FGCD: a database of fungal gene clusters related to secondary metabolism.

Fungal secondary metabolites are not necessary for growth, but they are important for fungal metabolism and ecology because they provide selective advantages for competition, survival and interactions with the environment. These various metabolites are widely used as medicinal precursors and insecticides. Secondary metabolism genes are commonly arranged in clusters along chromosomes, which allow for the coordinate control of complete pathways. In this study, we created the Fungal Gene Cluster Database to store, retrieve, and visualize secondary metabolite gene cluster information across fungal species. The database was created by merging data from RNA sequencing, Basic Local Alignment Search Tool, genome browser, enrichment analysis and the R Shiny web framework to visualize and query putative gene clusters. This database facilitated the rapid and thorough examination of significant gene clusters across fungal species by detecting, defining and graphically displaying the architecture, organization and expression patterns of secondary metabolite gene clusters. In general, this genomic resource makes use of the tremendous chemical variety of the products of these ecologically and biotechnologically significant gene clusters to our further understanding of fungal secondary metabolism. Database URL: https://www.hebaubioinformatics.cn/FungalGeneCluster/.

The color of biodegradable mulch films is associated with differences in peanut yield and bacterial communities.

Biodegradable mulch films (BDMs) are increasingly used in agricultural production as desirable alternatives to the current widespread use of polyethylene (PE) mulch films in China. However, potential effects of different colors of BDMs on field crop production and microbiomes remain unexplored. Here, the differences in bacterial communities of peanut rhizosphere soil (RS) and bulk soil (BS) under non-mulching (CK), PE, and three different colors of BDMs were studied. The results indicated that all treatments could increase the soil temperature, which positively affected the growth of the peanut plants. Moreover, mulching affected the bacterial community structure in RS and BS compared to CK. Furthermore, certain BDM treatments significantly enriched N-fixing bacteria (Bradyrhizobium and Mesorhizobium) and functional groups, increased the closeness of bacterial networks, and harbored more beneficial bacteria as keystone taxa in the RS. This in turn facilitated the growth and development of the peanut plants under field conditions. Our study provides new insights into the micro-ecological effects of mulch films, which can be affected by both the mulch type and color. The observed effects are likely caused by temperature and prevalence of specific microbial functions under the employed films and could guide the development of optimized mulching materials.

Identification and expression analysis of maize NF-YA subunit genes.

NF-YAs encode subunits of the nuclear factor-Y (NF-Y) gene family. NF-YAs represent a kind of conservative transcription factor in plants and are involved in plant growth and development, as well as resistance to biotic and abiotic stress. In this study, 16 maize (Zea mays) NF-YA subunit genes were identified using bioinformatics methods, and they were divided into three categories by a phylogenetic analysis. A conserved domain analysis showed that most contained a CCAAT-binding transcription factor (CBFB) _NF-YA domain. Maize NF-YA subunit genes showed very obvious tissue expression characteristics. The expression level of the NF-YA subunit genes significantly changed under different abiotic stresses, including Fusarium graminearum infection and salicylic acid (SA) or jasmonic acid (JA) treatments. After inoculation with Setosphaeria turcica and Cochliobolus heterostrophus, the lesion areas of nfya01 and nfya06 were significantly larger than that of B73, indicating that ZmNFYA01 and ZmNFYA06 positively regulated maize disease resistance. ZmNFYA01 and ZmNFYA06 may regulated maize disease resistance by affecting the transcription levels of ZmPRs. Thus, NF-YA subunit genes played important roles in promoting maize growth and development and resistance to stress. The results laid a foundation for clarifying the functions and regulatory mechanisms of NF-YA subunit genes in maize.

Global analysis of lysine 2-hydroxyisobutyrylation during Fusarium graminearum infection in maize.

Proteins post-translational modification (PTMs) is necessary in the whole life process of organisms. Among them, lysine 2-hydroxyisobutyrylation (Khib) plays an important role in protein synthesis, transcriptional regulation, and cell metabolism. Khib is a newly identified PTM in several plant species. However, the function of Khib in maize was unclear. In this study, western blotting results showed that Khib modification level increased significantly after Fusarium graminearum infection, and 2,066 Khib modified sites on 728 proteins were identified in maize, among which 24 Khib sites occurred on core histones. Subcellular localization results showed that these Khib modified proteins were localized in cytoplasm, chloroplast, and nucleus. Then, comparative proteomic analysis of the defense response to F. graminearum infection showed that Khib modification participated in plant resistance to pathogen infection by regulating glycolysis, TCA cycle, protein synthesis, peroxisome, and secondary metabolic processes, such as benzoxazinoid biosynthesis, phenylpropanoid biosynthesis, jasmonic acid synthesis, and tyrosine and tryptophan biosynthesis. In addition, we also demonstrated that lysine 2-hydroxyisobutyrylation sites on histones were involved in the gene expression of pathogenesis-related proteins. Our results provide a new perspective for the study of plant disease resistance, and had directive significance of maize disease resistance for molecular breeding.

BTB and TAZ domain protein BT4 positively regulates the resistance to Botrytis cinerea in Arabidopsis.

BT4 gene was identified to play an important role in Arabidopsis resistance to pst DC3000 in preliminary studies. However, the specific function and molecular mechanism of BT4 gene in regulation of Arabidopsis resistance to Botrytis cinerea had not been described to date. In this study, we found that the expression of BT4 was induced by wounding and B. cinerea inoculation in Arabidopsis. After inoculated with B. cinerea, T-DNA insertion mutants of the BT4 gene, bt4, showed significant susceptibility symptoms, whereas no significant symptoms were found in wild-type (WT), the complemented transgenic plants (CE), and the overexpression transgenic plants (OE). After inoculated with B. cinerea, the expression levels of JAR1 and PDF1.2 genes in bt4 mutant were induced; however, the expression levels of these genes in bt4 mutant were significantly lower than those in the WT, CE, and OE. These results indicated that the BT4 positively regulate the expression of genes in JA/ET signaling pathways. Therefore, the BT4 may be involved in the regulation of JA/ET signaling pathways to affect Arabidopsis resistance to B. cinerea.

Comparative Proteomic Analysis of the Defense Response to Gibberella Stalk Rot in Maize and Reveals That ZmWRKY83 Is Involved in Plant Disease Resistance.

Fusarium graminearum is the causal agent of Gibberella stalk rot in maize stem, resulting in maize lodging, yield, quality, and mechanical harvesting capacity. To date, little is known about the maize stem defense mechanism in response to the invasion of F. graminearum. This study represents a global proteomic approach to document the infection by F. graminearum. A total of 1,894 differentially expressed proteins (DEPs) were identified in maize stem with F. graminearum inoculation. Functional categorization analysis indicated that proteins involved in plant-pathogen interaction were inducible at the early stages of infection. We also found that the expression of proteins involved in phenylpropanoid, flavonoid, and terpenoid biosynthesis were upregulated in response to F. graminearum infection, which may reflect that these secondary metabolism pathways were important in the protection against the fungal attack in maize stem. In continuously upregulated proteins after F. graminearum infection, we identified a WRKY transcription factor, ZmWRKY83, which could improve the resistance to plant pathogens. Together, the results show that the defense response of corn stalks against F. graminearum infection was multifaceted, involving the induction of proteins from various immune-related pathways, which had a directive significance for molecular genetic breeding of maize disease-resistant varieties.

In silico analysis of maize HDACs with an emphasis on their response to biotic and abiotic stresses.

Histone deacetylases (HDACs) are key epigenetic factors in regulating chromatin structure and gene expression in multiple aspects of plant growth, development, and response to abiotic or biotic stresses. Many studies on systematic analysis and molecular function of HDACs in Arabidopsis and rice have been conducted. However, systematic analysis of HDAC gene family and gene expression in response to abiotic and biotic stresses has not yet been reported. In this study, a systematic analysis of the HDAC gene family in maize was performed and 18 ZmHDACs distributed on nine chromosomes were identified. Phylogenetic analysis of ZmHDACs showed that this gene family could be divided into RPD3/HDA1, SIR2, and HD2 groups. Tissue-specific expression results revealed that ZmHDACs exhibited diverse expression patterns in different tissues, indicating that these genes might have diversified functions in growth and development. Expression pattern of ZmHDACs in hormone treatment and inoculation experiment suggested that several ZmHDACs might be involved in jasmonic acid or salicylic acid signaling pathway and defense response. Interestingly, HDAC genes were downregulated under heat stress, and immunoblotting results demonstrated that histones H3K9ac and H4K5ac levels were increased under heat stress. These results provide insights into ZmHDACs, which could help to reveal their functions in controlling maize development and responses to abiotic or biotic stresses.

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.

Molecular Cloning and Expression Analysis of Eight PgWRKY Genes in Panax ginseng Responsive to Salt and Hormones.

Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS) technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e) based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C2H2-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA) showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA), abscisic acid (ABA), and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress-inducible genes responding to both salt and hormones.

Transcriptomics-based identification of WRKY genes and characterization of a salt and hormone-responsive PgWRKY1 gene in Panax ginseng.

WRKY proteins belong to a transcription factor (TF) family and play dynamic roles in many plant processes, including plant responses to abiotic and biotic stresses, as well as secondary metabolism. However, no WRKY gene in Panax ginseng C.A. Meyer has been reported to date. In this study, a number of WRKY unigenes from methyl jasmonate (MeJA)-treated adventitious root transcriptome of this species were identified using next-generation sequencing technology. A total of 48 promising WRKY unigenes encoding WRKY proteins were obtained by eliminating wrong and incomplete open reading frame (ORF). Phylogenetic analysis reveals 48 WRKY TFs, including 11 Group I, 36 Group II, and 1 Group III. Moreover, one MeJA-responsive unigene designated as PgWRKY1 was cloned and characterized. It contains an entire ORF of 1077 bp and encodes a polypeptide of 358 amino acid residues. The PgWRKY1 protein contains a single WRKY domain consisting of a conserved amino acid sequence motif WRKYGQK and a C2H2-type zinc-finger motif belonging to WRKY subgroup II-d. Subcellular localization of PgWRKY1-GFP fusion protein in onion and tobacco epidermis cells revealed that PgWRKY1 was exclusively present in the nucleus. Quantitative real-time polymerase chain reaction analysis demonstrated that the expression of PgWRKY1 was relatively higher in roots and lateral roots compared with leaves, stems, and seeds. Importantly, PgWRKY1 expression was significantly induced by salicylic acid, abscisic acid, and NaCl, but downregulated by MeJA treatment. These results suggested that PgWRKY1 might be a multiple stress-inducible gene responding to hormones and salt stresses.

Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng.

The v-myb avian myeloblastosis viral oncogene homolog (MYB) family constitutes one of the most abundant groups of transcription factors and plays vital roles in developmental processes and defense responses in plants. A ginseng (Panax ginseng C.A. Meyer) MYB gene was cloned and designated as PgMYB1. The cDNA of PgMYB1 is 762 base pairs long and encodes the R2R3-type protein consisting 238 amino acids. Subcellular localization showed that PgMYB1-mGFP5 fusion protein was specifically localized in the nucleus. To understand the functional roles of PgMYB1, we investigated the expression patterns of PgMYB1 in different tissues and under various conditions. Quantitative real-time polymerase chain reaction and western blot analysis showed that PgMYB1 was expressed at higher level in roots, leaves, and lateral roots than in stems and seeds. The expression of PgMYB1 was up-regulated by abscisic acid, salicylic acid, NaCl, and cold (chilling), and down-regulated by methyl jasmonate. These results suggest that PgMYB1 might be involved in responding to environmental stresses and hormones.

Transcriptome analysis of methyl jasmonate-elicited Panax ginseng adventitious roots to discover putative ginsenoside biosynthesis and transport genes.

The Panax ginseng C.A. Meyer belonging to the Araliaceae has long been used as an herbal medicine. Although public databases are presently available for this family, no methyl jasmonate (MeJA) elicited transcriptomic information was previously reported on this species, with the exception of a few expressed sequence tags (ESTs) using the traditional Sanger method. Here, approximately 53 million clean reads of adventitious root transcriptome were separately filtered via Illumina HiSeq™2000 from two samples treated with MeJA (Pg-MeJA) and equal volumes of solvent, ethanol (Pg-Con). Jointly, a total of 71,095 all-unigenes from both samples were assembled and annotated, and based on sequence similarity search with known proteins, a total of 56,668 unigenes was obtained. Out of these annotated unigenes, 54,920 were assigned to the NCBI non-redundant protein (Nr) database, 35,448 to the Swiss-prot database, 43,051 to gene ontology (GO), and 19,986 to clusters of orthologous groups (COG). Searching in the Kyoto encyclopedia of genes and genomes (KEGG) pathway database indicated that 32,200 unigenes were mapped to 128 KEGG pathways. Moreover, we obtained several genes showing a wide range of expression levels. We also identified a total of 749 ginsenoside biosynthetic enzyme genes and 12 promising pleiotropic drug resistance (PDR) genes related to ginsenoside transport.

Plant pleiotropic drug resistance transporters: transport mechanism, gene expression, and function.

Pleiotropic drug resistance (PDR) transporters belonging to the ABCG subfamily of ATP-binding cassette (ABC) transporters are identified only in fungi and plants. Members of this family are expressed in plants in response to various biotic and abiotic stresses and transport a diverse array of molecules across membranes. Although their detailed transport mechanism is largely unknown, they play important roles in detoxification processes, preventing water loss, transport of phytohormones, and secondary metabolites. This review provides insights into transport mechanisms of plant PDR transporters, their expression profiles, and multitude functions in plants.

Isolation and characterization of LHT-type plant amino acid transporter gene from Panax ginseng Meyer.

A lysine histidine transporter (LHT) cDNA was isolated and characterized from the roots of Panax ginseng, designated PgLHT. The cDNA is 1,865 bp with an open reading frame that codes for a protein with 449 amino acids and a calculated molecular mass of 50.6 kDa with a predicted isoelectric point of 8.87. Hydropathy analysis shows that PgLHT is an integral membrane protein with 9 putative membrane-spanning domains. Multiple sequence alignments show that PgLHT shares a high homology with other plant LHTs. The expression profile of the gene was investigated by real-time quantitative polymerase chain reaction during various chemical treatments. PgLHT was up-regulated in the presence of abscisic acid, salicylic acid, methyl jasmonate, NaCl, and amino acids. To further explore the function of PgLHT gene, full-length cDNA of PgLHT was introduced into P. ginseng by Agrobacterium rhizogenes A4. The overexpression of PgLHT in the hairy roots led to an obviously increase of biomass compared to the controls, and after addition of the amino acids, the overexpressed-PgLHT hairy roots grew more rapidly than untreated controls during early stage of the culture cycle. The results suggested that the PgLHT isolated from ginseng might have role in the environmental stresses and growth response.

Molecular cloning and expression analysis of PDR1-like gene in ginseng subjected to salt and cold stresses or hormonal treatment.

The plant pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters is potentially involved in diverse biological processes. Currently, little is known about their actual physiological functions. A Panax ginseng PDR transporter gene (PgPDR1) was cloned and the cDNA has an open reading frame of 4344 bp. The deduced amino acid sequence contained the characteristic domains of PDR transporters: Walker A, Walker B, and ABC signature. Genomic DNA hybridization analysis indicated that one copy of PgPDR1 gene was present in P. ginseng. Subcellular localization showed that PgPDR1-GFP fusion protein was specifically localized in the cell membrane. Promoter region analysis revealed the presence of cis-acting elements, some of which are putatively involved in response to hormone, light and stress. To understand the functional roles of PgPDR1, we investigated the expression patterns of PgPDR1 in different tissues and under various conditions. Quantitative real-time PCR (qRT-PCR) and Western blotting analysis showed that PgPDR1 was expressed at a high level in the roots and leaves compared to seeds and stems. The expression of PgPDR1 was up-regulated by salicylic acid (SA) or chilling, down-regulated by ABA, and regulated differently at transcript and protein levels by MeJA. These results suggest that PgPDR1 might be involved in responding to environmental stresses and hormones.