A synthetic agent ameliorates polycystic kidney disease by promoting apoptosis of cystic cells through increased oxidative stress.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development.

XBP1 Activation Reduces Severity of Polycystic Kidney Disease due to a Nontruncating Polycystin-1 Mutation in Mice.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1 α -XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1 . METHODS: We engineered a Pkd1 REJ domain missense murine model, Pkd1 R2216W , on the basis of the orthologous human hypomorphic allele Pkd1 R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression. RESULTS: Expression of active XBP1 in cultured cells bearing PC1 R2216W mutations increased levels and ciliary trafficking of PC1 R2216W . Mice homozygous for Pkd1 R2216W or heterozygous for Pkd1 R2216Win trans with a conditional Pkd1 fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters. CONCLUSIONS: Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations.

Risk assessment and molecular mechanism of Sclerotium rolfsii resistance to boscalid.

Boscalid, a widely used SDHI fungicide, has been employed in plant disease control for over two decades. However, there is currently no available information regarding its antifungal activity against Sclerotium rolfsii and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 100 S. rolfsii strains collected from five different regions in China during 2018-2019 to boscalid using mycelial growth inhibition method and assessed the risk of resistance development. The EC50 values for boscalid ranged from 0.2994 µg/mL to 1.0766 µg/mL against the tested strains, with an average EC50 value of 0.7052 ± 0.1473 µg/mL. Notably, a single peak sensitivity baseline was curved, indicating the absence of any detected resistant strains. Furtherly, 10 randomly selected strains of S. rolfsii were subjected to chemical taming to evaluate its resistance risk to boscalid, resulting in the successful generation of six stable and inheritable resistant mutants. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and virulence compared to their respective parental strains. Cross-resistance tests revealed a correlation between boscalid and flutolanil, benzovindiflupyr, pydiflumetofen, fluindapyr, and thifluzamide; however, no cross-resistance was observed between boscalid and azoxystrobin. Thus, we conclude that the development risk of resistance in S. rolfsii to boscalid is low. Boscalid can be used as an alternative fungicide for controlling peanut sclerotium blight when combined with other fungicides that have different mechanisms of action. Finally, the target genes SDHB, SDHC, and SDHD in S. rolfsii were initially identified, cloned and sequenced to elucidate the mechanism of S. rolfsii resistance to boscalid. Two mutation genotypes were found in the mutants: SDHD-D111H and SDHD-H121Y. The mutants carrying SDHD-H121Y exhibited moderate resistance, while the mutants with SDHD-D111H showed low resistance. These findings contribute to our comprehensive understanding of molecular mechanisms underlying plant pathogens resistance to SDHI fungicides.

The intrinsic resistance of Fusarium solani to the Fusarium-specific fungicide phenamacril is attributed to the natural variation of both T218S and K376M in myosin5.

Fusarium solani is responsible for causing root rot in various crops, resulting in wilting and eventual demise. Phenamacril, a specific inhibitor of myosin5 protein, has gained recognition as an effective fungicide against a broad spectrum of Fusarium species. It has been officially registered for controlling Fusarium diseases through spray application, root irrigation, and seed dipping. In this study, phenamacril was observed to exhibit negligible inhibitory effects on F. solani causing crop root rot, despite the absence of prior exposure to phenamacril. Considering the high selectivity of phenamacril, this phenomenon was attributed to intrinsic resistance and further investigated for its underlying mechanism. Sequence alignment analysis of myosin5 proteins across different Fusarium species revealed significant differences at positions 218 and 376. Subsequent homology modeling and molecular docking results indicated that substitutions T218S, K376M, and T218S&K376M impaired the binding affinity between phenamacril and myosin5 in F. solani. Mutants carrying these substitutions were generated via site-directed mutagenesis. A phenamacril-sensitivity test showed that the EC50 values of mutants carrying T218S, K376M, and T218S&K376M were reduced by at least 6.13-fold, 9.66-fold, and 761.90-fold respectively compared to the wild-type strain. Fitness testing indicated that mutants carrying K376M or T218S&K376M had reduced sporulation compared to the wild-type strain. Additionally, mutants carrying T218S exhibited an enhanced virulence compared to the wild-type strain. However, there were no significant differences observed in mycelial growth rates between the mutants and the wild-type strain. Thus, the intrinsic differences observed at positions 218 and 376 in myosin5 between F. solani and other Fusarium species are specifically associated with phenamacril resistance. The identification of these resistance-associated positions in myosin5 of F. solani has significantly contributed to the understanding of phenamacril resistance mechanisms, thereby discouraging the use of phenamacril for controlling F. solani.

Occurrence and Chemical Control Strategy of Wheat Brown Foot Rot Caused by Microdochium majus.

Wheat brown foot rot (WBFR), caused by a variety of phytopathogenic fungi, is an important soilborne and seedborne disease of wheat. WBFR causes wheat lodging and seedling dieback, which seriously affect the yield and quality of wheat. In this study, 64 isolates of WBFR were isolated from different wheat fields in Yancheng city, Jiangsu Province, China. The internal transcribed spacer, elongation factor 1α, and RNA polymerase II subunit were amplified and the sequencing results of the fragments were analyzed with BLAST in NCBI. Through morphological and molecular identification, all of the isolates were identified as Microdochium majus. Verification by Koch's postulates confirmed that M. majus was the pathogen causing WBFR. The antifungal activities of fludioxonil and prochloraz against 64 isolates of M. majus were determined based on mycelial growth inhibition method. The results showed that fludioxonil and prochloraz had good antifungal activity against M. majus. The mean 50% effective concentration values of fludioxonil and prochloraz against M. majus were 0.2956 ± 0.1285 µg/ml and 0.0422 ± 0.0157 µg/ml, respectively. Control efficacy for seed-coating treatments conducted in a greenhouse indicated that M. majus severely damaged the normal growth of wheat, while seed coating with fludioxonil or prochloraz significantly reduced the disease incidence and improved the seedling survival rates. At fludioxonil doses of 7.5 g per 100 kg and prochloraz doses of 15 g per 100 kg, the incidence was reduced by 22.26 and 25.33%, seedling survival rates increased by 25.37 and 22.66%, and control efficacy reached 70.02 and 72.30%, respectively. These findings provide vital information for the accurate diagnosis and effective management of WBFR.

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.

Genotypes and Characteristics of Phenamacril-Resistant Mutants in Fusarium asiaticum.

Fusarium asiaticum is a critical pathogen of Fusarium head blight (FHB) in the southern part of China. The fungicide phenamacril has been extensively used for controlling FHB in recent years, which reduced both FHB severity and mycotoxin production. Our previous report indicated that resistance of F. asiaticum to phenamacril was related to mutations in myosin5. A recent article revealed that the resistance level of phenamacril-resistant mutants was associated with the genotypes of myosin5 in these mutants. In total, we obtained 239 resistant isolates by fungicide domestication, and 82 resistant mutants were randomly selected for further study. Of these mutants, 25.6, 7.3, and 67.1% showed low resistance (LR), moderate resistance (MR), and high resistance (HR), respectively, to phenamacril determined by 50% effective concentration values. Point mutations A135T, V151M, P204S, I434M, A577T, R580G/H, or I581F led to LR. Point mutations S418R, I424R, and A577G were responsible for MR and point mutations K216R/E, S217P/L, or E420K/G/D conferred HR. Interestingly, all of the mutations concentrated in the myosin5 motor domain and mutations conferring HR occurred at codon 217 and 420, which we called the core region. Homology modeling revealed that mutations far from the core region led to a lower resistance degree. Phenotype assays revealed that the most highly resistant mutants did not significantly change pathogenicity but decreased conidia production compared with the wild type, which may slow down the formation of the resistant pathogen population in the fields.

Loop-Mediated Isothermal Amplification for the Rapid Detection of the F200Y Mutant Genotype of Carbendazim-Resistant Isolates of Sclerotinia sclerotiorum.

The point mutation at codon 200 (TTC→TAC, F200Y) confers moderate resistance to carbendazim in Sclerotinia sclerotiorum. This mutant genotype (F200Y) has been detected mainly by determining the minimum inhibitory concentration (MIC), which requires 3 to 5 days. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum. Specific LAMP primers were designed and concentrations of LAMP components were optimized. The optimal reaction conditions were 62 to 63°C for 45 min. The new LAMP assay requires no special equipment and is highly sensitive and specific (the i.e., it generated positive results with F200Y mutant genotype but generated negative results with other carbendazim-resistant mutants and with a variety of carbendazim-resistant mutants of Botrytis cinerea and Fusarium graminearum). Inclusion of the loop backward (LB) primer reduced the reaction time to 15 min. Results were identical with LAMP and MIC determinations. The advantages of the LB-accelerated LAMP assay for detection of the F200Y mutant genotype were demonstrated by assaying sclerotia produced on rape stems that were artificially inoculated in the field. The results indicated that the new LAMP assay represents an improved way to detect the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum.

Genome-wide association discoveries of alcohol dependence.

OBJECTIVE: To report the genome-wide significant and/or replicable risk variants for alcohol dependence and explore their potential biological functions. METHODS: We searched in PubMed for all genome-wide association studies (GWASs) of alcohol dependence. The following three types of the results were extracted: genome-wide significant associations in an individual sample, the combined samples, or the meta-analysis (p < 5 × 10(-8) ); top-ranked associations in an individual sample (p < 10(-5) ) that were nominally replicated in other samples (p < .05); and nominally replicable associations across at least three independent GWAS samples (p < .05). These results were meta-analyzed. cis-eQTLs in human, RNA expression in rat and mouse brains and bioinformatics properties of all of these risk variants were analyzed. RESULTS: The variants located within the alcohol dehydrogenase (ADH) cluster were significantly associated with alcohol dependence at the genome-wide level (p < 5 × 10(-8) ) in at least one sample. Some associations with the ADH cluster were replicable across six independent GWAS samples. The variants located within or near SERINC2, KIAA0040, MREG-PECR or PKNOX2 were significantly associated with alcohol dependence at the genome-wide level (p < 5 × 10(-8) ) in meta-analysis or combined samples, and these associations were replicable across at least one sample. The associations with the variants within NRD1, GPD1L-CMTM8 or MAP3K9-PCNX were suggestive (5 × 10(-8) < p < 10(-5) ) in some samples, and nominally replicable in other samples. The associations with the variants at HTR7 and OPA3 were nominally replicable across at least three independent GWAS samples (10(-5) < p < .05). Some risk variants at the ADH cluster, SERINC2, KIAA0040, NRD1, and HTR7 had potential biological functions. CONCLUSION: The most robust risk locus was the ADH cluster. SERINC2, KIAA0040, NRD1, and HTR7 were also likely to play important roles in alcohol dependence. PKNOX2, MREG, PECR, GPD1L, CMTM8, MAP3K9, PCNX, and OPA3 might play less important roles in risk for alcohol dependence based on the function analysis. This conclusion will significantly contribute to the post-GWAS follow-up studies on alcohol dependence.

Resistance Risk and Molecular Mechanism of Tomato Wilt Pathogen Fusarium oxysporum f. sp. lycopersici to Pyraclostrobin.

Tomato wilt disease caused by Fusarium oxysporum f. sp. lycopersici (Fol) results in a decrease in tomato yield and quality. Pyraclostrobin, a typical quinone outside inhibitor (QoI), inhibits the cytochrome bc1 complex to block energy transfer. However, there is currently limited research on the effectiveness of pyraclostrobin against Fol. In this study, we determined the activity of pyraclostrobin against Fol and found the EC50 values for pyraclostrobin against 100 Fol strains (which have never been exposed to QoIs before). The average EC50 value is 0.3739 ± 0.2413 µg/mL, indicating a strong antifungal activity of pyraclostrobin against Fol, as shown by unimodal curves of the EC50 values. Furthermore, we generated five resistant mutants through chemical taming and identified four mutants with high-level resistance due to the Cytb-G143S mutation and one mutant with medium-level resistance due to the Cytb-G137R mutation. The molecular docking results indicate that the Cytb-G143S or Cytb-G137R mutations of Fol lead to a change in the binding mode of Cytb to pyraclostrobin, resulting in a decrease in affinity. The resistant mutants exhibit reduced fitness in terms of mycelial growth (25 and 30 °C), virulence, and sporulation. Moreover, the mutants carrying the Cytb-G143S mutation suffer a more severe fitness penalty compared to those carrying the Cytb-G137R mutation. There is a positive correlation observed among azoxystrobin, picoxystrobin, fluoxastrobin, and pyraclostrobin for resistant mutants; however, no cross-resistance was detected between pyraclostrobin and pydiflumetofen, prochloraz, or cyazofamid. Thus, we conclude that the potential risk of resistance development in Fol toward pyraclostrobin can be categorized as ranging from low to moderate.

The G143S mutation in cytochrome b confers high resistance to pyraclostrobin in Fusarium pseudograminearum.

BACKGROUND: Wheat crown rot (WCR), primarily caused by Fusarium pseudograminearum has become more and more prevalent in winter wheat areas in China. However, limited fungicides have been registered for the control of WCR in China so far. Pyraclostrobin is a representative quinone outside inhibitor (QoI) with excellent activity against Fusarium spp. There is currently limited research on the resistance risk and resistance mechanism of F. pseudograminearum to pyraclostrobin. RESULTS: Here, we determined the activity of pyraclostrobin against F. pseudograminearum. The EC50 values ranged from 0.022 to 0.172 µg mL-1 with an average EC50 value of 0.071 ± 0.030 µg mL-1. Four highly pyraclostrobin-resistant mutants were obtained from two sensitive strains by ultraviolet (UV) mutagenesis in the laboratory. The mutants showed decreased mycelial growth rate and virulence as compared with the corresponding wild-type strains, indicating that pyraclostrobin resistance suffered a fitness penalty in F. pseudograminearum. It was found that the high resistance of four mutants was caused by the G143S mutation in Cytb. Molecular docking analysis also further confirms that the G143S mutation in Cytb decreased the binding affinity between pyraclostrobin and Cytb. CONCLUSION: The resistance risk of F. pseudograminearum to pyraclostrobin could be low to medium. Although a mutation at the G143S position of Cytb could potentially occur, this mutation decreases the fitness of the mutant, which may reduce its survival in the environment. Therefore, the negative consequences of a possible mutation are lower. This makes pyraclostrobin a good candidate for controlling crown rot in wheat. © 2024 Society of Chemical Industry.

Glis2 is an early effector of polycystin signaling and a target for therapy in polycystic kidney disease.

Mouse models of autosomal dominant polycystic kidney disease (ADPKD) show that intact primary cilia are required for cyst growth following the inactivation of polycystin-1. The signaling pathways underlying this process, termed cilia-dependent cyst activation (CDCA), remain unknown. Using translating ribosome affinity purification RNASeq on mouse kidneys with polycystin-1 and cilia inactivation before cyst formation, we identify the differential 'CDCA pattern' translatome specifically dysregulated in kidney tubule cells destined to form cysts. From this, Glis2 emerges as a candidate functional effector of polycystin signaling and CDCA. In vitro changes in Glis2 expression mirror the polycystin- and cilia-dependent changes observed in kidney tissue, validating Glis2 as a cell culture-based indicator of polycystin function related to cyst formation. Inactivation of Glis2 suppresses polycystic kidney disease in mouse models of ADPKD, and pharmacological targeting of Glis2 with antisense oligonucleotides slows disease progression. Glis2 transcript and protein is a functional target of CDCA and a potential therapeutic target for treating ADPKD.

Mutations in SEC63 cause autosomal dominant polycystic liver disease.

Mutations in PRKCSH, encoding the beta-subunit of glucosidase II, an N-linked glycan-processing enzyme in the endoplasmic reticulum (ER), cause autosomal dominant polycystic liver disease. We found that mutations in SEC63, encoding a component of the protein translocation machinery in the ER, also cause this disease. These findings are suggestive of a role for cotranslational protein-processing pathways in maintaining epithelial luminal structure and implicate noncilial ER proteins in human polycystic disease.

The G143A/S substitution of mitochondrially encoded cytochrome b (Cytb) in Magnaporthe oryzae confers resistance to quinone outside inhibitors.

BACKGROUND: Rice blast, caused by Magnaporthe oryzae, is a destructive disease threatening the production of staple foods worldwide. Quinone outside inhibitors (QoIs) are a group of chemicals exhibiting excellent activity against a majority of plant pathogens, with the disadvantage that pathogens can easily develop resistance to QoIs. RESULTS: Here, we investigated the activity of picoxystrobin against M. oryzae, which showed a great inhibitory effect on 100 strains of M. oryzae with half-maximal effective concentrations (EC50 ) ranging from 0.0251 to 0.1337 µg ml-1 . The EC50 values showed a continuous unimodal distribution that was identical to the normal distribution, suggesting the potency of our study to represent baseline sensitivity. In addition, nine resistant mutants were obtained by exposing M. oryzae to a high dosage of picoxystrobin in the laboratory; all of them showed cross-resistance to the other five QoI fungicides. Although some mutants showed a decreased resistance factor after ten successive cultures on fungicide-free medium, the resistance to picoxystrobin was still inheritable. Amino acid substitution of G143S was detected in eight of nine picoxystrobin-resistant mutants, and G143A was detected in only one of nine mutants. A fitness penalty was found in the mutants carrying G143S rather than G143A. CONCLUSION: Our findings suggested that M. oryzae had a mid to high risk of resistance to picoxystrobin. Considering this, we should be vigilant to the resistance risk and apply picoxystrobin sensibly in the field. © 2022 Society of Chemical Industry.

Evolution of Benzimidazole Resistance Caused by Multiple Double Mutations of ß-Tubulin in Corynespora cassiicola.

Cucumber target leaf spot caused by Corynespora cassiicola has devastated greenhouse cucumber production. In our previous study, the resistance monitoring of C. cassiicola to carbendazim was carried out, and a large number of resistant populations carrying various mutations (M163I&E198A, F167Y&E198A, F200S&E198A, or E198A) in ß-tubulin were detected. However, the single-point mutations M163I, F167Y, and F200S have remained undetected. To investigate the evolutionary mechanism of double mutations in ß-tubulin of C. cassiicola resistance to benzimidazoles, site-directed mutagenesis was used to construct alleles with corresponding mutation genotypes in ß-tubulin. Through PEG-mediated protoplast transformation, all the mutants except for the M163I mutation were obtained and conferred resistance to benzimidazoles. It was found that the mutants conferring the E198A or double-point mutations showed high resistance to carbendazim and benomyl, but the mutants conferring the F167Y or F200S mutations showed moderate resistance. Except, the F200S mutants showed low resistance, the resistance level of the other mutants to thiabendazole seemed no difference. In addition, compared to the other mutants, the F167Y and F200S mutants suffered a more severe fitness penalty in mycelial growth, sporulation, and virulence. Thus, combined with the resistance level, fitness, and molecular docking results, we concluded that the field double mutations (F167Y&E198A and F200S&E198A) evolved from the single mutations F167Y and F200S, respectively.

Controlling Shock-Induced Energy Release Characteristics of PTFE/Al by Adding Oxides.

Polytetrafluoroethylene (PTFE)/aluminum (Al)-based energetic material is a kind of energetic material with great application potential. In this research, the control of the shock-induced energy release characteristics of PTFE/Al-based energetic material by adding oxides (bismuth trioxide, copper oxide, molybdenum trioxide, and iron trioxide) was studied by experimentation and theoretical analysis. Ballistic impact experiments with impact velocity of 735~1290 m/s showed that the oxides controlled the energy release characteristics by the coupling of impact velocities and oxide characteristics. In these experiments, the overpressure characteristics, including the quasi-static overpressure peak, duration, and impulse, were used to characterize the energy release characteristics. It turned out that when the nominal impact velocity was 735 m/s, the quasi-static overpressure peak of PTFE/Al/MoO3 (0.1190 MPa) was 1.99 times higher than that of PTFE/Al (0.0598 MPa). Based on these experimental results, an analytical model was developed indicating that the apparent activation energy and impact shock pressure dominated the energy release characteristic of PTFE/Al/oxide. This controlling mechanism indicated that oxides enhanced the reaction after shock wave unloading, and the chemical and physical properties of the corresponding thermites also affected the energy release characteristics. These conclusions can guide the design of PTFE-based energetic materials, especially the application of oxides in PTFE-based reactive materials.

Mechanism of Fusarium graminearum Resistance to Ergosterol Biosynthesis Inhibitors: G443S Substitution of the Drug Target FgCYP51A.

Fusarium head blight (FHB), caused by the Fusarium graminearum species complex, is a devastating fungal disease resulting in substantial yield and quality losses. Ergosterol biosynthesis inhibitors (EBIs) are the most popular chemicals for controlling FHB. Recently, the resistance of F. graminearum to EBIs has emerged in the field, and an amino acid substitution (G443S) of the sterol 14α-demethylase FgCYP51A was detected in the field resistant strains. To further illustrate the resistance mechanism of F. graminearum to EBIs, site-directed mutants conferring the G443S substitution of FgCYP51A were generated from the progenitor strain PH-1 via genetic transformation with site-directed mutagenesis. We found that the FgCYP51A-G443S substitution significantly decreased the sensitivity of F. graminearum to EBIs with EC50 values ranging from 0.1190 to 0.2302 µg mL-1 and EC90 values ranging from 1.3420 to 9.1119 µg mL-1 for tebuconazole. Furthermore, the FgCYP51A-G443S substitution decreased sexual reproduction and virulence, which will reduce the initial infection source of pathogen populations in the field, while the increase of sporulation capability may enhance the frequencies of the disease cycle, thereby contributing to epidemics of FHB disease. Surprisingly, the FgCYP51A-G443S substitution accelerated DON biosynthesis by upregulating TRI5 expression and enhancing the fluorescence intensity of TRI1-GFP, the marker protein of Fusarium toxisomes. Thus, we concluded that the FgCYP51A-G443S substitution regulates EBI-fungicide resistance and DON biosynthesis, increasing the risk of fungicide resistance development in the field, thereby threatening the control efficacy of EBIs against FHB.

Cytochrome bc1 Complex: Potential Breach to Improve the Activity of Phenazines on Xanthomonas.

The effects of the natural pesticides, phenazines, were reported to be limited by some tolerant metabolism processes within Xanthomonas. Our previous studies suggested that the functional cytochrome bc1 complex, the indispensable component of the respiration chain, might participate in tolerating phenazines in Xanthomonas. In this study, the cytochrome bc1 mutants of Xanthomonas campestris pv. campestris (Xcc) and Xanthomonas oryzae pv. oryzae (Xoo), which exhibit different tolerance abilities to phenazines, were constructed, and the cytochrome bc1 complex was proven to partake a critical and conserved role in tolerating phenazines in Xanthomonas. In addition, results of the cytochrome c mutants suggested the different functions of the various cytochrome c proteins in Xanthomonas and that the electron channeled by the cytochrome bc1 complex to cytochrome C4 is the key to reveal the tolerance mechanism. In conclusion, the study of the cytochrome bc1 complex provides a potential strategy to improve the activity of phenazines against Xanthomonas.

Polycystin-2 activation by inositol 1,4,5-trisphosphate-induced Ca2+ release requires its direct association with the inositol 1,4,5-trisphosphate receptor in a signaling microdomain.

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP(3)R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.

Polycystin-2 is an intracellular calcium release channel.

Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.