Rho2 involved in development, stress response and pathogenicity of Fusarium oxysporum.

Rho GTPases regulate the activity of cell wall biosynthesis, actin assembly and polar cell secretion. However, the function of Rho GTPase in filamentous fungi is poorly understood. To understand the role of Rho2 GTPase in Fusarium oxysporum, which is one of root rot pathogens of Panax notoginseng, △rho2 mutant was constructed. Phenotypes of △rho2, including conidiation, germination of spores, stresses (osmotic-, cell membrane-, cell wall disturbing-, metal-, and high temperature-) tolerance and pathogenicity were analyzed. The results showed that the growth of △rho2 was destroyed under cell wall disturbing stress and high temperature stress, suggesting that Rho2 regulated the response of F. oxysporum to cell wall synthesis inhibitors and high temperature stress. Germination of spores and pathogenicity to P. notoginseng were reduced in △rho2 mutant. Western blot results showed that rho2 deletion increased the phosphorylation level of Mpk1. To identify genes regulated by Rho2, transcriptome sequencing was carried out. 2477 genes were identified as upregulated genes and 2177 genes were identified as downregulated genes after rho2 was deleted. These genes provide clues for further study of rho2 function.

14-3-3 proteins regulate the HCHO stress response by interacting with AtMDH1 and AtGS1 in tobacco and Arabidopsis.

Formaldehyde (HCHO) is one of the most essential common carcinogenic environmental pollutants. While 14-3-3 proteins are known to regulate the response of plants to HCHO stress, the regulatory mechanisms responsible for a tolerant phenotype remain unclear. We first performed qPCR analysis of HCHO-treated Arabidopsis and tobacco and determined that the expression of At14-3-3PSI and Nt14-3-3C genes was rapidly upregulated after HCHO stress. Furthermore, overexpression of 14-3-3, AtMDH1 or AtGS1 genes enhanced plant HCHO absorption capacity and resistance, and knockdown or knockout of 14-3-3, AtMDH1 or AtGS1 genes reduced plant HCHO absorption capacity and resistance. However, overexpression of the AtGS1 and AtMDH1 genes in the At14-3-3 psi mutant restored HCHO uptake and resistance in Arabidopsis. Moreover, 14-3-3 bound to the N-terminus of AtMDH1 and the C-terminus of AtGS1, respectively, and repressed and enhanced their expression. The 13C NMR results of HCHO stress mutants Atgs1 and Atmdh1 showed that the metabolites Glu and Asp rapidly increased, indicating that AtGS1 and AtMDH1 were indeed indispensable for Arabidopsis to metabolize HCHO. In conclusion, we uncovered a HCHO stress response mechanism mediated by 14-3-3, which enhances the plant's ability to absorb HCHO, deepening our understanding of how plants respond to HCHO stress.

[Effects of propiconazole on physiological and biochemical properties of Panax notoginseng and dietary risk assessment].

To study the residue and dietary risk of propiconazole in Panax notoginseng and the effects on physiological and bioche-mical properties of P. notoginseng, we conducted foliar spraying of propiconazole on P. notoginseng in pot experiments. The physiolo-gical and biochemical properties studied included leaf damage, osmoregulatory substance content, antioxidant enzyme system, non-enzymatic system, and saponin content in the main root. The results showed that at the same application concentration, the residual amount of propiconazole in each part of P. notoginseng increased with the increase in the times of application and decreased with the extension of harvest interval. After one-time application of propiconazole according to the recommended dose(132 g·hm~(-2)) for P. ginseng, the half-life was 11.37-13.67 days. After 1-2 times of application in P. notoginseng, propiconazole had a low risk of dietary intake and safety threat to the population. The propiconazole treatment at the recommended concentration and above significantly increased the malondialdehyde(MDA) content, relative conductivity, and osmoregulatory substances and caused the accumulation of reactive oxygen species in P. notoginseng leaves. The propiconazole treatment at half(66 g·hm~(-2)) of the recommended dose for P. ginseng significantly increased the activities of superoxide dismutase(SOD), peroxidase(POD), and catalase(CAT) in P. notoginseng leaves. The propiconazole treatment at 132 g·hm~(-2) above inhibited the activities of glutathione reductase(GR) and glutathione S-transferase(GST), thereby reducing glutathione(GSH) content. Proconazole treatment changed the proportion of 5 main saponins in the main root of P. notoginseng. The treatment with 66 g·hm~(-2) propiconazole promoted the accumulation of saponins, while that with 132 g·hm~(-2) and above propiconazole significantly inhibited the accumulation of saponins. In summary, using propiconazole at 132 g·hm~(-2) to prevent and treat P. notoginseng diseases will cause stress on P. notoginseng, while propiconazole treatment at 66 g·hm~(-2) will not cause stress on P. notoginseng but promote the accumulation of saponins. The effect of propiconazole on P. notoginseng diseases remains to be studied.

Enhancement production of lipid and unsaturation of fatty acids in Cryptococcus humicola via addition of calcium ion.

Lipids synthesized by oleaginous yeasts are considered to be the best candidates for biodiesel production. Cryptococcus humicola as an oleaginous yeast accumulated lipid in cells. In order to optimize the conditions for lipid production, different carbon and nitrogen sources were used and metals were added into the medium. Ca2+ addition increased the lipid production greatly. Xylose and peptone were optimal carbon source and nitrogen source, respectively for lipid accumulation. Response surface experiment results revealed that the accumulation of lipid could be maximized when the xylose, peptone and Ca2+ concentration was 61 g/L, 4.31 g/L, 0.67 mmol/L. C16 and C18 fatty acid account for about 91% of the total fatty acids. The most abundant fatty acid was oleic acid (42.68%), followed by palmitic acid (29.7%) and stearic acid (13.87%). The addition of Ca2+ increased the content of unsaturated fatty acids (such as C16:1 and C18:1) and improved the unsaturation of fatty acids. Quantitative real time PCR analysis revealed that expression of genes related to lipid biosynthesis showed up-regulated by Ca2+ treatment. This study provided a strategy for increase in lipid production and content of unsaturated fatty acids.

Rapid determination and dietary intake risk assessment of 249 pesticide residues in Panax notoginseng.

UPLC-MS/MS and GC-MS/MS were used to establish a method to simultaneously determine various pesticide residues in Panax notoginseng. Results showed that the limits of detection of 249 pesticides were all 5-10 µg/kg. The detection rate of pesticides in 121 P. notoginseng samples was 93.39%, and 19 pesticides were detected. According to the US Code of Federal Regulations, the Chinese Pharmacopoeia recommended algorithm, and the Japanese "positive list system", the pass rates of pesticide residues were 100%, 99.17%, and 89.26%, respectively. The chronic risk quotient (ADI%) and acute risk quotient (ARfD%) of P. notoginseng were 0.00-0.12% and 0.00-0.15%, respectively. In summary, the detection method established in this study can be used for routine analysis of various P. notoginseng pesticide residues. The pesticide residues in the main root samples of P. notoginseng were at a safe level and unlikely pose health risks to consumers.

Transcription Factor Crz1 from Cryptococcus humicola Conferred Aluminum Resistance and Interacted with Calcineurin.

Calcineurin was activated by aluminum stress and increased aluminum resistance. To investigate how the calcineurin pathway regulates aluminum stress in Cryptococcus humicola, the expressions of Crz1 under stresses were analyzed by quantitative real-time PCR. Calcium, cadmium, and aluminum induced the expression of Crz1. Cna1, calcineurin catalytic subunit A (CNA1) encoding gene, was constructed into pGBKT7 and Crz1 gene was constructed into pGADT7. The resultant plasmids, pGADT7-Crz1 and pGBKT7-Cna1, were transformed into Y2HGold and Y187 yeast strain, respectively. Yeast two-hybridization results showed an interaction between CNA1 and Crz1. The role of Crz1 gene in stresses resistance including hydrogen peroxide, calcium, cadmium, and aluminum was assayed by constructing transgenic yeast. The growth of Crz1 transgenic yeasts was much better than that of the control yeast under these stress conditions. These results suggested that Crz1 participated in resistance to stresses and Crz1 showed an interaction with CNA1.

Transcriptome analysis of Cryptococcus humicola under aluminum stress revealed the potential role of the cell wall in aluminum tolerance.

Aluminum (Al) toxicity is one of the most important limiting factors for crop yield in acidic soils. Bound Al gets converted into a toxic ionic state (Al3+) in acidic soil. Recent studies have shown that Al can act on the cell walls, cell membranes, organelles, and nuclei of microorganisms and affect substance and energy metabolism. To explore the gene expression at the transcriptional level under Al stress, we sequenced the transcriptome of Cryptococcus humicola, which is a highly Al-resistant yeast strain isolated from acidic soil and tolerates up to 200 mM Al3+. The screening conditions for genes from the control and experimental group were a false discovery rate (FDR) <0.05 and log 2|FC| > 1. A total of 4760 genes were differentially expressed, among which 3066 were upregulated and 1694 were downregulated. These genes control glycometabolism, protein synthesis, lipid metabolism and signalling pathways. Eleven selected differentially expressed genes were further validated using qRT-PCR. The results suggested that Al stress leads to complex responses in C. humicola. The effects of Al on the ß-d-glucan and mannose contents and Al accumulation in the cell wall were determined. With an increase in the Al treatment time and concentration, the contents of ß-d-glucan and mannose showed a trend of first increasing and then decreasing. Under Al treatment, the Al content of the cell wall also showed a trend of first increasing and then decreasing. These results suggested that Al accumulates in the cell wall and the cell wall plays a vital role in the Al resistance of C. humicola. The differentially expressed genes provide a foundation for the further study of Al tolerance in C. humicola.

Inhibition of Polyunsaturated Fatty Acids Synthesis Decreases Growth Rate and Membrane Fluidity of Rhodosporidium kratochvilovae at Low Temperature.

The intention of this study was to investigate the role of polyunsaturated fatty acids (PUFA) in the cold adaptation of Rhodosporidium kratochvilovae YM25235 by knockout of the Δ12/Δ15-fatty acid desaturase gene (RKD12) to inactivate Δ12/Δ15-fatty acid desaturase. Polymerase chain reaction (PCR) amplification was used to detect the genomic structure of RKD12 gene in YM25235. The RKD12 gene was knocked out by DNA homologous recombination to inhibit the biosynthesis of PUFA. Then, the contents of linoleic acid (LNA) and α-linolenic acid (ALA) after gene knockout were investigated using a gas chromatography-mass spectrometer, followed by determination of the growth rate and membrane fluidity of YM25235 at low temperature. After PCR amplification, a 1611 bp genomic fragment was amplified from YM25235. When the RKD12 gene was knocked out, the contents of LNA and ALA in YM25235 significantly decreased. The growth rate and membrane fluidity of YM25235 decreased significantly at low temperature. Inhibition of PUFA biosynthesis by RKD12 gene knockout influenced cold adaptation of YM25235 by decreasing the PUFA content in cell membranes and reducing the growth rate and membrane fluidity of YM25235 at low temperature.

Characterization of calcineurin from Cryptococcus humicola and the application of calcineurin in aluminum tolerance.

BACKGROUND: Calcineurin (CaN) is a Ca2+- and calmodulin (CaM)-dependent serine/threonine phosphatase. Previous studies have found that CaN is involved in the regulation of the stress responses. RESULTS: In this study, the growth of Cryptococcus humicola was inhibited by the CaN inhibitor tacrolimus (FK506) under aluminum (Al) stress. The expression of CNA encoding a catalytic subunit A (CNA) and its interaction with CaM were upregulated when the concentration of Al was increased. A CaM-binding domain and key amino acids responsible for interaction with CaM were identified. ∆CNAb with a deletion from S454 to A639 was detected to bind to CaM, while ∆CNAa with a deletion from R436 to A639 showed no binding to CaM. The binding affinities of CNA1 and CNA2, in which I439 or I443 were replaced by Ala, were decreased relative to wild-type CNA. The phosphatase activities of ∆CNAa, CNA1 and CNA2 were lower than the wild-type protein. These results suggest that the region between R436 and S454 is essential for the interaction with CaM and I439, I443 are key amino acids in this region. The ability of the CNA transgenic yeast to develop resistance to Al was significantly higher than that of control yeast. Residual Al in the CNA transgenic yeast culture media was significantly lower than the amount of Al originally added to the media or the residual Al remaining in the control yeast culture media. These findings suggest that CNA confers Al tolerance, and the mechanism of Al tolerance may involve absorption of active Al. CONCLUSIONS: Al stress up-regulated the expression of CNA. CaM-binding domain and key amino acids responsible for interaction with CaM were identified and both are required for phosphatase activities. CNA conferred yeast Al resistance indicating that the gene has a potential to improve Al-tolerance through gene engineering.

Effect of aluminum stress on the expression of calmodulin and the role of calmodulin in aluminum tolerance.

Calmodulin (CaM) is a calcium ion-binding protein that regulates a variety of cellular functions through its downstream target proteins. Previous studies have reported that overexpression of CaM enhances tolerance to stress, including resistance to salt, heat, cold, drought and plant pathogens. In this study, the growth of Cryptococcus humicola was inhibited by the CaM inhibitor, trifluoperazine, under aluminum (Al) stress. The expression of CaM of C. humicola (ChCaM) was upregulated when the concentration and treatment time with Al was increased. These results indicate that Al stress affects the transcription and translation of ChCaM and that ChCaM may play an important role in Al tolerance. Transgenic ChCaM Saccharomyces cerevisiae was constructed and designated as Sc-ChCaM. The ability of Sc-ChCaM to develop resistance to Al was significantly higher than that of control yeast containing the empty vector pYES3/CT designated as Sc. The residual Al content in the medium containing ChCaM transgenic yeast in culture was significantly lower than the initial amount of Al added in the medium or the residual Al content in the medium containing the control yeast in culture. This finding suggests that ChCaM confers Al tolerance in transgenic yeast, and the absorption of active Al from the culture may be one reason for Al tolerance. These results indicate that ChCaM may be a candidate gene for Al tolerance in engineered plants.

Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of Cryptococcus humicola response to aluminum stress.

Cryptococcus humicola is a highly aluminum (Al) tolerant yeast strain isolated from a tea field. Here the relative changes of protein expression in C. humicola undergoing aluminum stress were analyzed to understand the genetic basis of aluminum tolerance. In this work, iTRAQ-based (isobaric tags for relative and absolute quantification) quantitative proteomic technology was used to detect statistically significant proteins associated with the response to aluminum stress. A total of 625 proteins were identified and were mainly involved in translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover/chaperones, energy production and conversion, and amino acid transport and metabolism. Of these proteins, 59 exhibited differential expression during aluminum stress. Twenty-nine proteins up-regulated by aluminum were mainly involved in translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover and chaperones, and lipid transport and metabolism. Thirty proteins down-regulated by aluminum were mainly associated with energy transport and metabolism, translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover/chaperones, and lipid transport and metabolism. The potential functions of some proteins in aluminum tolerance are discussed. These functional changes may be beneficial for cells to protect themselves from aluminum toxic conditions.

Overexpression of the formaldehyde dehydrogenase gene from Brevibacillus brevis to enhance formaldehyde tolerance and detoxification of tobacco.

The faldh gene coding for a putative Brevibacillus brevis formaldehyde dehydrogenase (FALDH) was isolated and then transformed into tobacco. A total of three lines of transgenic plants were generated, with each showing 2- to 3-fold higher specific formaldehyde dehydrogenase activities than wild-type tobacco, a result that demonstrates the functional activity of the enzyme in formaldehyde (HCHO) oxidation. Overexpression of faldh in tobacco confers a high tolerance to exogenous HCHO and an increased ability to take up HCHO. A (13)C-nuclear magnetic resonance technique revealed that the transgenic plants were able to oxidize more aqueous HCHO to formate than the wild-type (WT) plants. When treated with gaseous HCHO, the transgenic tobacco exhibited an enhanced ability to transform more HCHO into formate, citrate acid, and malate but less glycine than the WT plants. These results indicate that the increased capacity of the transgenic tobacco to take up, tolerate, and metabolize higher concentrations of HCHO was due to the overexpression of B. brevis FALDH, revealing the essential function of this enzyme in HCHO detoxification. Our results provide a potential genetic engineering strategy for improving the phytoremediation of HCHO pollution.

Physiological and transcriptional analysis of the effects of aluminum stress on Cryptococcus humicola.

Aluminum (Al) toxicity is a major factor that limits crop productivity in acid soils. The toxic effects of Al exposure on Al-tolerant Cryptococcus humicola were analyzed at the physiological level. The exposure to 20 mM Al led to a clear increase in malondialdehyde content and a significant decrease in the levels of protein carbonyls, suggesting that Al stress caused oxidative damage to membrane lipids but not to proteins. Suppression subtractive hybridization (SSH) results showed that when C. humicola was exposed to 20 mM Al, a total of 141 ESTs were differentially expressed. Of them, 27 had unknown functions and 48 were newly identified in this study. The genes with known functions were categorized into seven groups. The largest group was related to metabolism and energy, followed by protein synthesis and processing, cell structure, signal transduction and transcription, transporters, stress and defense. Reverse transcription (RT)-PCR analysis of certain genes was performed to confirm the reliability of the SSH data. Nine selected genes were upregulated by Al in a time-dependent manner. The potential functions of some genes involved in Al-tolerance were predicted and are discussed. The diversity of the putative functions of these genes indicates that Al stress results in a complex response in C. humicola.

Isolation of transposon mutants and characterization of genes involved in biofilm formation by Pseudomonas fluorescens TC222.

Biofilm formation mutants are often found to have defective or altered motility. The motility phenotype was exploited to identify Pseudomonas fluorescens biofilm formation mutants. Fourteen motility mutants were obtained from P. fluorescens isolate TC222 and eight stable mutants were studied further. The eight transposon insertion mutants showed altered ability to form biofilm compared with the parent. Five Tn5-inserted genes from these mutants were cloned and sequenced. Genetic analysis showed that two insertions were located in genes affecting multiple cell surface characteristics, including lipopolysaccharide (rfbD) and polar flagella (fliR). Three genes encoding for a putative Mig-14 family protein (epsB), a probable bacteriophage signal peptide protein (bspA) and a soluble pyridine nucleotide transhydrogenase (pyrA) were reported for the first time to be involved in biofilm formation. Complementation experiments of rfbD and epsB genes proved that biofilm formation of the corresponding mutants could be restored. Further semi-quantitative reverse transcription-PCR analysis showed that both rfbD and epsB can express their transcripts much higher in the complemented strains than that in wild-type strains. The transcripts of both genes in their mutants could not be detected.