作者机构：
[Zheng, Yong; Guo, Jilin; Wang, Hongbin; Tian, Zhongping; Li, Jin] Xinjiang Normal Univ, Xinjiang Key Lab Special Species Conservat & Regu, Coll Life Sci, Urumqi 830054, Peoples R China.;[Yuan, Yongze; Zheng, Yong; Zheng, Y; Deng, Lingfu] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.
通讯机构：
[Zheng, Yong] X;[Zheng, Y; Yuan, YZ] C;Xinjiang Normal Univ, Xinjiang Key Lab Special Species Conservat & Regu, Coll Life Sci, Urumqi 830054, Peoples R China.;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.
摘要：
The traditional Chinese desert shrub Lycium ruthenicum is widely distributed in arid environments such as north-west China, exhibiting ideal salt tolerance to cope with soil desertification, salinity, and alkalinity. However, the salt-tolerance mechanism of L. ruthenicum, especially of its young seedlings at early vegetative stages, remains largely unknown. In the present study, we collected whole-seedling samples from Lycium ruthenicum at a-pair-leaf stage with and without a mild salt (75 mM sodium chloride) treatment, and then performed transcriptome profiling to compare their gene expression patterns. The de novo assembly achieved 94,651 unigenes with 55,156 annotated. Among them, 199 differentially expressed genes (DEGs) were identified between salt-treated seedlings and control, with 41 up-regulated and 158 down-regulated. These DEGs were highly enriched into gene ontology (GO) classifications 'metabolic process' and 'catalytic activity', into Clusters of Orthologous Groups (COG) function classifications 'translation, ribosomal structure and biogenesis' and 'energy production and conversion', and into Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways 'ribosome' and 'oxidative phosphorylation'. Specifically, genes involved in energy metabolism (oxidative phosphorylation) and related energy-consuming metabolisms, including ribosome-associated biogenesis and biosynthesis of organic nitrogen-derived compatible solutes (i.e., arginine and proline), were generally down-regulated. Specific genes involved in abscisic acid (ABA) biosynthesis and signaling pathway were simultaneously up-regulated. Changes in the transcript levels of salt-responsive DEGs selected from the transcriptomic profiling were further validated by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Based on these results, salinity-adaptive strategies for the L. ruthenicum early seedlings are discussed.
摘要：
Penicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified. The present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6 h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2, ERG6 and EGR11 (CYP51A), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1, and Ca2+/calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2. Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes (i.e., Bck1 and Slt2) required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms (CYP51A/B/C), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses. The present findings provided transcriptomic evidence on P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.
摘要：
Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum (P. italicum). In this study, we isolated and characterized a new double-stranded RNA (dsRNA) virus, designated Penicillium italicum chrysovirus 1 (PiCV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that PiCV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1DeltapksP defective in the polyketide synthase gene (pksP), which is involved in pigment biosynthesis, and these mutants formed albino (white) colonies. Then we applied hyphal anastomosis method to horizontally transmit PiCV1 from the white virus-donors (i.e., HSPi-YN1 mutants) to wild-type recipients (i.e., P. italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable PiCV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic dsRNA profile analysis. This blue-white colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized PiCV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen PiCV1-infected isogenic recipients.
摘要：
Penicillium sp. are damaging to a range of foods and fruits including citrus. To date, double-stranded (ds)RNA viruses have been reported in most Penicillium species but not in citrus pathogen P. crustosum. Here we report a novel dsRNA virus, designated as Penicillium crustosum chrysovirus 1 (PcCV1) and isolated from P. crustosum strain HS-CQ15. PcCV1 genome comprises four dsRNA segments, referred to as dsRNA1, dsRNA2, dsRNA3, and dsRNA4, which are 3600, 3177, 3078, and 2808 bp in length, respectively. Sequence analysis revealed the presence of four open reading frames (ORFs) in the PcCV1 genome. ORF1 in dsRNA1 encodes a putative RNA-dependent RNA polymerase (RdRp) and ORF2 in dsRNA2 encodes a putative coat protein (CP). The two remaining ORFs, ORF3 in dsRNA3 and ORF4 in dsRNA4, encode proteins of unknown function. Phylogenetic analysis based on RdRp sequences showed that PcCV1 clusters with other members of the genus Chrysovirus, family Chrysoviridae. Transmission electron microscope (TEM) analysis revealed that the PcCV1 visions are approximately 40 nm in diameter. Regarding biological effects of PcCV1, HS-CQ15 harboring the chrysovirus exhibited no obvious difference in colony morphology under fungicide-free conditions but decreased resistance to demethylation inhibitor (DMI)-fungicide prochloraz, as compared to PcCV1-cured strain. Here we provide the first evidence of a virus present in citrus pathogenic fungus P. crustosum and the chrysovirus-induced change in fungicide-resistance of its host fungus.
作者机构：
[Yuan, Yongze; Liu, Deli; Niu, Yuhui; Mao, Jiali; Wang, Menglan; Zhang, Tingfu; Yang, Zhu; Geng, Hui; Liu, DL] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.;[Zheng, Yongliang] Huanggang Normal Univ, Coll Life Sci, Huanggang 438000, Hubei, Peoples R China.
通讯机构：
[Yuan, YZ; Liu, DL] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.
摘要：
To date, partitiviruses, including gammapartitiviruses, have been extensively studied in various fungal hosts but have not been reported in Penicillium digitatum (also called green mold, the pathogenic fungus infecting citrus). In the present work, we isolated and molecularly characterized a double-stranded RNA (dsRNA) partitivirus from citrus green mold, which we have named "Penicillium digitatum gammapartitivirus 1" (PdGV1). The bisegmented genome of PdGV1 contains two dsRNA segments (dsRNA1 and dsRNA2) with a length of 1795 bp and 1622 bp, respectively. Each of the two genomic dsRNAs contains a single open reading frame encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. Phylogenetic analysis based on RdRp and CP sequences showed that PdGV1 clustered with mycoviruses belonging to the genus Gammapartitivirus, family Partitiviridae, e.g., Penicillium stoloniferum virus S. The 5'- and 3'-untranslated regions (UTRs) of the PdGV1 genomic dsRNAs both contained unique conserved RNA motifs that have never been found in any other partitivirus. This is the first report of a new gammapartitivirus that infects the citrus-pathogenic fungus P. digitatum.
作者机构：
[Yuan, Yongze; Cao, Qianwen; Liu, Deli; Wang, Shengqiang; Niu, Yuhui; Mao, Jiali; Zhang, Tingfu; Yang, Zhu] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.
通讯机构：
[Liu, Deli] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.
摘要：
Pathogenic fungi including Penicillium digitatum and Penicillium italicum are the main destructive pathogens in the citrus industry, causing great losses during postharvest process. To our knowledge, only one mycovirus from P. digitatum has been reported, and the prevalence of such mycoviruses against citrus postharvest pathogenic fungi and their genotyping were still under investigation. In the present study, we showed that 39 of 152 Penicillium isolates from main citrus-growing areas in China were infected with various mycoviruses belonging to polymycoviruses, Narna-like viruses, and families Totiviridae, Partitivirdae and Chrysoviridae. The next generation sequencing (NGS) towards virus genome library and the following molecular analysis revealed two novel mycoviruses Penicillium digitatum polymycovirus 1 (PdPmV1) and Penicillium digitatum Narna-like virus 1 (PdNLV1), coexisting in P. digitatum strain HS-RH2. The fungicide-resistant P. digitatum strains HS-F6 and HS-E9 coinfected by PdPmV1 and PdNLV1 exhibited obvious reduction in triazole drug prochloraz resistance by mycelial growth analysis on both PDA plates and citrus fruit epidermis with given prochloraz concentration. This report at the first time characterized two novel mycoviruses from P. digitatum and revealed the mycovirus-induced reduction of fungicide resistance.
摘要：
Sterol 14α-demethylases from Cytochrome P450 family (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. The 3D structure of CYP51A from Penicillium italicum (PiCYP51A) was constructed through homology modeling based on the crystal structure of human CYP51A (PDB: 3LD6). Molecular dynamics (MD) simulation was operated to relax the initial model and followed by quality assessment using PROCHECK program. On the basis of the docking information on the currently available CYP51s with the patent demethylase inhibitors (DMIs), pharmacophore-based virtual screening combined with docking analysis was performed to pick out twelve new compounds from ZINC database. Six hits revealed in the ligand database suggested potential ability to inhibit PiCYP51A. Compared to patent fungicide triazolone, the top three lead compounds had similar or higher affinity with the target enzyme, and accordingly, exhibited comparable or lower EC50 values to P. italicum isolates. The results could provide references for de novo antifungal drug design.
摘要：
Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 between nitrogen (N) and oxygen (O) in their structures, including triethanolamine (TEA), diethanolamine, monoethanolamine, 1-amino-2-propanol, 2-amino-2-methyl-1-propanol, and triisopropanolamine. In contrast, much lower or no rate-enhancing effects were observed in the adding of amines, alcohols, amine/alcohol mixtures, or 3-amino-1-propanol (C3 between N and O). The 300 mM TEA further increased DFP-degrading activities of OPH mutants F132Y and L140Y, the previously reported OPH mutants with desirable activities towards DFP. However, the treatment of ethylenediaminetetraacetate (EDTA) markedly abolished the TEA-induced activation of OPH. The product fluoride effectively inhibited OPH-catalyzed hydrolysis of DFP by a linear mixed inhibition (inhibition constant Ki ~ 3.21 mM), which was partially released by TEA adding at initial or later reaction stage. The obtained results indicate the activation of OPH by aminoalcohol buffers could be attributed to the reduction of fluoride inhibition, which would be beneficial to the hydrolase-based detoxification of organophosphofluoridate.
作者机构：
[Yuan, Y. Z.; Li, D. D.; Chen, S. L.; Zhang, Y. Z.; Liu, D. L.; Niu, Y. H.; Li, S. X.; Liu, J.; Geng, H.; Li, N.] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.;[Li, N.] Honghe Univ, Key Lab Crops High Qual & Efficient Cultivat & Se, Yunnan Higher Educ Inst, Coll Life Sci & Technol, Mengzi 661100, Peoples R China.
通讯机构：
[Liu, D. L.] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.
作者机构：
[Yuan, Y. Z.; Li, D. D.; Zhang, YZ; Liu, DL; Chen, S. L.; Zhang, Y. Z.; Liu, D. L.; Niu, Y. H.; Li, S. X.; Liu, J.; Geng, H.; Li, N.] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.;[Li, N.] Honghe Univ, Coll Life Sci & Technol, Yunnan Higher Educ Inst, Key Lab Crops High Qual & Efficient Cultivat & Se, Mengzi 661100, Peoples R China.
通讯机构：
[Zhang, YZ; Liu, DL] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.
摘要：
Cytochrome P450nor catalyzes an unusual reaction that transfers electrons from NADP/NADPH to bound heme directly. To improve the expression level of P450nor2 from Cylindrocarpon tonkinense (C.P450nor2), Escherichia coli system was utilized to substitute the yeast system we constructed for expression of the P450nor2 gene, and the protein was purified in soluble form using Ni+-NTA affinity chromatography. In contrast to P450nor from Fusarium oxysporum (F.P450nor) and P450nor1 from Cylindrocarpon tonkinense (C.P450nor1), C.P450nor2 shows a dual specificity for using NADH or NADPH as electron donors. The present study developed a computational approach in order to illustrate the coenzyme specificity of C.P450nor2 for NADH and NADPH. This study involved homology modeling of C.P450nor2 and docking analyses of NADH and NADPH into the crystal structure of F.P450nor and the predictive model of C.P450nor2, respectively. The results suggested that C.P450nor2 and F.P450nor have different coenzyme specificity for NADH and NADPH; whilst the space around the B'-helix of the C.P450nor2, especially the Ser79 and Gly81, play a crucial role for the specificity of C.P450nor2. In the absence of the experimental structure of C.P450nor2, we hope that our model will be useful to provide rational explanation on coenzyme specificity of C.P450nor2.
作者机构：
[Yuan, Yongze; Liu, Jing; Liu, Deli; Wang, Shengqiang; Niu, Yuhui; Zhang, Tingfu] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.;[Zhu, Ying] Wuhan Univ, Coll Life Sci, State Key Lab Virol, Wuhan 430072, Peoples R China.
通讯机构：
[Liu, Deli] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.
关键词：
dsRNA mycovirus;Penicillium digitatum;PdV1;Victorivirus in Totiviridae;Transfection;Hypovirulence
摘要：
A novel double-stranded RNA virus designated Penicillium digitatum virus 1 (PdV1) was isolated from the citrus fruit rot pathogen P. digitatum (HS-RH1). The full-length cDNA sequence of the dsRNA/PdV1 (5211 bp) possesses two partially overlapping open reading frames, which encode a coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on multiple alignments of the amino acid sequences of the RdRp and CP indicated that PdV1 tentatively belongs to the genus Victorivirus in the Totiviridae family. Electron micrographs of negatively stained viral particles purified from the peak fraction of sucrose density gradient centrifugation showed spherical particles similar to 35 nm in diameter. Transfection experiments with purified virions indicated that PdV1 could reduce the vegetative growth and virulence of P. digitatum strain HS-F6. In summary, we report the first isolation and characterization of a mycovirus from P. digitatum that contributes to the hypovirulence phenotypes of the host strain. (C) 2016 Elsevier Inc. All rights reserved.
摘要：
Sucrose has been known as pivotal carbon source for plant nutrient metabolism including ammonium (NH4
+) assimilation. However, the correlation between rice root NH4
+ assimilation and gene expression responsible for sucrose allocation has not been investigated. Here, we reported the transcriptional regulation of OsSUTs and OsSPSs by exogenously supplied sucrose and the response of NH4
+ assimilation in rice roots. Spraying sucrose to mature leaves of rice (Oryza sativa L. cv. indica 9311) up-regulated transcript abundances of leaf OsSUT1, OsSUT2, OsSUT3, OsSUT4, OsSPS6, and OsSPS11, down-regulated mRNA expression of leaf OsSUT5, OsSPS1, OsSPS2, and OsSPS8, increased soluble sugar contents in leaves and roots, and promoted root NH4
+ assimilation. The similar responses, except for leaf OsSPS1 mRNA expression, were observed when sucrose was fed to hydroponic media. Rice (indica 9311) treated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) exhibited altered transcript abundances of leaf OsSUTs and OsSPSs, lower contents of soluble sugars in leaves and roots, and reduced capacity of root NH4
+ assimilation, which was partially reversed by sucrose supply. The results indicated that the exogenously supplied sucrose coordinately regulated leaf OsSUTs and OsSPSs mRNA expression and root NH4
+ assimilation in rice seedlings.
