Thermal Asymmetric Interlaced PCR Research Articles

Rhamnogalacturonan lyase 1 (RGL1), as a suppressor of E3 ubiquitin ligase Arabidopsis thaliana ring zinc finger 1 (AtRZF1), is involved in dehydration response to mediate proline synthesis and pectin rhamnogalacturonan-I composition.

Proline metabolism plays a crucial role in both environmental stress responses and plant growth. However, the specific mechanism by which proline contributes to abiotic stress processes remains to be elucidated. In this study, we utilized atrzf1 (Arabidopsis thaliana ring zinc finger 1) as a parental line for T-DNA tagging mutagenesis and identified a suppressor mutant of atrzf1, designated proline content alterative 31 (pca31). The pca31 mutant suppressed the insensitivity of atrzf1 to dehydration stress during early seedling growth. Using Thermal Asymmetric Interlaced-PCR, we found that the T-DNA of pca31 was inserted into the promoter region of the At2g22620 gene, which encodes the cell wall enzyme rhamnogalacturonan lyase 1 (RGL1). Enzymatic assays indicated that RGL1 exhibited rhamnogalacturonan lyase activity, influencing cell wall pectin composition. The decrease in RGL1 gene expression suppressed the transcriptomic perturbation of the atrzf1 mutant. Silencing of the RGL1 gene in atrzf1 resulted in a sensitive phenotype similar to pca31 under osmotic stress conditions. Treatment with mannitol, salt, hydrogen peroxide, and abscisic acid induced RGL1 expression. Furthermore, we uncovered that RGL1 plays a role in modulating root growth and vascular tissue development. Molecular, physiological, and genetic experiments revealed that the positive modulation of RGL1 during abiotic stress was linked to the AtRZF1 pathway. Taken together, these findings establish that pca31 acts as a suppressor of atrzf1 in abiotic stress responses through proline and cell wall metabolisms.

Isolation of actin regulatory region from medicinal plants by thermal asymmetric interlaced PCR (TAIL PCR) and its bioinformatic analysis

Isolation of actin regulatory region from medicinal plants by thermal asymmetric interlaced PCR (TAIL PCR) and its bioinformatic analysis

A new l-serine binding orphan SerBP affects indole synthesis in Pantoea ananatis.

Indole is traditionally known as a metabolite of l-tryptophan and now as an important signaling molecule in bacteria, however, the understanding of its upstream synthesis regulation is very limited. Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa), can produce indole to regulate various physiological and biochemical behaviors. We constructed a mutant library of YJ76 using the mTn5 transposon insertion mutation method, from which an indole-deficient mutant was screened out. Via high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), the transposon was determined to be inserted in a gene (RefSeq: WP014605468.1) of unknown function that is highly conserved at the intraspecific level. Bioinformatics analysis implied that the protein (Protein ID: WP089517194.1) encoded by the mutant gene is most likely to be a new orphan substrate-binding protein (SBP) for amino acid ABC transporters. Amino acid supplement cultivation experiments and surface plasmon resonance revealed that the protein could bind tol-serine (KD = 6.149 × 10-5 M). Therefore, the SBP was named as SerBP. This is the first case that a SBP responds tol-serine ABC transports. As a precursor of indole synthesis, the transmembrane transportedl-serine was directly correlated with indole signal production and the mutation of serBP gene weakened the resistance of YJ76 to antibiotics, alkali, heavy metals, and starvation. This study provided a new paradigm for exploring the upstream regulatory pathway for indole synthesis of bacteria.

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization.

Penicillium brocae strain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated by Agrobacterium tumefaciens-mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation of A. tumefaciens and P. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA from Aspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi and Saccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene of P. brocae is involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.

Combined transcriptome and proteome analysis of Bcfrp1 involved in regulating the biosynthesis of abscisic acid and growth in Botrytis cinerea TB-31.

Abscisic acid (ABA) is an important sesquiterpene compound that regulates the stress resistance of plants. Botrytis cinerea can synthesize ABA via the mevalonic acid pathway. To identify the functional genes that are involved in the biosynthesis of ABA, we performed insertion mutagenesis into B. cinerea TB-31. We obtained the ABA-reduced mutant E154 by insertion mutagenesis, and we identified the insertion site was located upstream of the gene bcfrp1 by Thermal asymmetric interlaced PCR. We performed a detailed phenotypic characterization of the bcfrp1 knockout and complementation mutants in TB-31. Furthermore, transcriptome and proteome analyses were conducted to explore how bcfrp1 affects the level of the ABA biosynthesis. The bcfrp1 gene encodes an F-box protein. The phenotypic results confirmed the positive contribution of bcfrp1 to the biosynthesis of ABA and growth. Between TB-31 and ΔBcfrp1, we obtained 4,128 and 1,073 differentially expressed genes and proteins, respectively. The impaired ABA biosynthesis in the ΔBcfrp1 mutants was primarily affected by the different levels of expression of the ABA biosynthetic gene cluster and the genes involved in the mevalonic acid pathway. In addition, we further characterized the differentially expressed genes and proteins that participated in the growth, secondary metabolism, and signal transduction in B. cinerea based on the transcriptome and proteome data. This research based on the transcriptome and proteome analyses to display the changes after the deletion of bcfrp1 in B. cinerea TB-31, will help us to explore the molecular mechanism of ABA biosynthesis in B. cinerea.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, andfliR), 4 curli fimbriae genes (csgD, csgA, csgF, andcsgG) and 3 type 1 fimbriae genes (fimA, fimD, andfimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, andcsgG) and other functional genes (fimC, glxK, yehE, 07045, and11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

Insertion Mutation of MSMEG_0392 Play an Important Role in Resistance of M. smegmatis to Mycobacteriophage SWU1.

PurposePhage is a new choice for the treatment of multi-drug-resistant bacteria, and phage resistance is also an issue of concern. SWU1 is a mycobacteriophage, and the mechanism of its resistance remain poorly understood.MethodsThe mutant strains which were stably resistant to SWU1 were screened by transposon mutation library. The stage of phage resistance was observed by transmission electron microscope (TEM). The insertion site of transposon was identified by thermal asymmetric interlaced PCR (TAIL-PCR). The possible relationship between insertion site and phage resistance was verified by gene knockout technique. The fatty acid composition of bacterial cell wall was analyzed by Gas Chromatography-Mass Spectrometer (GC-MS). Through the amplification and sequencing of target genes and gene complement techniques to find the mechanism of SWU1 resistance.ResultsThe transposon mutant M12 which was stably resistant to mycobacteriophage SWU1 was successfully screened. It was confirmed that resistance occurred in the adsorption stage of bacteriophage. It was verified that the insertion site of the transposon was located in the MSMEG_3705 gene, but after knocking out the gene in the wild type M. smegmatis mc2 155, the resistance of the knockout strain to SWU1 was not observed. Through the amplification and sequencing of the target gene MSMEG_0392, it was found that there was an adenine insertion mutation at position 817. After complementing MSMEG_0392 in M12, it was found that M12 returned to sensitivity to SWU1.ConclusionWe confirmed that the resistance of M12 to SWU1 was related to the functional inactivation of MSMEG_0392 and this phenomenon may be caused by the change of cell wall of M. smegmatis.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda.

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase). In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Development of an Agrobacterium tumefaciens-mediated transformation system for Tilletia controversa Kühn

Dwarf bunt of wheat caused by Tilletia controversa Kühn has been identified an international quarantine disease, which replace the grain material into millions of teliospores. Agrobacterium tumefaciens-mediated transformation (ATMT) system is a powerful tool for fungi transformation with significant advantages of simple operation, high efficiency, and genetic stability of transformants. In this study, we constructed ATMT system for T. controversa. All the transformants were tested using Acetosyringone (AS) concentration at 150 μmol/l, hygromycin B at 25 μg/ml, 1 × 106 T. controversa hypha cells/ml, A. tumefaciens with OD600 of 0.5 co-cultivation at 16 °C for 48 h and culture was incubated at 16 °C for 20 days. Using the ATMT method, we cultivated 8 generations of transformants on complete medium (CM) containing hygromycin B antibiotic and validated by PCR, which indicate that T-DNA had been successfully inserted into each of T. controversa transformants. In addition, thermal asymmetric interlaced PCR (TAIL-PCR) evaluated the Ti element inserts were at random sites in the fungal genome. Thus, ATMT approach is an efficient tool for insertional mutagenesis of T. controversa.

Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize.

The insertion position of the exogenous fragment sequence in a genetically modified organism (GMO) is important for the safety assessment and labeling of GMOs. SK12-5 is a newly developed transgenic maize line transformed with two trait genes [i.e., G10evo-5-enolpyrul-shikimate-3-phosphate synthase (EPSPS) and Cry1Ab/Cry2Aj] that was recently approved for commercial use in China. In this study, we tried to determine the insertion position of the exogenous fragment for SK12-5. The transgene–host left border and right border integration junctions were obtained from SK12-5 genomic DNA by using the thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and next-generation Illumina sequencing technology. However, a Basic Local Alignment Search Tool (BLAST) analysis revealed that the flanking sequences in the maize genome are unspecific and that the insertion position is located in a repetitive sequence area in the maize genome. To locate the fine-scale insertion position in SK12-5, we combined the methods of genetic mapping and nanopore-based sequencing technology. From a classical bulked-segregant analysis (BSA), the insertion position in SK12-5 was mapped onto Bin9.03 of chromosome 9 between the simple sequence repeat (SSR) markers umc2337 and umc1743 (26,822,048–100,724,531 bp). The nanopore sequencing results uncovered 10 reads for which one end was mapped onto the vector and the other end was mapped onto the maize genome. These observations indicated that the exogenous T-DNA fragments were putatively integrated at the position from 82,329,568 to 82,379,296 bp of chromosome 9 in the transgenic maize SK12-5. This study is helpful for the safety assessment of the novel transgenic maize SK12-5 and shows that the combined method of genetic mapping and the nanopore-based sequencing technology will be a useful approach for identifying the insertion positions of transgenic sequences in other GM plants with relatively large and complex genomes.

Ectopic expression of antifreeze protein gene from Ammopiptanthus nanus confers chilling tolerance in maize

Improved chilling tolerance is important for maize production. Previous efforts in transgenics and marker-assisted breeding have not achieved practical results. In this study, the antifreeze protein (AnAFP) from the super-xerophyte Ammopiptanthus nanus was aligned to KnS-type dehydrins. Phosphorylation in vitro and subcellular localization showed that AnAFP was phosphorylated by maize casein kinase II and translocated from nucleus to cytoplasm under chilling stress. AnAFP also increased lactate dehydrogenase activity. A parent line of commercial maize hybrids was transformed with the AnAFP gene. Based on thermal asymmetric interlaced PCR, one hemizygous and two homozygous integration sites were identified in one T1 line. Ectopic expression of AnAFP in transgenic lines was confirmed by quantitative real-time PCR, RNA-seq, and Western blotting. After chilling treatment, the transgenic lines showed significantly improved phenotype, higher kernel production, survival rate and biomass, and lower relative electrolyte leakage and superoxide dismutation than the untransformed line. Thus, ectopic expression of AnAFP gene improved chilling tolerance in the transgenic lines, which could be used to apply for further safety assessment for commercial breeding.

CHARACTERISATION OF AmCCR1 AND pseudoCOMTAm PROMOTERS IN Acacia mangium

Acacia mangium is one of the most important species in forest plantation industry in Southeast Asia. Lignin is a complex polymer found in plant which has to be removed using harsh and toxic chemicals for the production of high quality paper. Understanding the complex pathway that underlies the regulation of lignin biosynthetic genes requires in depth knowledge of the genes involved and it’s regulatory elements. Using Thermal Asymmetric Interlaced PCR, a 770bp promoter of pseudoCOMTAm and 619bp of AmCCR1 promoter were isolated. Bioinformatics analysis revealed the presence of cis acting elements commonly found in lignin biosynthetic genes such as TATA box, CAAT box, W box, AC-I and AC-11 elements on both promoters.

Genome-Wide Investigation of 2,4-Diacetylphloroglucinol Protection Genes in Arabidopsis thaliana.

The compound 2,4-diacetylphloroglucinol (DAPG) is a well-known secondary metabolite produced by Pseudomonas spp. that are used as biocontrol agents. DAPG displays a remarkably broad spectrum of toxic activity against pathogens of plants. Yet high concentrations of DAPG may also have negative effect on plants, but the phytotoxicity of DAPG is not clearly understood. Here, we used genome-wide activation, tagging Arabidopsis plants as the model plant to investigate the plant response to DAPG. A total of 15 lines were selected as DAPG-tolerant plants from among 62,000 lines investigated. The DAPG-responsible genes were then identified via thermal asymmetric interlaced PCR and quantitative reverse transcription PCR, and the gene ontology analysis showed the distribution of these genes having different biological processes, cellular regulations, and molecular functional properties. Collectively, these findings suggest that plants may rely on several pathways to prevent DAPG phytotoxicity.

Characterization of Arabidopsis thaliana Root-Related Mutants Reveals ABA Regulation of Plant Development and Drought Resistance

The plant hormone abscisic acid (ABA) regulates many processes of plant growth and development. ABA receptors have been identified in studies of the ABA response of stomatal movement, but the underlying mechanisms of ABA-regulated root growth and development are unclear. To address these questions, we screened for Arabidopsis thaliana mutants based on the sensitivity of root growth to exogenous ABA, using ethyl methanesulfonate-mutagenized (EMS) and T-DNA insertion mutant libraries. We identified 11 mutants, termed roa1–roa11, with Root growth Overly sensitive to ABA (ROA) phenotypes, and cloned two of the ROA genes, one by thermal asymmetric interlaced PCR technology (ROA3) and the other by map-based cloning (ROA9). The roa mutants were also found to have defects in other major ABA responses, including ABA-mediated seed germination and drought resistance. The roa mutants provide crucial genetic material for further studies of ABA signaling and regulatory mechanisms in root growth and development.

An optimized Agrobacterium tumefaciens-mediated transformation system for random insertional mutagenesis in Fonsecaea monophora

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi, is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens-mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future.

Abnormal tapetum development in hermaphrodites of an androdioecious tree, Tapiscia sinensis.

Tapiscia sinensis Oliv. (Tapisciaceae) has been proven to be a functional androdioecious species with both male and hermaphroditic individuals, and the pollen viability of males is far higher than that of hermaphrodites. To better understand the causes of the low pollen viability in hermaphroditic flowers, different stages of anther development were observed. We found that hermaphroditic flowers exhibit abnormal tapetum development, resulting in low pollen viability. To clarify the underlying molecular mechanism of abnormal tapetum development in hermaphrodites, quantitative real-time PCR analyses were performed. The results revealed that the expression levels of an important transcription factor for tapetum development and function, T. sinensis DYSFUNCTIONAL TAPETUM1 (TsDYT1), and its potential downstream regulatory genes T. sinensis DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1 (TsTDF1), T. sinensis ABORTED MICROSPORE (TsAMS) and T. sinensis MALE STERILITY 1 (TsMS1) were all significantly downregulated in hermaphrodites compared with males at some key stages of anther development. The amino acid sequence similarity, expression pattern, gene structure and subcellular localization of these genes were analyzed, and the results indicated functional conservation between T. sinensis and homologues in Arabidopsis thaliana. Next, rapid amplification of cDNA end and thermal asymmetric interlaced PCR were employed to clone the full-length cDNA and promoter sequences of these genes, respectively. In addition, results of yeast two-hybrid analysis showed that TsDYT1 can form heterodimers with TsAMS, and yeast one-hybrid analysis demonstrated that TsDYT1 directly binds to the promoter regions of TsTDF1 and TsMS1. TsTDF1 can directly regulate expression of TsAMS, suggesting that a functionally conserved pathway exists between A. thaliana and T. sinensis to regulate tapetum development. In conclusion, the results suggest that abnormal expression of core transcription factors for tapetum development, including TsDYT1, TsTDF1, TsAMS and TsMS1, plays an important role in the abnormal development of the tapetum in T. sinensis hermaphrodites. Furthermore, a hermaphroditic tapetum with abnormal function causes the low pollen viability of hermaphroditic trees. Our results provide new insight into our understanding of the underlying mechanism of why pollen viability is much higher in males than hermaphrodites of the androdioecious tree T. sinensis.

Genome-wide analysis of flanking sequences reveals thatTnt1 insertion is positively correlated with gene methylation in Medicago truncatula.

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.

An Efficient System for Ds Transposon Tagging in Brachypodium distachyon.

Transposon tagging is a powerful tool that has been widely applied in several species for insertional mutagenesis in plants. Several efforts have aimed to create transfer-DNA (T-DNA) insertional mutant populations in Brachypodium distachyon, a monocot plant used as a model system to study temperate cereals, but there has been a lack of research aimed at using transposon strategies. Here, we describe the application of a maize (Zea mays) Dissociation (Ds) transposon tagging system in B distachyon The 35S::AcTPase cassette and Ds element were constructed within the same T-DNA and transformed into B distachyon plants. The Ds element was readily transposed to other chromosomes or to the same chromosome under the function of Activator (Ac) transposase. Through homologous chromosome synapsis, recombination, and segregation, the Ds element separated from the Ac element. We selected stable Ds-only plants using G418 and GFP assays and analyzed 241 T0 lines, some of which were highly efficient at producing Ds-only progeny. Through thermal asymmetric interlaced PCR, we isolated 710 independent Ds flanking sequences from Ds-only plants. Furthermore, we identified a large collection of mutants with visible developmental phenotypes via this transposon tagging system. The system is relatively simple and rapid in comparison to traditional T-DNA insertion strategies, because once efficiency lines are obtained they can be reused to generate more lines from nontransposed plants without the use of time-consuming tissue culture steps.

A Myb transcription factor represses conidiation and cephalosporin C production in Acremonium chrysogenum

Acremonium chrysogenum is the industrial producer of cephalosporin C (CPC). We isolated a mutant (AC554) from a T-DNA inserted mutant library of A. chrysogenum. AC554 exhibited a reduced conidiation and lack of CPC production. In consistent with it, the transcription of cephalosporin biosynthetic genes pcbC and cefEF was significantly decreased in AC554. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed and sequence analysis indicated that a T-DNA was inserted upstream of an open reading frame (ORF) which was designated AcmybA. On the basis of sequence analysis, AcmybA encodes a Myb domain containing transcriptional factor. Observation of red fluorescent protein (RFP) tagged AcMybA showed that AcMybA is naturally located in the nucleus of A. chrysogenum. Transcriptional analysis demonstrated that the AcmybA transcription was increased in AC554. In contrast, the AcmybA deleted mutant (ΔAcmybA) overproduced conidia and CPC. To screen the targets of AcmybA, we sequenced and compared the transcriptome of ΔAcmybA, AC554 and the wild-type strain at different developmental stages. Twelve differentially expressed regulatory genes were identified. Taken together, our results indicate that AcMybA negatively regulates conidiation and CPC production in A. chrysogenum.

The Kynurenine 3-Monooxygenase Encoding Gene, BcKMO, Is Involved in the Growth, Development, and Pathogenicity of Botrytis cinerea.

A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea. A novel pathogenicity-related gene BcKMO, which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO-complementing mutant (BCG183/BcKMO) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/BcKMO. Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H2O2, and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1, Pka2, PkaR, Bcg2, Bcg3, bmp1, and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea. Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea.