C2H2 Zinc Finger Research Articles

The response to cabbage looper infestation in Arabidopsis is altered by lowering levels of Zat18 a Q-type C2H2 zinc finger protein

ABSTRACT Q-type C2H2 zinc finger proteins (ZFPs) play a role in plant stress response. Overexpression of individual ZFPs enhances tolerance to abiotic stresses such as drought or salt. Overexpression of ZFP, Zat18 enhances tolerance to drought, whereas plants with lower amounts are less resilient. The effect of Zat18 loss on gene expression has not been characterized. Overexpression of an ortholog of Zat18 in potato, StZFP2 enhances tolerance to infestation by Manduca sexta. This work focuses on two Zat18 mutant lines, a knockout and knockdown. The two lines differ in response to infestation. Typically, a chewing insect induces the jasmonic acid (JA) pathway. While infestation induces JA pathway genes, Zat18 mutant lines downregulate abscisic acid signaling and induce ethylene signaling genes ERF1 and EBF2 as well as salicylic acid response gene NPR3. Knockdown Zat18, cause far more changes in the infestation response compared with Col-0 or the Zat18 knockout line.

Zinc Finger Protein LipR Represses Docosahexaenoic Acid and Lipid Biosynthesis in Schizochytrium sp.

The heterotrophic marine microalgae Schizochytrium sp. is an important industrial producer of docosahexaenoic acid (DHA). Increased production of DHA and lipids in Schizochytrium sp. has been achieved by standard fermentation optimization and metabolic engineering methods; however, regulatory mechanisms for DHA and lipid biosynthesis remain unknown. In this study, the C2H2 zinc finger protein LipR was identified in Schizochytrium sp. ATCC 20888 by transcriptional analysis. Deletion of the lipR gene significantly (P < 0.001) increased production of total lipids and DHA by 33% and 48%, respectively. LipR repressed DHA and lipid production by directly inhibiting transcription of polyunsaturated fatty acid (PUFA) and fatty acid synthase (FAS) genes (pfa1, pfa2, pfa3, and fas). Specific binding of LipR to 9-bp recognition sequence 5'-(C/A)(A/G)CCATCTT-3' in upstream regions of target genes was demonstrated by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays. Expression of several key genes (acc, acl, ampD, fabD, mae, zwf, and dga1) related to levels of precursors and NADPH, and to triacylglycerol storage rate, were also directly repressed by LipR. Our findings, taken together, indicate that the evolutionarily unique regulator LipR is an essential repressor of DHA and saturated fatty acid biosynthesis in Schizochytrium sp. IMPORTANCE Regulatory mechanisms for DHA and saturated fatty acid biosynthesis in the heterotrophic marine microalgae Schizochytrium sp. are unclear. We demonstrate here that deletion of the gene (lipR) encoding the C2H2 zinc finger protein LipR promotes DHA and saturated fatty acid production in this genus. LipR acts as a key repressor of such production by binding to 9-bp consensus sequence 5'-(C/A)(A/G)CCATCTT-3' in the upstream regions of polyunsaturated fatty acid and fatty acid synthase genes (pfa1, pfa2, pfa3, and fas), and genes related to levels of precursors and NADPH (acc, acl, ampD, fabD, mae, and zwf), and to triacylglycerol storage rate (dga1). This is the first demonstration that a regulator inhibits synthesis of DHA and lipids in Schizochytrium sp. by directly controlling transcription of PUFA synthase and fas genes. Manipulation of the lipR gene provides a potential strategy for enhancing accumulation of polyunsaturated fatty acids and lipids in thraustochytrids.

Roles of Myc-associated zinc finger protein in malignant tumors.

As an important transcription factor that is widely expressed in most tissues of the human body, Myc-associated zinc finger protein (MAZ) has been reported highly expressed in many malignant tumors and thought to be a promising therapeutic target for cancer treatment. In this review, we aim to offer a comprehensive understanding of MAZ regulation in malignant tumors. The carboxy terminal of MAZ protein contains six C2H2 zinc fingers, and its regulation of transcription is based on the interaction between the GC-rich DNA binding sites of target genes and its carboxy-terminal zinc finger motifs. MAZ protein has been found to activate or inhibit the transcriptional initiation process of many target genes, as well as play an important role in the transcriptional termination process of some target genes, so MAZ poses dual regulatory functions in the initiation and termination process of gene transcription. Through the transcriptional regulation of c-myc and Ras gene family, MAZ poses an important role in the occurrence and development of breast cancer, pancreatic cancer, prostate cancer, glioblastoma, neuroblastoma, and other malignant tumors. Our review shows a vital role of MAZ in many malignant tumors and provides novel insight for cancer diagnosis and treatment.

A novel HD-Zip I/C2H2-ZFP/WD-repeat complex regulates the size of spine base in cucumber.

Fruit spine is an important trait in cucumber, affecting not only commercial quality, but also fruit smoothness, transportation and storage. Spine size is determined by a multi-cellular base. However, the molecular mechanism underlying the regulation of cucumber spine base remains largely unknown. Here, we report map-based cloning and characterization of a spine base size 1 (SBS1) gene, encoding a C2H2 zinc-finger transcription factor. Near-isogenic lines of cucumber were used to map, identify and quantify cucumber spine base size 1 (CsSBS1). Yeast-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP) and RNA-sequencing assays were used to explore the molecular mechanism of CsSBS1 in regulating spine base size development. CsSBS1 was specifically expressed in cucumber ovaries with particularly high expression in fruit spines. Overexpression of CsSBS1 resulted in large fruit spine base, while RNA-interference silencing of CsSBS1 inhibited the expansion of fruit spine base. Sequence analysis of natural cucumber accessions revealed that CsSBS1 was lost in small spine base accessions, resulting from a 4895bp fragment deletion in CsSBS1 locus. CsSBS1 can form a trimeric complex with two positive regulators CsTTG1 and CsGL1 to regulate spine base development through ethylene signaling. A novel regulator network is proposed that the CsGL1/CsSBS1/CsTTG1 complex plays a significant role in regulating spine base formation and size, which offers a strategy for cucumber breeders to develop smooth fruit.

H and HL synergistically regulate jasmonate-triggered trichome formation in tomato.

The development of trichomes, which protect plants against herbivores, is affected by various stresses. In tomato, previous studies showed that stress-triggered jasmonic acid (JA) signaling influences trichome formation, but the underlying mechanism is not fully resolved. Here, we found that two C2H2 zinc finger proteins synergistically regulate JA-induced trichome formation in tomato. The naturally occurring mutations in the H gene and its close homolog H-like in a spontaneous mutant, LA3172, cause severely affected trichome development. Compared with the respective single mutant, the h/hl double mutant displayed more severe trichome defects in all tissues. Despite their partially redundant function, the H and HL genes regulate trichome formation in a spatially distinct manner, with HL more involved in hypocotyls and leaves while H is more involved in stems and sepals. Furthermore, the activity of H/HL is essential for JA-triggered trichome formation. The JA signaling inhibitor SlJAZ2 represses the activity of H and HL via physical interaction, resulting in the activation of THM1, a negative regulator of trichome formation. Our results provide novel insight into the mechanism of trichome formation in response to stress-induced JA signaling in tomato.

Genome-wide identification and expression analyses of C2H2 zinc finger transcription factors in Pleurotus ostreatus.

The C2H2-type zinc finger proteins (C2H2-ZFPs) regulate various developmental processes and abiotic stress responses in eukaryotes. Yet, a comprehensive analysis of these transcription factors which could be used to find candidate genes related to the control the development and abiotic stress tolerance has not been performed in Pleurotus ostreatus. To fill this knowledge gap, 18 C2H2-ZFs were identified in the P. ostreatus genome. Phylogenetic analysis indicated that these proteins have dissimilar amino acid sequences. In addition, these proteins had variable protein characteristics, gene intron-exon structures, and motif compositions. The expression patterns of PoC2H2-ZFs in mycelia, primordia, and young and mature fruiting bodies were investigated using qRT-PCR. The expression of some PoC2H2-ZFs is regulated by auxin and cytokinin. Moreover, members of PoC2H2-ZFs expression levels are changed dramatically under heat and cold stress, suggesting that these genes may participate in abiotic stress responses. These findings could be used to study the role of P. ostreatus-derived C2H2-ZFs in development and stress tolerance.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. This modification, thereby, alters the affinity and specificity of DNA-binding proteins; some of them favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events that impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the SET- and RING finger-associated domain (SRA), and (3) some of TF families, such as the C2H2 zinc finger domain, basic helix-loop-helix (bHLH), basic leucine-zipper (bZIP), and homeodomain proteins. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and the difference in the recognition mechanisms renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this chapter, we summarize structural and biochemical analyses elucidating the mechanisms for recognition of DNA methylation and correlate this information with emerging genomic and functional data.

The WKRY transcription factor MdWRKY75 regulates anthocyanins accumulation in apples (Malus domestica).

Anthocyanins play important roles in plant secondary metabolism. Although previous studies have identified many transcription factors (TFs) that participate in the synthetic pathway of anthocyanins, the regulation mechanism of the pathway remain poorly understood. In this study, we identified a WRKY Group IIc TF, MdWRKY75, which contained a typical WRKYGQK heptapeptide sequence and a C2 H2 -zinc finger structure. Subcellular localisation assays found that MdWRKY75 was located in the nucleus. Overexpression of MdWRKY75 promoted the accumulation of anthocyanins in apple (Malus domestica L.) 'Orin' calli. MdWRKY75 mainly stimulated the accumulation of anthocyanins by binding to the promoter of MYB transcription factor, MdMYB1 . Our research could provide new insights into how WRKY TFs regulate the accumulation of anthocyanins in apples.

Development of PDE6D and CK1α Degraders through Chemical Derivatization of FPFT-2216.

Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1α. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1α, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

The MaC2H2-like zinc finger protein is involved in ripening and ripening disorders caused by chilling stress via the regulation of softening-related genes in ‘Fenjiao’ banana

As one of the largest transcription factor families, C2H2 zinc finger proteins contain a zinc finger motif and a conserved DNA-binding homeodomain. Although C2H2 zinc finger proteins have been found to play an essential role in ethylene biosynthetic and cold stress in banana fruit, their transcriptional regulatory mechanisms involved in the softening of ‘Fenjiao’ banana remain largely elusive. In this study, a new MaC2H2-like gene was identified, which is closely related to fruit softening. MaC2H2-like localizes in the nucleus. MaC2H2-like activated the transcription of four starch (MaBAM4, MaBAM6, MaISA2 and MaPWD1) and five cell wall (MaEXP-A2, MaEXP-A8, MaSUR14, MaSUR14-like and MaXYL32) degradation-related genes, by specifically interacting with their promoters. Protein-protein interaction assays showed that MaC2H2-like cooperates with ethylene-insensitive 3-binding F-box protein (MaEBF1) in vitro and vivo, and their interaction enhanced the binding and activated the promoter activities of MaBAM4, MaPWD1, MaISA2, MaEXP-A2, MaSUR14-like and MaXYL32. The transient overexpression of MaC2H2-like in ‘Fenjiao’ banana fruit promoted fruit ripening and softening by accelerating ethylene production, decreasing fruit firmness and inducing the transcription of starch and cell wall degradation-related genes. Collectively, we uncovered a novel module of MaC2H2-like-MaEBF1 that cooperates in actively impacting starch and cell wall degradation by up-regulating the transcript level of genes related to the degradation of starch and cell walls.

Zinc Fingers 10 and 11 of Miz-1 undergo conformational exchange to achieve specific DNA binding

Miz-1 (ZBTB17) is a poly-zinc finger BTB/POZ transcription factor with 12 consecutive C2H2 zinc fingers (ZFs) that binds transcriptional start sites (TSSs) to regulate the expression of genes involved in cell development and proliferation. As of now, it is not known which of the 12 consecutive ZFs are responsible for the recognition of the 24 base pair consensus sequence found at these TSSs. Evidence suggests ZFs 7-12 plays this role. We provide validation for this and describe the structural and dynamical characterization of unprecedented conformational exchange in the linker between ZFs 10 and 11. This conformational exchange uncouples ZFs 7-10 from 11 and 12 and promotes a scanning-recognition mechanism through which the two segments cooperate to bind two sub-sites at both ends of the consensus. We further show that this can result in the coiling of TSSs as part of Miz-1's mechanism of transcriptional transactivation.

SALL Proteins; Common and Antagonistic Roles in Cancer.

Simple SummaryTranscription factors play essential roles in regulating gene expression, impacting the cell phenotype and function, and in the response of cells to environmental conditions. Alterations in transcription factors, including gene amplification or deletion, point mutations, and expression changes, are implicated in carcinogenesis, cancer progression, metastases, and resistance to cancer treatments. Not surprisingly, transcription factor activity is altered in numerous cancers, representing a unique class of cancer drug targets. This review updates and integrates information on the SALL family of transcription factors, highlighting the synergistic and/or antagonistic functions they perform in various cancer types.SALL proteins are a family of four conserved C2H2 zinc finger transcription factors that play critical roles in organogenesis during embryonic development. They regulate cell proliferation, survival, migration, and stemness; consequently, they are involved in various human genetic disorders and cancer. SALL4 is a well-recognized oncogene; however, SALL1–3 play dual roles depending on the cancer context and stage of the disease. Current reviews of SALLs have focused only on SALL2 or SALL4, lacking an integrated view of the SALL family members in cancer. Here, we update the recent advances of the SALL members in tumor development, cancer progression, and therapy, highlighting the synergistic and/or antagonistic functions they perform in similar cancer contexts. We identified common regulatory mechanisms, targets, and signaling pathways in breast, brain, liver, colon, blood, and HPV-related cancers. In addition, we discuss the potential of the SALL family members as cancer biomarkers and in the cancer cells’ response to therapies. Understanding SALL proteins’ function and relationship will open new cancer biology, clinical research, and therapy perspectives.

A chromosome-level genome of Portunus trituberculatus provides insights into its evolution, salinity adaptation and sex determination.

Portunus trituberculatus (Crustacea: Decapoda: Brachyura), commonly known as the swimming crab, is of major ecological importance, as well as being important to the fisheries industry. P. trituberculatus is also an important farmed species in China due to its rapid growth rate and high economic value. Here, we report the genome sequence of the swimming crab, which was assembled at the chromosome scale, covering ~1.2Gb, with 79.99% of the scaffold sequences assembled into 53 chromosomes. The contig and scaffold N50values were 108.7kb and 15.6Mb, respectively, with 19,981 protein-coding genes. Based on comparative genomic analyses of crabs and shrimps, the C2H2 zinc finger protein family was found to be the only gene family expanded in crab genomes, suggesting it was closely related to the evolution of crabs. The combination of transcriptome and bulked segregant analysis provided insights into the genetic basis of salinity adaptation and rapid growth in P. trituberculatus. In addition, the specific region of the Y chromosome was located for the first time in the genome of P. trituberculatus, and three genes were preliminarily identified as candidate genes for sex determination in this region. Decoding the swimming crab genome not only provides a valuable genomic resource for further biological and evolutionary studies, but is also useful for molecular breeding of swimming crabs.

Pseudomonas syringae activates ZAT18 to inhibit salicylic acid accumulation by repressing EDS1 transcription for bacterial infection.

Phytopathogens can manipulate plant hormone signaling to counteract immune responses; however, the underlying mechanism is mostly unclear. Here, we report that Pseudomonas syringae pv tomato (Pst) DC3000 induces expression of C2H2 zinc finger transcription factor ZAT18 in a jasmonic acid (JA)-signaling-dependent manner. Biochemical assays further confirmed that ZAT18 is a direct target of MYC2, which is a very important regulator in JA signaling. CRISPR/Cas9-generated zat18-cr mutants exhibited enhanced resistance to Pst DC3000, while overexpression of ZAT18 resulted in impaired disease resistance. Genetic characterization of ZAT18 mutants demonstrated that ZAT18 represses defense responses by inhibiting the accumulation of the key plant immune signaling molecule salicylic acid (SA), which is dependent on its EAR motif. ZAT18 exerted this inhibitory effect by directly repressing the transcription of Enhanced Disease Susceptibility 1 (EDS1), which is the key signaling component of pathogen-induced SA accumulation. Overexpression of ZAT18 resulted in decreased SA content, while loss of function of ZAT18 showed enhanced SA accumulation upon pathogen infection. Furthermore, enhanced resistance and SA content in zat18-cr mutants was abolished by the mutation in EDS1. Our data indicate that pathogens induce ZAT18 expression to repress the transcription of EDS1, further antagonising SA accumulation for bacterial infection.

Genome-Wide Identification of the Q-type C2H2 Transcription Factor Family in Alfalfa (Medicago sativa) and Expression Analysis under Different Abiotic Stresses.

Q-type C2H2 zinc-finger protein (C2H2-ZFP) transcription factors are associated with many plant growth development and environmental stress responses. To date, there have been few analyses of the Q-type C2H2-ZFP gene family in alfalfa (Medicago sativa subsp. sativa). In this study, we identified 58 Q-type C2H2-ZFPs across the entire alfalfa genome, and the gene structure, motif composition, chromosomal mapping, and cis-regulatory elements were explored, as well as the expression profiles of specific tissues and the response under different abiotic stresses. According to their phylogenetic features, these 58 MsZFPs were divided into 12 subgroups. Synteny analysis showed that duplication events play a vital role in the expansion of the MsZFP gene family. The collinearity results showed that a total of 26 and 42 of the 58 MsZFP genes were homologous with Arabidopsis and M. truncatula, respectively. The expression profiles showed that C2H2-ZFP genes played various roles in different tissues and abiotic stresses. The results of subsequent quantitative real-time polymerase chain reaction (qRT-PCR) showed that the nine selected MsZFP genes were rapidly induced under different abiotic stresses, indicating that C2H2-ZFP genes are closely related to abiotic stress. This study provides results on MsZFP genes, their response to various abiotic stresses, and new information on the C2H2 family in alfalfa.

Comparative analysis of physiological, agronomic and transcriptional responses to drought stress in wheat local varieties from Mongolia and Northern China

Drought is one of the major abiotic stresses that threaten wheat production worldwide, especially in the Mongolian Plateau and adjacent regions. This study aims to find local wheat varieties with high yields and drought resistance at various developmental stages based on agronomic traits and drought resistance indices analysis and explore the underlining molecular mechanisms by transcriptome analysis. Our results revealed that drought stress started at the seedling stage has a greater impact on crop yields. Four types of drought responses were found among the tested varieties. Type 1 and type 2 show low tolerance to drought stress despite high or low yield in control condition, type 3 exhibits high yield under control condition but dropped significantly after drought, and type 4 displays relatively high and stable yields under control and drought conditions. Transcriptome analysis performed with the representative varieties of the four types revealed GO terms and KEGG pathways enriched among drought-triggered differential expressed genes (DEGs). A network containing 18 modules was constructed using weighted gene co-expression analysis (WGCNA). Ten modules were significantly correlated to yield by module-trait correlation, and 3 modules showed Darkhan 144 specific gene expression patterns. C2H2 zinc finger factor-recognized motifs were identified from the promoters of genes in these modules. qRT-PCR confirmed several key DEGs with specific expression patterns and physiological measurements validated the relatively low oxidative damage and high antioxidant capacity in the drought tolerant variety Dankhan 144. These findings provide an important basis for local agriculture and breeding of drought-tolerant high yield wheat varieties.

In Silico Functional Prediction and Expression Analysis of C2H2 Zinc-Finger Family Transcription Factor Revealed Regulatory Role of ZmZFP126 in Maize Growth.

The C2H2-zinc finger proteins (ZFP) comprise a large family of transcription factors with various functions in biological processes. In maize, the function regulation of C2H2- zine finger (ZF) genes are poorly understood. We conducted an evolution analysis and functional prediction of the maize C2H2-ZF gene family. Furthermore, the ZmZFP126 gene has been cloned and sequenced for further favorable allelic variation discovery. The phylogenetic analysis of the C2H2-ZF domain indicated that the position and sequence of the C2H2-ZF domain of the poly-zinc finger gene are relatively conserved during evolution, and the C2H2-ZF domain with the same position is highly conserved. The expression analysis of the C2H2-ZF gene family in 11 tissues at different growth stages of B73 inbred lines showed that genes with multiple transcripts were endowed with more functions. The expression analysis of the C2H2-ZF gene in P1 and P2 inbred lines under drought conditions showed that the C2H2-ZF genes were mainly subjected to negative regulation under drought stress. Functional prediction indicated that the maize C2H2-ZF gene is mainly involved in reproduction and development, especially concerning the formation of important agronomic traits in maize yield. Furthermore, sequencing and correlation analysis of the ZmZFP126 gene indicated that this gene was significantly associated with the SDW-NAP and TDW-NAP. The analysis of the relationship between maize C2H2-ZF genes and C2H2-ZF genes with known functions indicated that the functions of some C2H2-ZF genes are relatively conservative, and the functions of homologous genes in different species are similar.

Cataloging Human PRDM9 Allelic Variation Using Long-Read Sequencing Reveals PRDM9 Population Specificity and Two Distinct Groupings of Related Alleles.

The PRDM9 protein determines sites of meiotic recombination in humans by directing meiotic DNA double-strand breaks to specific loci. Targeting specificity is encoded by a long array of C2H2 zinc fingers that bind to DNA. This zinc finger array is hypervariable, and the resulting alleles each have a potentially different DNA binding preference. The assessment of PRDM9 diversity is important for understanding the complexity of human population genetics, inheritance linkage patterns, and predisposition to genetic disease. Due to the repetitive nature of the PRDM9 zinc finger array, the large-scale sequencing of human PRDM9 is challenging. We, therefore, developed a long-read sequencing strategy to infer the diploid PRDM9 zinc finger array genotype in a high-throughput manner. From an unbiased study of PRDM9 allelic diversity in 720 individuals from seven human populations, we detected 69 PRDM9 alleles. Several alleles differ in frequency among human populations, and 32 alleles had not been identified by previous studies, which were heavily biased to European populations. PRDM9 alleles are distinguished by their DNA binding site preferences and fall into two major categories related to the most common PRDM9-A and PRDM9-C alleles. We also found that it is likely that inter-conversion between allele types is rare. By mapping meiotic double-strand breaks (DSBs) in the testis, we found that small variations in PRDM9 can substantially alter the meiotic recombination landscape, demonstrating that minor PRDM9 variants may play an under-appreciated role in shaping patterns of human recombination. In summary, our data greatly expands knowledge of PRDM9 diversity in humans.

The Piragua Gene Is not Essential for Drosophila Development.

Proteins with clusters of C2H2 zinc finger domains (C2H2-proteins) constitute the most abundant class of transcription factors in higher eukaryotes. N-terminal ZAD (zinc finger-associated domain) dimerization domain has been identified in a large group of C2H2-proteins mostly in insects. The piragua gene encodes one of these proteins, Fu2. We have generated CRISPR/Cas9-mediated deletion of the piragua gene that has no phenotype. We have used φC31-mediated attP/attB recombination to generate a transgenic line expressing Fu2 protein fused with HA epitope. This line will be useful for analysis of DNA binding profile and functions of Fu2 protein.

Coriolopsis trogii hydrophobin genes favor a clustering distribution and are widely involved in mycelial growth and primordia formation.

Hydrophobins are small, secreted proteins with important physiological functions in mycelial growth and fungal development. Here, 1 nucleus-specific and 35 allelic hydrophobin genes were identified in the genome of a white rot fungus, Coriolopsis trogii. Among these, 22 were eight-cysteine class I hydrophobin genes and the other 14 were uncommon six-cysteine hydrophobin genes. The six-cysteine hydrophobins were speculated to have originated from a common ancestor. The hydrophobin genes favored a clustering distribution and two recent duplication pairs were identified. The genes had conserved gene structures with three exons and two introns. Cthyd18, Cthyd19, and Cthyd32 were constitutively highly expressed in all developmental stages. Cthyd20, Cthyd21, Cthyd22, Cthyd28, Cthyd30, Cthyd31, and Cthyd33 were highly expressed in mycelia, and Cthyd12 and Cthyd35 in the reproductive stages. Sixteen hydrophobin genes were regulated differently in the transition from mycelia to primordia; Cthyd35 showed maximal upregulation of 1922-fold, and Cthyd23 showed maximal downregulation of 552-fold. Most (32) hydrophobin genes showed significant differential expression between mycelia cultured in different media (potato dextrose agar or broth). Weighted gene co-expression network analysis and promoter analysis revealed that C2H2 zinc finger proteins may regulate hydrophobin genes. These results may support further research into the function and evolution of hydrophobins.