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1.
Proc Natl Acad Sci U S A ; 119(50): e2214096119, 2022 12 13.
Article in English | MEDLINE | ID: mdl-36469771

ABSTRACT

Mycovirus-infected fungi can suffer from poor growth, attenuated pigmentation, and virulence. However, the molecular mechanisms of how mycoviruses confer these symptoms remain poorly understood. Here, we report a mycovirus Stemphylium lycopersici alternavirus 1 (SlAV1) isolated from a necrotrophic plant pathogen Stemphylium lycopersici that causes altered colony pigmentation and hypovirulence by specifically interfering host biosynthesis of Altersolanol A, a polyketide phytotoxin. SlAV1 significantly down-regulates a fungal polyketide synthase (PKS1), the core enzyme of Altersolanol A biosynthesis. PKS1 deletion mutants do not accumulate Altersolanol A and lose pathogenicity to tomato and lettuce. Transgenic expression of SlAV1 open-reading frame 3 (ORF3) in S. lycopersici inhibits fungal PKS1 expression and Altersolanol A accumulation, leading to symptoms like SlAV1-infected fungal strains. Multiple plant species sprayed with mycelial suspension of S. lycopersici or S. vesicarium strains integrating and expressing ORF3 display enhanced resistance against virulent strains, converting the pathogenic fungi into biocontrol agents. Hence, our study not only proves inhibiting a key enzyme of host phytotoxin biosynthesis as a molecular mechanism underlying SlAV1-mediated hypovirulence of Stemphylium spp., but also demonstrates the potential of mycovirus-gene integrated fungi as a potential biocontrol agent to protect plants from fungal diseases.


Subject(s)
Ascomycota , Fungal Viruses , Plant Diseases/microbiology , Fungal Viruses/genetics , Plants
2.
Planta ; 253(6): 122, 2021 May 18.
Article in English | MEDLINE | ID: mdl-34003383

ABSTRACT

MAIN CONCLUSION: The rice OsFAH gene functions identically to that of Arabidopsis SSCD1 encoding FAH. Loss of OsFAH causes rice sterility. Fumarylacetoacetate hydrolase (FAH) is the last enzyme in the tyrosine (Tyr) degradation pathway that is crucial for animals. By genetic analysis of the mutant of Short-day Sensitive Cell Death 1 gene encoding Arabidopsis FAH, we first found the pathway also plays a critical role in plants (Han et al., Plant Physiol 162:1956-1964, 2013). To further understand the role of the Tyr degradation pathway in plants, we investigated a biological function of the rice FAH. Firstly, the cDNA of rice FAH gene (OsFAH) was cloned and confirmed to be able to rescue the Arabidopsis Short-day Sensitive Cell Death 1 mutant defective in the FAH. Then, we identified the OsFAH T-DNA insertion mutant and generated the OsFAH RNA interference lines, and found that loss of OsFAH results in rice sterility. Furthermore, we analyzed expression of the OsFAH gene in roots, stems, leaves and young panicles at booting stage of rice and found that its transcript level was highest in young panicles and lowest in roots. In addition, the expression analysis of ß-glucuronidase driven by OsFAH promoter in transgenic Arabidopsis showed that the OsFAH promoter was highly active in aerial tissues in vegetative stage, and sepals, filaments and stigma in reproductive stage. These results suggested that FAH plays an important role in rice fertility.


Subject(s)
Arabidopsis , Oryza , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Fertility , Gene Expression Regulation, Plant , Hydrolases/genetics , Hydrolases/metabolism , Oryza/genetics , Oryza/metabolism , Plants, Genetically Modified/metabolism
3.
Mycobiology ; 46(2): 85-91, 2018.
Article in English | MEDLINE | ID: mdl-29963309

ABSTRACT

Endophytic fungi strains (n = 81) were isolated from the leaves, barks, and fruits of Camellia oleifera from Hunan province (China) to delineate their species composition and potential as biological control agents of C. oleifera anthracnose. The fungi were identified by morphological and phylogenetic analyses. Fungal colonization rates of the leaves, barks, and fruits were 58.02, 27.16, and 14.81%, respectively. The isolates were identified as 14 genera, belonging to two subdivisions, Deuteromycotina and Ascomycotina; 87.65% of all isolates belonged to Deuteromycotina. The dominant species, occurring with a high relative frequency, were Pestalotiopsis sp. (14.81%), Penicillium sp. (14.81%), and Fusarium sp. (12.35%). The Simpson's and Shannon's diversity indices revealed the highest species diversity in the leaves, followed by the barks and fruits. The similarity index for the leaves versus barks comparison was the highest, indicating that the number of endophytic fungal species shared by the leaves and barks was higher than barks and fruits or leaves and fruits. Based on the results of dual culture experiments, only five strains exhibited antifungal activity against C. oleifera anthracnose pathogen, with isolate ty-64 (Oidium sp.) generating the broadest inhibition zones. Our results indicate that the endophytes associated with C. oleifera could be employed as natural agents controlling C. oleifera anthracnose.

4.
Planta ; 248(2): 499-511, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29785518

ABSTRACT

MAIN CONCLUSION: Fumarylacetoacetate hydrolase participates in positive regulation of salt stress in Arabidopsis. Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolysis of fumarylacetoacetate into fumarate and acetoacetate, the final step in the Tyr degradation pathway that is essential to animals. However, the Tyr degradation pathway is not well understood in plants. Previously, we found that mutation of the SHORT-DAY SENSITIVE CELL DEATH 1 (SSCD1) gene encoding FAH in Arabidopsis causes spontaneous cell death under short day, which first indicated that the Tyr degradation pathway also plays an important role in plants. In this study, we found that the SSCD1 gene was up-regulated by salt stress, and the sscd1 mutant was hypersensitive to salt stress. However, the double mutant of SSCD1 and HOMOGENTISATE DIOXYGENASE, in which intermediates of the Tyr degradation pathway could not be produced, displayed a normal response to salt stress. Furthermore, the sscd1 mutant showed more accumulation of reactive oxygen species (ROS) and less up-regulation of some ROS-scavenging genes such as ASCORBATE PEROXIDASE 2 and COPPER/ZINC SUPEROXIDE DISMUTASE 1 compared with wild type under salt stress. In addition, SSCD1 expression was also up-regulated by H2O2, and the sscd1 mutant exhibited hypersensitivity to oxidative stress compared with wild type. Taken together, we concluded that loss of FAH in sscd1 leads to the accumulation of Tyr degradation intermediates, which impairs the up-regulation of some ROS-scavenging genes under salt stress, causing more accumulation of ROS, resulting in the hypersensitivity of sscd1 to salt stress.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Gene Expression Regulation, Plant , Hydrolases/metabolism , Reactive Oxygen Species/metabolism , Stress, Physiological , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Ascorbate Peroxidases/metabolism , Cell Death , Homogentisate 1,2-Dioxygenase/metabolism , Hydrogen Peroxide/metabolism , Hydrolases/genetics , Mutation , Oxidative Stress , Salt Tolerance , Up-Regulation
5.
Front Plant Sci ; 8: 2122, 2017.
Article in English | MEDLINE | ID: mdl-29312386

ABSTRACT

The hypersensitive response (HR) is a mechanism by which plants prevent the spread of pathogen. Despite extensive study, the molecular mechanisms underlying HR remain poorly understood. Lesion mimic mutants (LMMs), such as LIL1 that was identified in an ethylmethane sulfonate mutagenized population of Indica rice (Oryza sativa L. ssp. Indica) 93-11, can be used to study the HR. Under natural field conditions, the leaves of LIL1 mutant plants exhibited light-induced, small, rust-red lesions that first appeared at the leaf tips and subsequently expanded throughout the entire leaf blade to the leaf sheath. Histochemical staining indicated that LIL1 lesions displayed an abnormal accumulation of reactive oxygen species (ROS) and resulted from programmed cell death (PCD). The LIL1 mutants also displayed increased expression of defense-related genes and enhanced resistance to rice blast fungus (Magnaporthe grisea). Genetic analysis showed that mutation of LIL1 created a semi-dominant allele. Using 1,758 individuals in the F2 population, LIL1 was mapped in a 222.3 kb region on the long arm of chromosome 7. That contains 12 predicted open reading frames (ORFs). Sequence analysis of these 12 candidate genes revealed a G to A base substitution in the fourth exon of LOC_Os07g30510, a putative cysteine-rich receptor-like kinase (CRK), which led to an amino acid change (Val 429 to Ile) in the LIL1 protein. Comparison of the transcript accumulation of the 12 candidate genes between LIL1 and 93-11 revealed that LOC_Os07g30510 was up-regulated significantly in LIL1. Overexpression of the LOC_Os07g30510 gene from LIL1 induced a LIL1-like lesion phenotype in Nipponbare. Thus, LIL1 is a novel LMM in rice that will facilitate the further study of the molecular mechanisms of HR and the rice blast resistance.

6.
Arch Virol ; 161(3): 725-9, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26650038

ABSTRACT

Here, we report a novel virus isolated from rice blast fungus, Magnaporthe oryzae, an important plant pathogen. This virus has an RNA genome of 3246 nucleotides. Its genome possesses two in-frame open reading frames (ORFs). The smaller ORF1 encodes a protein with significant similarity to a protein encoded by the ssRNA mycovirus Diaporthe ambigua RNA virus 1 (DaRV1). The larger ORF2 encodes a protein with similarity to RNA-dependent RNA polymerases (RdRp) of DaRV1 and other plant viruses of the family Tombusviridae. In silico analysis and comparisons with DaRV1 genome expression suggest that ORF2 is translated via a readthrough mechanism together with ORF1. Based upon results of this study, this virus, for which the provisional name Magnaporthe oryzae virus A (MoVA) is proposed, belongs to a new virus species. Furthermore, MoVA along with DaRV1 belong to a new taxon of mycoviruses that are evolutionarily related to plant viruses belonging to the family Tombusviridae.


Subject(s)
Magnaporthe/virology , RNA Viruses/classification , RNA Viruses/isolation & purification , Tombusviridae/genetics , Cluster Analysis , Molecular Sequence Data , Open Reading Frames , Oryza/microbiology , Phylogeny , Plant Diseases/microbiology , RNA Viruses/genetics , RNA, Viral/genetics , Sequence Analysis, DNA , Sequence Homology
7.
Virus Genes ; 51(1): 167-70, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26116286

ABSTRACT

Here we present the genome sequence of a novel dsRNA virus we designed as Rhizoctonia solani RNA virus HN008 (RsRV-HN008) from a filamentous fungus R. solani. Its genome (7596 nucleotides) contains two non-overlapping open reading frames (ORF1 and ORF2). ORF1 encoded a 128 kDa protein that showed no significant identity to any other virus sequence in the NCBI database. ORF2 encoded a protein with a molecular weight of 140 kDa and shared a low percentage of sequence identity to the RdRps of unclassified dsRNA viruses. Sequence analysis revealed that RsRV-HN008 may be a member of a novel unclassified family of mycoviruses.


Subject(s)
Fungal Viruses/genetics , Fungal Viruses/isolation & purification , Genome, Viral , RNA Viruses/genetics , RNA Viruses/isolation & purification , RNA, Viral/genetics , Rhizoctonia/virology , Cluster Analysis , Fungal Viruses/classification , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Phylogeny , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Viral Proteins/chemistry , Viral Proteins/genetics
8.
Arch Virol ; 160(7): 1827-30, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25951967

ABSTRACT

In an effort to discover new mycoviruses from phytopathogenic fungi, a dsRNA molecule of 10,290 nt, resembling those associated with the viruses belonging to the family Endornaviridae, was isolated from Alternaria brassicicola, one of the causal agents of rapeseed black spot disease. Genome analysis revealed the presence of a single open reading frame coding for a polyprotein of 3400 aa containing conserved viral methyltransferase (MTR), viral RNA helicase 1 (Hel-1), and RNA-dependent RNA polymerase (RdRp) domains. In addition, a cysteine-rich region (CRR) with conserved CXCC motifs, shared among several endornaviruses, was also identified between the MTR and Hel-1 domains. Phylogenetic analysis based on the RdRp sequence strongly suggested that the virus infecting A. brassicicola should be considered a representative of a novel endornavirus species, and this virus was designated as Alternaria brassicicola endornavirus 1 (AbEV1).


Subject(s)
Alternaria/virology , Genome, Viral , Plant Diseases/microbiology , RNA Viruses/genetics , Amino Acid Sequence , Base Sequence , Brassica rapa/microbiology , Molecular Sequence Data , Phylogeny , RNA Viruses/classification , RNA Viruses/isolation & purification , Sequence Alignment , Viral Proteins/chemistry , Viral Proteins/genetics
9.
Virus Genes ; 48(2): 329-33, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24510355

ABSTRACT

In this study, three dsRNA segments from the rice false smut fungus Ustilaginoidea virens, the causal agent of a serious disease in rice, with molecular size ranging from 1.3 to 5 Kb, were isolated and named as dsRNA-L, dsRNA-M, and dsRNA-S. The complete nucleotide sequences of dsRNA-M and dsRNA-S were determined and analyzed. The dsRNA-M putatively encodes an RNA-dependent RNA polymerase, which is similar to that of the partitiviruses in the family Partitiviridae. Although the protein encoded by dsRNA-S showed less similarity to the typical coat protein of the virus in the family Partitiviridae, the structural analysis results indicated that the dsRNA-S might function as the capsid protein. We propose that the virus is Ustilaginoidea virens partitivirus 2-Uv0901, a new member, but distantly related to the newly proposed genus Gammapartitivirus with a distinct sequence pattern of capsid protein.


Subject(s)
Genome, Fungal , Oryza/microbiology , Ustilaginales/genetics , Amino Acid Sequence , Molecular Sequence Data , Sequence Homology, Amino Acid , Ustilaginales/virology
10.
Plant Signal Behav ; 6(4): 575-7, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21445012

ABSTRACT

Jasmonates (JAs) induce leaf senescence in many plant species. The Arabidopsis F-box protein coronatine insensitive 1 (COI1) is required for various JA-regulated plant responses including plant fertility, defense responses and leaf senescence. However, the molecular basis for COI1-dependent JA-induced leaf senescence remains unknown. In our Plant Physiology paper, we identified a COI1-dependent JA-repressed protein, Rubisco activase (RCA) in Arabidopsis. Further genetic and physiological analyses showed that the COI1-dependent JA repression of RCA correlated with JA-induced leaf senescence, and that loss of RCA led to typical senescence-associated features. Therefore, we suggested that the COI1-dependent JA repression of RCA played an important role in JA-induced leaf senescence. In this addendum, we made a relatively deep discussion on RCA function in JA-induced leaf senescence and JA-mediated defense responses. We also discussed the possible role of JA in plant natural senescence.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cellular Senescence/drug effects , Cyclopentanes/pharmacology , Oxylipins/pharmacology , Plant Leaves/metabolism , Acetates/pharmacology , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cellular Senescence/genetics , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/genetics , Plant Leaves/drug effects , Plant Leaves/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/drug effects , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism
12.
Plant Physiol ; 151(3): 1412-20, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19741050

ABSTRACT

The F-box protein CORONATINE INSENSITIVE1 (COI1) plays a central role in jasmonate (JA) signaling and is required for all JA responses in Arabidopsis (Arabidopsis thaliana). To dissect JA signal transduction, we isolated the partially suppressing coi1 (psc1) mutant, which partially suppressed coi1 insensitivity to JA inhibition of root growth. The psc1 mutant partially restored JA sensitivity in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background. Genetic mapping, sequence analysis, and complementation tests revealed that psc1 is a leaky mutation of DWARF4 (DWF4) that encodes a key enzyme in brassinosteroid (BR) biosynthesis. Physiological analysis showed that an application of exogenous BR eliminated the partial restoration of JA sensitivity by psc1 in coi1-2 background and the JA hypersensitivity of psc1 in wild-type COI1 background. Exogenous BR also attenuated JA inhibition of root growth in the wild type. In addition, the expression of DWF4 was inhibited by JA, and this inhibition was dependent on COI1. These results indicate that (1) BR is involved in JA signaling and negatively regulates JA inhibition of root growth, and (2) the DWF4 is down-regulated by JA and is located downstream of COI1 in the JA-signaling pathway.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Cyclopentanes/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Oxylipins/pharmacology , Plant Growth Regulators/pharmacology , Plant Roots/growth & development , Amino Acid Sequence , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Base Sequence , Chromosome Mapping , Cloning, Molecular , Cytochrome P-450 Enzyme System/genetics , Gene Expression Regulation, Plant , Genetic Complementation Test , Molecular Sequence Data , Mutation , Plant Roots/drug effects , Sequence Analysis, DNA , Signal Transduction
13.
J Integr Plant Biol ; 50(5): 630-7, 2008 May.
Article in English | MEDLINE | ID: mdl-18713432

ABSTRACT

The transcription factor WRKY70 was previously reported to be a common component in salicylic acid (SA) and jasmonate (JA) mediated signal pathways in Arabidopsis. Here, we present that the inactivation of the WRKY70 gene in wrky70-1 mutant does not alter the responses of both JA and SA, and that wrky70 mutation is unable to restore the coi1 mutant in JA responses. However, overexpression of WRKY70 reduces JA responses such as expression of JA-induced genes and JA-inhibitory root growth, and activates expression of SA-inducible PR1. These data indicate that the WRKY70 is important but not indispensable for JA and SA signaling, and that other regulators may display the redundant role with WRKY70 in modulation of JA and SA responses in Arabidopsis. Furthermore, we showed that JA inhibits expression of WRKY70 and PR1 by both COI1-dependent and COI1-independent pathways.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/drug effects , Arabidopsis/metabolism , Cyclopentanes/pharmacology , Oxylipins/pharmacology , Salicylic Acid/pharmacology , Signal Transduction/drug effects , Transcription Factors/metabolism , Arabidopsis Proteins/genetics , Blotting, Northern , DNA, Bacterial , Fertility/drug effects , Flowers/drug effects , Flowers/physiology , Gene Expression Regulation, Plant/drug effects , Mutagenesis, Insertional , Mutation/genetics , Transcription Factors/genetics
14.
Sheng Wu Gong Cheng Xue Bao ; 24(2): 239-44, 2008 Feb.
Article in Chinese | MEDLINE | ID: mdl-18464607

ABSTRACT

A cDNA, named Dd-ace-2, encoding an acetylcholinesterase (AChE, EC3.1.1.7), was isolated from sweet-potato-stem nematode, Ditylenchus destructor. The nucleotide and amino acid sequences among different nematode species were compared and analyzed with DNAMAN5.0, MEGA3.0 softwares. The results showed that the complete nucleotide sequence of Dd-ace-2 gene of Ditylenchus destructor contains 2425 base pairs from which deduced 734 amino acids (GenBank accession No. EF583058). The homology rates of amino acid sequences of Dd-ace-2 gene between Ditylenchus destructor and Meloidogyne incognita, Caenorhabditis elegans, Dictyocaulus viviparous were 48.0%, 42.7%, 42.1% respectively. The mature acetylcholinesterase sequences of Ditylenchus destructor may encode by the first 701 residues of deduced 734 amino acids.The conserved motifs involved in the catalytic triad, the choline binding site and 10 aromatic residues lining the catalytic gorge were present in the Dd-ace-2 deduced protein. Phylogenetic analysis based on AChEs of other nematodes and species showed that the deduced AChE formed the same cluster with ACE-2s.


Subject(s)
Acetylcholinesterase/genetics , Genes, Helminth/genetics , Ipomoea batatas/parasitology , Nematoda/enzymology , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/genetics , Molecular Sequence Data , Nematoda/genetics , Plant Stems/parasitology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, Protein
15.
Curr Microbiol ; 56(5): 442-6, 2008 May.
Article in English | MEDLINE | ID: mdl-18259812

ABSTRACT

In order to improve the insecticidal activity, the chitinase gene from tobacco (Nicotiana tabacum) endochitinase and the cry1Ac gene from Bacillus thuringiensis were cloned into the vector pHT315 and designated as pHUAccB5 plasmid. The constructed transcriptional fusion was attempted under the control of the native cry1Ac promoter. Plasmid pHUAccB5 was introduced into B. thuringiensis acrystalliferous by electroporation. Analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot, the transformant XBU-HUAccB5 produced 130-kDa Cry1Ac protein and 30-kDa chitinase protein. During the chitinase active analysis, the transformant, XBU-HUAccB5 chitinase active, reached 7.5 U/mL at 72 h, and was 5 times higher than the HTX-42 and 6 times higher than the parent strains. When the insecticidal activity of the transformant was evaluated against Helicoverpa armigera Hubner, the XBU-HUAccB5 toxicity was 11.30 times higher than the transformant HTX-42 expressed single cry1Ac at 48 h and was 18.76 times higher at 72 h.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Chitinases/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Pest Control, Biological , Animals , Bacillus thuringiensis/enzymology , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Electroporation , Endotoxins/metabolism , Gene Expression Regulation, Bacterial , Genetic Vectors , Hemolysin Proteins/metabolism , Moths , Plasmids/genetics , Nicotiana/enzymology , Nicotiana/genetics
16.
Zhongguo Zhong Yao Za Zhi ; 32(8): 664-7, 2007 Apr.
Article in Chinese | MEDLINE | ID: mdl-17608213

ABSTRACT

OBJECTIVE: To study viruses infecting Pinellia ternata in China. METHOD: Symptom observation, DAS-ELISA and RT-PCR detection were applied. RESULT AND CONCLUSION: During a survey in early spring, SMV and CMV were both commonly distributed as main viruses infecting P. ternata collected from different areas in China. But DsMV was the virus which infected P. ternate in natural condition. The infection ratio of cultivated P. ternate by SMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang province; 61.9% and 33.3% for 21 samples from Hebei province; 50.0% and 41.7% for 12 samples from Anhui province; 16.7% and 16.7% for 12 samples from Sichuan province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by SMV and CMV were both 20.0%.


Subject(s)
Cucumovirus/isolation & purification , Mosaic Viruses/isolation & purification , Pinellia/virology , Plants, Medicinal/virology , China , Cluster Analysis , Cucumovirus/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Mosaic Viruses/classification , Mosaic Viruses/genetics , Plant Diseases/virology , Sequence Analysis, DNA
17.
Wei Sheng Wu Xue Bao ; 47(6): 1002-8, 2007 Dec.
Article in Chinese | MEDLINE | ID: mdl-18271254

ABSTRACT

A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process, the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein, there were some bipy ramidal crystals with a size of 1.5 x 3.0 microm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10 micro/mL and 11.34 micro/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with high toxicity.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Chitinases/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Nicotiana/enzymology , Recombinant Fusion Proteins/genetics , Animals , Bacillus thuringiensis Toxins , Blotting, Western , Cloning, Molecular , Drug Synergism , Insecticides/pharmacology , Plasmids , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/pharmacology
18.
Plant J ; 42(4): 514-24, 2005 May.
Article in English | MEDLINE | ID: mdl-15860010

ABSTRACT

The SKP1-Cullin/Cdc53-F-box protein ubiquitin ligases (SCF) target many important regulatory proteins for degradation and play vital roles in diverse cellular processes. In Arabidopsis there are 11 Cullin members (AtCUL). AtCUL1 was demonstrated to assemble into SCF complexes containing COI1, an F-box protein required for response to jasmonates (JA) that regulate plant fertility and defense responses. It is not clear whether other Cullins also associate with COI1 to form SCF complexes, thus, it is unknown whether AtCUL1, or another Cullin that assembles into SCF(COI1) (even perhaps two or more functionally redundant Cullins), plays a major role in JA signaling. We present genetic and physiological data to directly demonstrate that AtCUL1 is necessary for normal JA responses. The homozygous AtCUL1 mutants axr6-1 and axr6-2, the heterozygous mutants axr6/AXR6, and transgenic plants expressing mutant AtCUL1 proteins containing a single amino acid substitution from phenylalanine-111 to valine, all exhibit reduced responses to JA. We also demonstrate that ax6 enhances the effect of coi1 on JA responses, implying a genetic interaction between COI1 and AtCUL1 in JA signaling. Furthermore, we show that the point mutations in AtCUL1 affect the assembly of COI1 into SCF, thus attenuating SCF(COI1) formation.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/physiology , Cell Cycle Proteins/physiology , Cullin Proteins/physiology , Cyclopentanes/metabolism , Plant Growth Regulators/physiology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cell Cycle Proteins/genetics , Cullin Proteins/genetics , DNA-Binding Proteins/physiology , Oxylipins , Phenotype , Plants, Genetically Modified , Point Mutation , Transcription Factors/physiology
19.
Sheng Wu Gong Cheng Xue Bao ; 20(5): 656-61, 2004 Sep.
Article in Chinese | MEDLINE | ID: mdl-15973985

ABSTRACT

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Recombinant Proteins/biosynthesis , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/biosynthesis , Bacterial Proteins/pharmacology , Cloning, Molecular , Endotoxins/biosynthesis , Endotoxins/pharmacology , Hemolysin Proteins/biosynthesis , Hemolysin Proteins/pharmacology , Microscopy, Atomic Force , Moths , Plasmids , Recombinant Proteins/pharmacology
20.
Ying Yong Sheng Tai Xue Bao ; 14(3): 443-6, 2003 Mar.
Article in Chinese | MEDLINE | ID: mdl-12836558

ABSTRACT

There may be several ecological impacts induced by transgenic cottons, apart from their direct impact on target pest. The interactions between target insect and transgenic cotton, and the way of toxic expressed by transgenic cotton varied with plant spatial parts and different growing stages are regarded as the main cause for insect to develop resistance. In transgenic cotton field, although chemicals applied to control major insect pest could be reduced greatly, insect community structure including insect pests and beneficial organisms is less stable than that of regular cotton field. It is much easier for minor insect pests developing to be major pest. In order to full utilization of transgenic cottons and keep their efficiency on target pest, methods including integrated pest management and breeding higher efficiency transgenic cottons by genetic engineering are proposed for management of pest resistance and non-target pests.


Subject(s)
Bacillus thuringiensis/genetics , Ecology , Gossypium/genetics , Pest Control, Biological , Plants, Genetically Modified
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