industry.

Plum blossom low molecular weight polypeptide protects HaCaT cells against UVB-induced oxidative damage in vitro and underlying mechanism.

BACKGROUND: Increasing amounts of ultraviolet radiation occur as ozone depletion causes the earth's ozone layer to be destroyed, making antioxidant efficacy a research hotspot. Previous studies on plum blossom have mostly focused on Volatile Oils, Flavonoids, Phenylpropanoids, and other compounds, whereas few studies have focused on low molecular weight polypeptide (LMWP) of plum blossom. This research provides a reference for the deep processing and utilization of plum blossom. OBJECTIVES: (a) Plum blossom low molecular weight polypeptides protect HaCaT cells against UVB-induced oxidative damage in vitro and the underlying mechanism. (b) Improve the theoretical basis for the intense processing and utilization of plum blossom. METHODS: The safe concentration of LMWP and the survival rate of HaCaT cells were determined using the CCK-8 experiment. The fluorescence intensity of reactive oxygen species (ROS) was identified using the dichlorofluorescin diacetate (DCFH-DA) method; Superoxide dismutase (SOD) and malondialdehyde (MDA) concentrations were measured in ruptured cells; Western blot analysis was used to examine the expression levels of three proteins: nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and benzoquinone oxidoreductase 1 (NQO-1). RESULTS: It was noted that a certain concentration of LMWP could promote cell proliferation. In oxidatively damaged HaCaT cells, SOD levels and survival rates were markedly reduced, but ROS and MDA levels were elevated. However, after treatment with LMWP, the survival rate of the cells and SOD levels were markedly increased, and the levels of ROS and MDA were markedly decreased. As shown by Western blotting, the model group exhibited lower levels of Nrf2, HO-1, and NQO-1 expression than the control group, whereas LMWP-treated cells had significantly higher levels of Nrf2, HO-1, and NQO-1 expression than their model-treated counterparts. CONCLUSIONS: LMMP can effectively protect HaCaT cells against oxidative damage in vitro induced by UVB, and the underlying mechanism is linked to the activation of the transcription factor Nrf2.

Bioconversion of food waste to crayfish feed using solid-state fermentation with yeast.

In order to realize the value-added utilization of food waste (FW), the preparation of crayfish (Procambarus clarkii) feed by yeast fermentation was investigated. Firstly, the suitable fermentation condition was obtained through a single factor experiment as follows: the initial moisture of the FW was adjusted to 60% with bran and inoculated with a 2% yeast mixture (Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica, 3:2:1) followed by aerobic solid-state fermentation for 7 days. The crude protein and acid-soluble protein contents in the fermented feed were 25.14% and 5.16%, which were increased by 8% and 140.67%, respectively. The crude fat content was 0.74%, decreased by 68.29%. The content of antioxidant glutathione (571.78 µg/g) increased 63.33%, and the activities of protease and amylase increased nearly 9 and 3 times, respectively. The maximum degradation rates of aflatoxin B1, zearalenone, and deoxynivalenol were 63.83%, 77.52%, and 80.16%, respectively. The fermented feeds were evaluated by substituting (0%, 10%, 30%, 50%, and 100%) commercial diet for crayfish (30-day culture period). When the replacement proportion was 30%, the weight gain of crayfish reached 44.87% (initial body weight 13.98 ± 0.41 g), which was significantly increased by 10.25% compared with the control (p = 0.0005). In addition, the lysozyme and SOD enzyme activities in crayfish hepatopancreas were also increased significantly. Our findings suggest that yeast-fermented feed from FW can replace 30% of crayfish's conventional diet, which may improve crayfish's antioxidant capacity and enhance non-specific immunity by providing molecules such as glutathione.

Stringent Starvation Protein SspA and Iron Starvation Sigma Factor PvdS Coordinately Regulate Iron Uptake and Prodiginine Biosynthesis in Pseudoalteromonas sp. R3.

Organisms need sufficient intracellular iron to maintain biological processes. However, cells can be damaged by excessive iron-induced oxidation stress. Therefore, iron homeostasis must be strictly regulated. In general, bacteria have evolved complex mechanisms to maintain iron homeostasis. In this study, we showed that Pseudoalteromonas sp. R3 has four sets of iron uptake systems. Among these, the siderophore pyoverdine-dependent iron uptake system and the ferrous iron transporter Feo system are more important for iron uptake and prodiginine biosynthesis. Stringent starvation protein SspA positively controls iron uptake and iron-dependent prodiginine biosynthesis by regulating the expression of all iron uptake systems. In turn, the expression of SspA can be induced and repressed by extracellular iron deficiency and excess, respectively. Interestingly, extracytoplasmic function sigma factor PvdS also regulates iron uptake and prodiginine production and responds to extracellular iron levels, exhibiting a similar phenomenon as SspA. Notably, not only do SspA and PvdS function independently, but they can also compensate for each other, and their expression can be affected by the other. All of these findings demonstrate that SspA and PvdS coordinate iron homeostasis and prodiginine biosynthesis in strain R3. More importantly, our results also showed that SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 have similar functions in iron uptake to their counterparts in Pseudoalteromonas, suggesting that coordination between SspA and PvdS on iron homeostasis could be conserved in typical Gram-negative bacteria. Since master regulation of iron homeostasis is extremely important for cell survival, this cross talk between SspA and PvdS may be environmentally significant. IMPORTANCE Both deficiency and excess of intracellular iron can be harmful, and thus, the iron homeostasis needs to be tightly regulated in organisms. At present, the ferric uptake regulator (Fur) is the best-characterized regulator involved in bacterial iron homeostasis, while other regulators of iron homeostasis remain to be further explored. Here, we demonstrated that the stringent starvation protein SspA and the extracytoplasmic function sigma factor PvdS coordinate iron uptake and iron-dependent prodiginine biosynthesis in Pseudoalteromonas sp. R3. These two regulators work independently, but their functions can compensate for the other and their expression can be affected by the other. Moreover, their expression can be activated and repressed by extracellular iron deficiency and excess, respectively. Notably, SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 exhibit similar functions in iron uptake to their counterparts in Pseudoalteromonas, suggesting that this novel fine-tuned mode of iron homeostasis could be conserved in typical Gram-negative bacteria.

SPARC plays an important role in the oviposition and nymphal development in Nilaparvata lugens Stål.

BACKGROUND: The brown planthopper (Nilaparvata lugens Stål)is a notorious rice pest in many areas of Asia. Study on the molecular mechanisms underlying its development and reproduction will provide scientific basis for effective control. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is one of structural component of the extracellular matrix, which influences a diverse array of biological functions. In this study, the gene for SPARC was identified and functionally analysed from N.lugens. RESULTS: The result showed that the NlSPARC mRNA was highly expressed in fat body, hemolymph and early embryo. The mortality increased significantly when NlSPARC was downregulated after RNA interference (RNAi) in 3 ~ 4th instar nymphs. Downregulation of NlSPARC in adults significantly reduced the number of eggs and offspring, as well as the transcription level of NlSPARC in newly hatched nymphs and survival rate in progeny. The observation with microanatomy on individuals after NlSPARC RNAi showed smaller and less abundant fat body than that in control. No obvious morphological abnormalities in the nymphal development and no differences in development of internal reproductive organ were observed when compared with control. CONCLUSION: NlSPARC is required for oviposition and nymphal development mainly through regulating the tissue of fat body in N.lugens. NlSPARC could be a new candidate target for controlling the rapid propagation of N.lugens population. Our results also demonstrated that the effect of NlSPARC RNAi can transfer to the next generation in N.lugens.

The stringent starvation protein SspA modulates peptidoglycan synthesis by regulating the expression of peptidoglycan synthases.

The peptidoglycan (PG) layer of bacterial cells is essential for maintaining the cell shape and survival of cells; therefore, the synthesis of PG needs to be spatiotemporally controlled. While it is well established that PG synthesis is mediated posttranslationally through interactions between PG synthases and their cognate partners, much less is known about the transcriptional regulation of genes encoding these synthases. Based on a previous finding that the Gram-negative bacterium Shewanella oneidensis lacking the prominent PG synthase exhibits impaired cell wall integrity, we performed genetic selections to isolate the suppressors. We discovered that disrupting the sspA gene encoding stringent starvation protein A (SspA) is sufficient to suppress compromised PG. SspA serves as a transcriptional repressor that regulates the expression of the two types of PG synthases, class A penicillin-binding proteins and SEDS/bPBP protein complexes. SspA is an RNA polymerase-associated protein, and its regulation involves interactions with the σ70 -RNAP complex and an antagonistic effect of H-NS, a global nucleoid-associated protein. We also present evidence that the regulation of PG synthases by SspA is conserved in Escherichia coli, adding a new dimension to the current understanding of PG synthesis and its regulation.

Development of Whole-Cell Biosensors for Screening of Peptidoglycan-Targeting Antibiotics in a Gram-Negative Bacterium.

There is an urgent need to develop novel antibiotics since antibiotic resistance is an increasingly serious threat to global public health. Whole-cell biosensors are one of the promising strategies for new antibiotic discovery. The peptidoglycan (PG) of the bacterial cell wall is one of the most important targets for antibiotics. However, the biosensors for the detection of PG-targeting antibiotics in Gram-negative bacteria have not been developed, mainly because of the lack of the regulatory systems that sense and respond to PG stress. Recently, we identified a novel two-component signal transduction system (PghKR) that is responsible for sensing and responding to PG damage in the Gram-negative bacterium Shewanella oneidensis. Based on this system, we developed biosensors for the detection of PG-targeting antibiotics. Using ampicillin as an inducer for PG stress and the bacterial luciferase LuxCDABE as the reporter, we found that the PghKR biosensors are specific to antibiotics targeting PG synthesis, including ß-lactams, vancomycin, and d-cycloserine. Deletion of genes encoding PG permease AmpG and ß-lactamase BlaA improves the sensitivity of the biosensors substantially. The PghKR biosensor in the background of ΔblaA is also functional on agar plates, providing a simple method for screening bacteria that produce PG-targeting antibiotics. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative bacteria urgently needs new strategies so that researchers can develop novel antibiotics. Microbial whole-cell biosensors are capable of sensing various stimuli with a quantifiable output and show tremendous potential for the discovery of novel antibiotics. As the Achilles' heel of bacteria, the synthesis of the peptidoglycan (PG) is targeted by many antibiotics. However, the regulatory systems that sense and respond to PG-targeting stress in Gram-negative bacteria are reported rarely, restricting the development of biosensors for the detection of PG-targeting antibiotics. In this study, we developed a highly sensitive and specific biosensor based on a novel two-component system in the Gram-negative bacterium Shewanella oneidensis that is responsible for the sensing and responding to PG stress. Our biosensors have great potential for discovering novel antibiotics and determining the mode of action of antibiotics.

The same genetic regulation strategy produces inconsistent effects in different Saccharomyces cerevisiae strains for 2-phenylethanol production.

A CRISPR/Cas9 system with gene editing efficiency of 100% in the industrial diploid Saccharomyces cerevisiae CWY-132 strain for 2-phenylethanol (2-PE) production was constructed. The effect of deletion of acetyltransferase gene ATF1 in the Ehrlich pathway on 2-PE synthesis was studied for the first time in S. cerevisiae. Laboratory and industrial strains were compared for the deletion effect of ATF1 and acetaldehyde dehydrogenase genes ALD2 and ALD3 involved in competing branches of the Ehrlich pathway on the 2-PE titer. The results showed that in 2-PE low-yielding haploid strain PK-2C, the ATF1∆ mutant produced 2-PE of 0.45 g/L, an increase of 114%, whereas in CWY-132, the 2-PE yield of ATF1∆ decreased significantly from 3.50 to 0.83 g/L. In PK-2C, the 2-PE yield of ALD2∆ increased from 0.21 to 1.20 g/L, whereas in CWY-132, it decreased from 3.50 to 3.02 and 2.93 g/L in ALD2∆ and ALD3∆ mutants, respectively, and to 1.65 g/L in ALD2∆ALD3∆. These results indicate that the same genetic manipulation strategy used for strains with different 2-PE yield backgrounds produces significantly different or even opposite effects. Moreover, we found that a supply of NADH or GSH increased the 2-PE production in S. cerevisiae. The correlation between the synthesis of 2-PE and ethanol was also revealed, and the tolerance of cells to 2-PE and ethanol was suggested to be a key limiting factor for further increase of 2-PE production in high-yielding strains. KEY POINTS: • Deletion of genes competing for 2-PE synthesis produces different effects in S. cerevisiae strains. • The ATF1∆, ALD2∆, or ALD3∆ increased 2-PE production in laboratory strains but not industrial strains. • The supply of NADH or GSH increased the titer of 2-PE in S. cerevisiae.

Exocyst subunit BcSec3 regulates growth, development and pathogenicity in Botrytis cinerea.

Botrytis cinerea is a saprophytic plant pathogenic fungus that can infect a variety of crops and cause gray mold, which leads to huge losses worldwide. The role of exocyst in fungal pathogenicity is being revealed. In this study, homologous recombination technology was used to knock out the exocyst subunit BcSec3 of B. cinerea, and it was found that the BcSec3 subunit plays a crucial role in the growth and pathogenicity of B. cinerea. Compared with the wild-type strain B05.10, the mycelial growth ability of the BcSec3 deletion strain was reduced by up to 49.8%, the conidia production capacity of the deletion strain was severely lost, and no sclerotia was formed. The polygalacturonase, is one of plant cell wall hydrolases, whose activity in BcSec3 deletion strain was significantly reduced. In the tomato leaves infection assay in vitro, the lesion area caused by the BcSec3 deletion strain was only 20% of the wild type after 5 days of infection. Observation by light microscope showed that the morphology of BcSec3 deletion strain mycelium was significantly changed, the mycelium became thinner and deformed, and the polarity growth was not obvious. Further observation with laser confocal microscopy and transmission electron microscopy was conducted. It was found that compared with the wild type, the number of vesicles in BcSec3 deleted cells reduced and localization and distribution of vesicles changed. In mutant cell, vesicles relatively concentrated in the cytoplasm, while in wild-type cell mainly concentrated inside the cell membrane. These evidences indicate that the exocyst subunit BcSec3 plays an important role in the growth, development and pathogenicity of B. cinerea.

Identification and functional analysis of five genes that encode distinct isoforms of protein phosphatase 1 in Nilaparvata lugens.

Ten distinct cDNAs encoding five different protein phosphatases 1 (PPP1) were cloned from Nilaparvata lugens. NlPPP1α and NlPPP1ß are highly conserved whereas NlPPP1-Y, NlPPP1-Y1 and NlPPP1-Y2 are lowly conserved among insects. NlPPP1α and NlPPP1ß exhibited a ubiquitous expression, while NlPPP1-Y, NlPPP1-Y1, and NlPPP1-Y2 were obviously detected from the 4th instar nymph to imago developmental stages in males, especially detected in internal reproductive organ and fat bodies of the male. Injection nymphs with dsRNA of NlPPP1α or NlPPP1ß was able to reduce the target gene expression in a range of 71.5-91.0%, inducing a maximum mortality rate of 95.2% or 97.2% at 10th day after injection and eclosion ratio down by 65.5-100.0%. Injection with dsNlPPP1Ys targeted to NlPPP1-Y, NlPPP1-Y1and NlPPP1-Y2 was able to induce a maximum mortality rate of 95.5% at 10th day after injection, eclosion ratio down by 86.4%. Knock-down one of the male-biased NlPPP1 genes has no effect on survival and eclosion ratio. Injection of 4th instar nymph with dsNlPPP1Ys led to reduced oviposition amount and hatchability, down by 44.7% and 19.6% respectively. Knock-down of NlPPP1-Y1 or NlPPP1-Y2 gene did not significantly affect oviposition amount but significantly affected hatchability. The results indicate that the male-biased NlPPP1 genes have overlapping functions in N. lugens development, and NlPPP1-Y1 and NlPPP1-Y2 may play important roles in spermatogenesis and fertilization. The dsNlPPP1ß and dsNlPPP1Ys in this study could be the preferred sequence in RNAi and low-conserved male-biased NlPPP1 genes could be potential target for N. lugens control.

Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli.

Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (P cas ) and suppresses the type I-E CRISPR-Cas system in Escherichia coli Although the H-NS paralogue StpA also binds P cas , its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of P cas Third, by comparing transcriptional activities of the intact P cas and the P cas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in P cas Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of P cas Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coliIMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

Functional analysis of the exocyst subunit BcExo70 in Botrytis cinerea.

Botrytis cinerea is one of the most important saprophytic plant pathogenic fungi. The exocyst complex and exocytosis was demonstrated to be involved in fungal development and plant infection. Here, we investigated the function of an exocyst subunit gene Bcexo70 in B. cinerea. The results show that knockout of the Bcexo70 gene significantly reduced the fungal growth and hindered the production of conidia and sclerotia. The Bcexo70 deletion strains showed a severe decrease in virulence toward tomato leaves and reduced secretion of cell wall-degrading enzyme. Confocal and electronic microscopic observation showed that the vesicles in the Bcexo70 mutants were enlarged and scattered in the cytoplasm compared to the regular distribution in the hyphal tip in wild-type strain. This study showed that the exocyst gene Bcexo70 is crucial for fungal growth, conidiation and pathogenicity in B. cinerea.

Molecular Characterization of Ca2+/Calmodulin-Dependent Protein Kinase II Isoforms in Three Rice Planthoppers-Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

This study reports the identification of splice variants for the calcium/calmodulin-dependent protein kinase II (CaMKII) gene from Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera. CaMKII is a multifunctional serine/threonine protein kinase that transduces Ca2+ signals in cells to control a range of cellular processes in the nervous system and muscular tissue. Sequence analysis showed that CaMKII was 99.0% identical at the amino acid level among three rice planthoppers, with the exception of a variable region located in the association domain. Four kinds of 20-81 amino acid "inserts" were found in the variable region. The phylogenetic tree of the deduced amino acid sequences showed that the NlCaMKII isoforms were more closely related to the LsCaMKII isoforms and were slightly distinct from SfCaMKII. CaMKII-E was the dominant type among the five main isoforms. CaMKII genes were constitutively expressed in various nymphal and adult stages and in tested tissues with the predominant transcription occurring in the head. There was no major tissue specificity of isoform expression, but the expression pattern and relative abundance of isoforms varied when compared with the RT-PCR between tissues. In addition, RNAi in N. lugens with dsRNA at a concentration of 200 ng nymph-1 induced a mortality of 77.7% on the 10th day and a reduction in the mRNA expression level of 67.2%. Unlike the holometabolous insect Helicoverpa armigera, the knockdown of NlCaMKII did not suppress the expression of 20E response genes, such as ECR, USP1, and HR3, in N. lugens. These results indicate that the role of CaMKII in hemimetabolous insects may be different from that in holometabolous insects.

Double-stranded RNA targeting calmodulin reveals a potential target for pest management of Nilaparvata lugens.

BACKGROUND: Calmodulin (CaM) is an essential protein in cellular activity and plays important roles in many processes in insect development. RNA interference (RNAi) has been hypothesized to be a promising method for pest control. CaM is a good candidate for RNAi target. However, the sequence and function of CaM in Nilaparvata lugens are unknown. Furthermore, the double-stranded RNA (dsRNA) target to CaM gene in pest control is still unavailable. RESULTS: In the present study, two alternatively spliced variants of CaM transcripts, designated NlCaM1 and NlCaM2, were cloned from N. lugens. The two cDNA sequences exhibited 100% identity to each other in the open reading frame (ORF), and only differed in the 3' untranslated region (UTR). NlCaM including NlCaM1 and NlCaM2 mRNA was detectable in all developmental stages and tissues of N. lugens, with significantly increased expression in the salivary glands. Knockdown of NlCaM expression by RNAi with different dsRNAs led to an inability to molt properly, increased mortality, which ranged from 49.7 to 92.5%, impacted development of the ovaries and led to female infertility. There were no significant reductions in the transcript levels of vitellogenin and its receptor or in the total vitellogenin protein level relative to the control group. However, a significant reduction in vitellogenin protein was detected in ovaries injected with dsNlCaM. In addition, a specific dsRNA of NlCaM for control of N. lugens was designed and tested. CONCLUSION: NlCaM plays important roles mainly in nymph development and uptake of vitellogenin by ovaries in vitellogenesis in N. lugens. dsRNA derived from the less conserved 3'-UTR of NlCaM shows great potential for RNAi-based N. lugens management. © 2018 Society of Chemical Industry.

Multiwall carbon nanotubes modulate paraquat toxicity in Arabidopsis thaliana.

Carbon nanotubes can be either toxic or beneficial to plant growth and can also modulate toxicity of organic contaminants through surface sorption. The complex interacting toxic effects of carbon nanotubes and organic contaminants in plants have received little attention in the literature to date. In this study, the toxicity of multiwall carbon nanotubes (MWCNT, 50 mg/L) and paraquat (MV, 0.82 mg/L), separately or in combination, were evaluated at the physiological and the proteomic level in Arabidopsis thaliana for 7-14 days. The results revealed that the exposure to MWCNT had no inhibitory effect on the growth of shoots and leaves. Rather, MWCNT stimulated the relative electron transport rate and the effective photochemical quantum yield of PSII value as compared to the control by around 12% and lateral root production up to nearly 4-fold as compared to the control. The protective effect of MWCNT on MV toxicity on the root surface area could be quantitatively explained by the extent of MV adsorption on MWCNT and was related to stimulation of photosynthesis, antioxidant protection and number and area of lateral roots which in turn helped nutrient assimilation. The influence of MWCNT and MV on photosynthesis and oxidative stress at the physiological level was consistent with the proteomics analysis, with various over-expressed photosynthesis-related proteins (by more than 2 folds) and various under-expressed oxidative stress related proteins (by about 2-3 folds). This study brings new insights into the interactive effects of two xenobiotics (MWCNT and MV) on the physiology of a model plant.

Ras-like family small GTPases genes in Nilaparvata lugens: Identification, phylogenetic analysis, gene expression and function in nymphal development.

Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRß, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRß could be served as a candidate target for dsRNA-based pesticides for N.lugens control.

Newly Isolated Penicillium ramulosum N1 Is Excellent for Producing Protease-Resistant Acidophilic Xylanase.

Penicillium ramulosum N1 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest activities of xylanases (250 U/ml) and carboxymethyl cellulose (CMCase; 6.5 U/ml) were produced when 1% barley straw was added as a carbon source. The optimum temperature and pH for xylanase activity was 55 and 3.0 °C, respectively. The xylanases exhibited strong protease resistance. CMCase revealed maximum activities at pH 3.0 and in the range of 60-70 °C. Filter paper activity was optimally active at pH 5.0 and 55 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that there are four bands of protein with xylanase activity and three bands of proteins with endoglucanase. The results revealed that P. ramulosum N1 is a promising acidophilic and protease-resistant xylanase-producing microorganism that has great potential to be used in animal feed and food industry applications.

BcMctA, a putative monocarboxylate transporter, is required for pathogenicity in Botrytis cinerea.

Monocarboxylate transporters have a central role in mammalian metabolism, but rarely reported in phytopathogenic fungi. In this study, a putative monocarboxylate transporter gene in Botrytis cinerea [B. cinerea MctA (BcMctA)] was identified in the research of a B. cinerea transfer DNA (T-DNA) insertional mutant (74). Disruption of the gene decreased the growth rate on the medium with monocarboxylate (acetate or pyruvate) as the sole carbon sources, but not affected on lactate. The pyruvate contents in BcmctA deletion mutants decreased about 35 % compared with the wild strain. Besides, the conidial yield was increased about two times in BcmctA disruption mutant. The pathogenicity assay indicated that disruption of BcmctA significantly reduced the virulence of B. cinerea on cucumber and tomato leaves. Our results demonstrated that BcMctA is related to pyruvate uptake and pathogenicity of B. cinerea on cucumber and tomato leaves.

Molecular cloning, expression of CPR gene from Rhizopus oryzae into Rhizopus nigericans and its application in the 11α-hydroxylation of 16α, 17-epoxy-progesterone.

The hydroxylations of the steroid skeleton structure are catalyzed by a family of enzymes, the cytochromes P450 (CYPs). In this study, the pCB1004-PgpdA plasmid was used for cloning the cytochrome P450 reductase (CPR) gene from Rhizopus oryzae into Rhizopus nigericans to strengthen the expression of CPR gene in R. nigericans with REMI (Restriction Enzyme Mediate Integration) mediated protoplast transformation. The conditions for the protoplast production of R. nigericans were optimized as follows: 75 µg/mL yatalase, 50 µg/mL lywallzyme, fungus age of 12h, digestion time of 3 h and digestion temperature of 30°C. REMI mediated protoplast transformation with plasmid pCB1004-PgpdA into R. nigericans was performed to construct the transformants. More than 30 transformants were successfully selected from the hygromycin B-resistant plates and 6 transformants had the abilities to improve the biotransformation of 16α, 17-epoxyprogesterone. The highest biotransformation rate of the transformants was 65.38%, which was 7.06% higher than that of the original strain.