Two-hybrid Assay Research Articles

Phytol-induced interplant signaling in maize facilitates EXP-A20-driven resistance through ACO31-dependent ethylene accumulation against Ostrinia furnacalis.

Plants have evolved sophisticated defense mechanisms against insect herbivores, including cell wall fortification through lignin biosynthesis. Insect attack primes systemic acquired resistance in plants, preparing them to respond more swiftly and vigorously to subsequent insect assaults. Here, we found that Beauveria bassiana-exposed maize plants can emit phytol upon infestation by Spodoptera frugiperda, inducing plant-to-plant (PTP) communication of alert signals for neighboring plants, and revealed the expansin protein EXP-A20 as a pivotal node mediating maize defense responses in neighboring plants against the destructive pest Ostrinia furnacalis via stimulation of ethylene (ET) synthesis and lignin production. Through virus-induced gene silencing, we showed that EXP-A20 is essential for maize resistance, while downregulating ET and lignin pathways. Critically, protein-protein interactions determined via luciferase complementation and yeast two-hybrid assays demonstrated that EXP-A20 binds to and likely activates the ET-forming enzyme gene ACO31 to initiate defense signaling cascades, representing a novel signaling modality for expansins. Treatment with the plant volatile phytol has known insecticidal/priming activity, but we found that its effectiveness requires EXP-A20. This finding highlights the importance of EXP-A20 upstream of hormone-cell wall crosstalk in defense activation by volatiles. Overall, our multifaceted dissection of EXP-A20 revealed key molecular intersections underlying inducible maize immunity against herbivores. Furthermore, we provide functional evidence that extensive cell growth processes directly stimulate defense programs in plants. Our work opens new avenues for enhancing durable, broad-spectrum pest resistance in maize through the use of volatile organic compounds and PTP interactions.

TaFAR9 and TaFAR10 synergistically regulate fertility conversion of photo-thermo-sensitive genic male sterility lines in wheat by modulating ROS homeostasis

Photo-thermo-sensitive genic male sterility (PTGMS), which exhibits varying fertility levels under different environmental conditions, is a crucial method for heterosis utilization in wheat. However, the mechanisms underlying fertility conversion remain unclear. In the study, three BS type PTGMS lines were analyzed to study fertility conversion characteristics. The results indicated that the fertility conversion occurred during meiosis and was accompanied by an increase in reactive oxygen species (ROS) under a sterile environment. TaFAR9 encoding a novel fatty acyl CoA reductase was identified using transcriptome sequencing. Expression analysis suggested that TaFAR9 was localized in the endoplasmic reticulum (ER), with high expression levels in anthers. Furthermore, the down-regulation of TaFAR9 expression displayed characteristics of male sterility, accompanied by the accumulation of ROS. Cytological analysis revealed abnormal development in the anther and pollen walls of TaFAR9-silenced lines. Additionally, TaFAR9 and TaFAR10 were confirmed to physically interact using molecular docking simulation, yeast two-hybrid, luciferase complementation, and bimolecular fluorescence complementation assays. The reduced expression of TaFAR10 also exhibited male sterility and ROS burst. Moreover, the co-silencing of TaFAR9 and TaFAR10 produced sterility phenotypes that were similar to those observed when silencing TaFAR9 or TaFAR10 individually. Transcriptome analysis suggested that the ROS burst in BSMV: TaFAR9/10 anthers can result in cellular metabolic disorders. These findings indicate that TaFAR9 and TaFAR10 may form heterodimers that synergistically regulate fertility conversion in PTGMS lines by modulating ROS metabolism. And this study offers a fresh insight into the regulatory processes involved in fertility conversion in PTGMS lines.

A Genome-Wide Characterization of Receptor-like Cytoplasmic Kinase IV Subfamily Members in Populus deltoides Identifies the Potential Role of PdeCRCK6 in Plant Osmotic Stress Responses.

The IV subfamily of receptor-like cytoplasmic kinase (RLCK-IV), known as calcium-binding receptor-like cytoplasmic kinases (CRCKs), plays a vital role in plant signal transduction, particularly in coordinating growth and responses to abiotic stresses. However, our comprehension of CRCK genes in Populus deltoides, a species characterized as fast-growing and pest-resistant but with drought intolerance, is limited. Here, we identify 6 members of the CRCK subfamily on a genome-wide scale in P. deltoides, denoted as PdeCRCK1-PdeCRCK6. An evolutionary and structural analysis revealed highly conserved kinase catalytic domains across all PdeCRCKs, characterized by calmodulin (CaM)-binding sites and serine (Ser)/threonine (Thr) phosphorylation sites. The cis-acting elements of promoters indicated the presence of responsive elements for plant hormones, abiotic stresses, and transcription factor binding sites, which is supported by the distinct transcriptional expression patterns of PdeCRCKs under abscisic acid (ABA), polyethylene glycol (PEG), and mannitol treatments. A transient overexpression of PdeCRCK3/5/6 in tobacco (Nicotiana benthamiana) leaves indicated their involvement in reactive oxygen species (ROS) scavenging, polyamine gene synthesis, and ABA signaling pathway modulation. Immunoprecipitation-Mass Spectrometry (IP-MS) and a yeast two-hybrid (Y2H) assay showed that PdeCRCK6 interacted with AAA-type ATPase proteins and ubiquitin, suggesting its potential function in being involved in chloroplast homeostasis and the 26S ubiquitin protease system. Taken together, these findings offer a comprehensive analysis of the RLCK-IV subfamily members in P. deltoides, especially laying a foundation for revealing the potential mechanism of PdeCRCK6 in response to osmotic stresses and accelerating the molecular design breeding of drought tolerance in poplar.

BPMs regulate Arabidopsis seedling development by promoting auxin-independent degradation of the Aux/IAA protein IAA10.

After germination, seedlings undergo etiolated development (skotomorphogenesis), enabling them to grow towards the soil surface. In Arabidopsis, etiolated seedlings exhibit rapid hypocotyl elongation, apical hook formation and closed cotyledons to protect the meristem. In this study, we found that high-order mutants in the BPM gene family displayed defects in seedling development, characterized by a shorter hypocotyl, early apical hook opening, and opened cotyledons in the dark. BPM1, BPM2, BPM4, and BPM5 exhibit distinct expression patterns and subcellular localization in etiolated seedlings. In a hypocotyl segment assay the bpm mutants showed defects in auxin response indicating impaired auxin signaling in the hypocotyl. Expression of the auxin reporter DR5:GFP was also altered in the bpm1,4,5 mutant in various tissues compared to the wild type. Furthermore, we showed that BPM1 and IAA10 interact in yeast two-hybrid, BiFC, and Co-IP assays. Experiments in protoplasts indicated that BPM1 promotes ubiquitylation and degradation of IAA10, and the level of IAA10 protein is greater in the bpm1,4,5 mutant. In addition, IAA10 over-expression resulted in phenotypes similar to the bpm mutants. These results indicate that the BPMs target the Aux/IAA proteins for ubiquitylation and degradation. Overall, our findings shed light on the key roles of the BPMs in auxin signaling during seedling development.

A C2H2-type zinc finger protein TaZFP8-5B negatively regulates disease resistance

BackgroundZinc finger proteins (ZFPs) are important regulators in abiotic and biotic stress tolerance in plants. However, the role of the ZFPs in wheat responding to pathogen infection is poorly understood.ResultsIn this study, we found TaZFP8-5B was down-regulated by Puccinia striiformis f. sp. tritici (Pst) infection. TaZFP8-5B possesses a single C2H2-type zinc finger domain with a plant-specific QALGGH motif, and an EAR motif (LxLxL) at the C-terminus. The EAR motif represses the trans-activation ability of TaZFP8-5B. Knocking down the expression of TaZFP8 by virus-induced gene silencing increased wheat resistance to Pst, whereas TaZFP8-5B-overexpressing reduced wheat resistance to stripe rust and rice resistance to Magnaporthe oryzae, suggesting that TaZFP8-5B plays a negative role in the modulation of plant immunity. Using bimolecular fluorescence complementation, split-luciferase, and yeast two-hybrid assays, we showed that TaZFP8-5B interacted with a wheat calmodulin-like protein TaCML21. Knock-down of TaCML21 reduced wheat resistance to Pst.ConclusionsThis study characterized the function of TaZFP8-5B and its interacting protein TaCML21. Our findings provide a new perspective on a regulatory module made up of TaCML21-TaZFP8-5B in plant immunity.

RMI1 is essential for maintaining rice genome stability at high temperature.

Heat is a critical environmental stress for plant survival.One of its harmful effects on the cells is the disruption of genome integrity. However, the mechanisms by which plants cope with heat-induced DNA damage remain largely unknown. RMI1, a component of the RTR (RECQ4-TOP3α-RMI1) complex, plays a pivotal role in maintaining genome stability. In this study, we identified the target gene RMI1by characterizing a high-temperature-sensitive mutant. The growth and development of rmi1-1 seedlings carrying a non-frameshift mutation in RMI1 were hindered at 38°C. Abnormal mitotic chromosome behaviours ultimately led to the cell death of root tips. Additionally, the presence of chromosome fragments during anaphase I caused pollen abortion and sterility in rmi1-1 plants. Yeast two-hybrid assays revealed that the interactions between RMI1-1 and RECQ4 or TOP3α were weakened with increasing temperature and entirely ceased at 36°C. In contrast, the functionalRMI1 maintained its interactions with RECQ4 or TOP3α under the same conditions. These results indicate that the non-frameshift mutation in RMI1 disrupts the formation of the RTR complex at high temperatures, leading to defects in DNA repair and increased sensitivity of rmi1-1 under heat stress. However, embryos of the rmi1-cr2 mutant with a frameshift mutation in RMI1 exhibited complete lethality. In addition, the overexpression of RMI1 enhanced the heat tolerance in rice. These findings provide insights into the molecular mechanisms that RMI1 responds to high temperatures by maintaining genome stability in rice.

Genome-Wide Analysis of GLK Gene Family in Four Cotton Species Provides Insights into Their Involvement in Cotton Abiotic Stress Response

Cotton is a crucial economic crop that supplies natural fibers for the textile industry, with fiber quality being greatly impacted by abiotic stress throughout its growth stages. The Golden2-Like (GLK) gene family plays a key role in plant development and adaptation to abiotic stress. However, the specific functions and regulatory mechanisms of GLK members in cotton remain largely unexplored. In this study, a thorough analysis of GLK in four cotton species (Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense) was conducted. A total of 198 GLK genes were identified in cotton. Conserved sequence analysis revealed that most GLK proteins contain two highly conserved domains: a MYB DNA-binding domain and a C-terminal (GCT) box. Promoter element analysis results show that the GLK gene family contains many stress response-related elements. Expression analysis demonstrated that GhGLK2, GhGLK11, GhGLK16, and GhGLK30 responded significantly to drought, salt, and temperature stresses. And GhGLK2, GhGLK13, GhGLK38, GhGLK42, and GhGLK46 responded significantly to cotton development. Yeast one-hybrid, yeast two-hybrid, and dual-luciferase assay results indicate that GhGLK2 interacts with GhGUN5, GhPIL6, GhNAC6, GhTPX2, and GhERF10. These findings suggest that these GhGLKs may play crucial roles in regulating the response to abiotic stress. Overall, this study provides a solid theoretical foundation for understanding the role of the GLK gene family in cotton’s response to abiotic stress.

Identification of the NAC Family and a Functional Analysis of NoNAC36a Under Flooding Stress in Watercress (Nasturtium officinale R.Br.)

Watercress (Nasturtium officinale R.Br.) is a cruciferous aquatic vegetable that possesses significant nutritional value. The NAC family is a transcription factor family specific to plants that play an important role in regulating plant responses to abiotic stress. In order to investigate the response of NAC genes to flooding stress in watercress, we conducted a study on the NoNAC family. In this study, a total of 119 NoNAC genes were obtained through genome-wide identification. Phylogenetic analysis indicated that the NoNAC family members can be categorized into ten subgroups. The results of gene structure analysis revealed that each branch within the subgroups exhibited similar motif composition and gene structure. The heatmap analysis showed that several NoNAC genes demonstrated tissue-specific expression patterns, suggesting their potential as regulators of associated tissue development. As an aquatic plant, watercress serves as a valuable material for investigating plant resistance to flooding stress. This study found that flooding can significantly increase the watercress plant height, which is a typical escape strategy under flooding. The analysis of the expression of NoNAC genes in the stem transcriptome after flooding indicated that only NoNAC36a consistently exhibited significant differential changes and down-regulated expression at the three time points of flooding treatment. This suggests that NoNAC36a may be involved in regulating watercress plant height increases under flooding stress. The utilization of a virus-induced gene silencing assay to investigate the biological function of NoNAC36a revealed that NoNAC36a silencing caused cell elongation and expansion, thus increasing watercress plant height. The yeast one-hybrid and dual luciferase assays demonstrated that NoNAC36a binds the promoter of NoXTH33 and inhibits its expression. Subsequently, the results of yeast two-hybrid, luciferase complementary, and pull-down assays revealed the interaction between NoMOB1A and NoNAC36a in vivo and in vitro. Sequence alignment indicated that NoMOB1A and AtMOB1A share an identical amino acid sequence. RT-qPCR analysis indicated that flooding prompted the expression of NoMOB1A in stems. Thus, it is speculated that NoMOB1A may exhibit functions similar to AtMOB1A and that the up-regulation of NoMOB1A expression in stems may facilitate an increase in plant height under flooding. In summary, the NoNAC family was analyzed, and revealed a regulatory network centered on NoNAC36a that facilitates watercress resistance to flooding stress. This study enhanced the understanding of the NoNAC genes and established a theoretical foundation for investigating plant flooding tolerance.

Orf116b Induces Pollen Abortion in a Novel Cotton (Gossypium hirsutum L.) Cytoplasmic Male Sterile Line J4A.

Cytoplasmic male sterile (CMS) systems have great potential in hybrid seed production in many plants. However, the incompatibility between the cytoplasmic and nuclear genes and the availability of fewer CMS lines limit the system's application in cotton heterosis. Therefore, the present study sequenced the mitochondrial (mt) genomes of a novel cotton (Gossypium hirsutum L.) CMS line J4A and its cytoplasmic nuclear homologous maintainer line J4B to investigate the mechanism underlying CMS and improve its application. A novel CMS gene, orf116b, was identified in the CMS line. Ectopic expression of orf116b in Escherichia coli suppressed growth, while its overexpression in Arabidopsis, rice, tobacco, and cotton led to complete or partial male sterility. Further analysis of anthers revealed mt disruption in J4A with higher levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA), but lower levels of ATP and ribosomal protein (RP) than in J4B. Finally, a membrane-based yeast two-hybrid (MYTH) assay and bimolecular fluorescence complementation (BiFC) assays demonstrated that orf116b probably interacts with an anther-specific protein, LAT52. These observations collectively proved that orf116b is associated with early and stable pollen abortion in cotton, providing a foundation for further research on cotton fertility restoration and heterosis breeding.

Use of the Puccinia sorghi haustorial transcriptome to identify and characterize AvrRp1-D recognized by the maize Rp1-D resistance protein.

The common rust disease of maize is caused by the obligate biotrophic fungus Puccinia sorghi. The maize Rp1-D allele imparts resistance against the P. sorghi IN2 isolate by initiating a defense response that includes a rapid localized programmed cell death process, the hypersensitive response (HR). In this study, to identify AvrRp1-D from P. sorghi IN2, we employed the isolation of haustoria, facilitated by a biotin-streptavidin interaction, as a powerful approach. This method proves particularly advantageous in cases where the genome information for the fungal pathogen is unavailable, enhancing our ability to explore and understand the molecular interactions between maize and P. sorghi. The haustorial transcriptome generated through this technique, in combination with bioinformatic analyses such as SignalP and TMHMM, enabled the identification of 251 candidate effectors. We ultimately identified two closely related genes, AvrRp1-D.1 and AvrRp1-D.2, which triggered an Rp1-D-dependent defense response in Nicotiana benthamiana. AvrRp1-D-induced Rp1-D-dependent HR was further confirmed in maize protoplasts. We demonstrated that AvrRp1-D.1 interacts directly and specifically with the leucine-rich repeat (LRR) domain of Rp1-D through yeast two-hybrid assay. We also provide evidence that, in the absence of Rp1-D, AvrRp1-D.1 plays a role in suppressing the plant immune response. Our research provides valuable insights into the molecular interactions driving resistance against common rust in maize.

A cell-penetrating NS5B-specific nanobody inhibits bovine viral diarrhea virus replication

Bovine viral diarrhea virus (BVDV) causes one of the significant devastating diseases for the cattle industry worldwide. The virus can cross the placenta and result in the persistent infection of the fetus, which has hampered the efficacy and the development of vaccines. Hence, efficient antiviral strategies are urgently needed. In our previous work, a specific nanobody Nb1 against nonstructural protein 5 (NS5B) was successfully isolated, and the replication of BVDV was significantly reduced in the MDBK cell line stably expressing Nb1. Nevertheless, the Nb1 protein itself cannot enter the cells autonomously, which has severely hampered the application of the Nb1. In this work, Nb1fuses with a trans-activating transduction (TAT) peptide to form the TAT-Nb1. The TAT-Nb1 was expressed in Escherichia coli, and then purified by Ni-nitrilotriacetic acid (Ni-NTA) resin. We showed that TAT successfully delivered Nb1 into the MDBK cells, and the TAT-Nb1 efficiently inhibited the replication of BVDV in a dose-and time-dependent manner. Furthermore, the recognition site of Nb1 to NS5B was identified as NS5Baa186-487 by the yeast two-hybrid assay. In summary, this study indicates that the TAT-Nb1 can potentially to be an antiviral drug against BVDV infection, and this research may accelerate the process of Nb1 for clinical use.

EXECUTER1 and singlet oxygen signaling: A reassessment of nuclear activity.

Chloroplasts are recognized as environmental sensors, capable of translating environmental fluctuations into diverse signals to communicate with the nucleus. Among the reactive oxygen species produced in chloroplasts, singlet oxygen (1O2) has been extensively studied due to its dual roles, encompassing both damage and signaling activities, and the availability of conditional mutants overproducing 1O2 in chloroplasts. In particular, investigating the Arabidopsis (Arabidopsis thaliana) mutant known as fluorescent (flu) has led to the discovery of EXECUTER1 (EX1), a plastid 1O2 sensor residing in the grana margin of the thylakoid membrane. 1O2-triggered EX1 degradation is critical for the induction of 1O2-responsive nuclear genes (SOrNGs). However, a recent study showed that EX1 relocates from chloroplasts to the nucleus upon 1O2 release, where it interacts with WRKY18 and WRKY40 (WRKY18/40) transcription factors to regulate SOrNG expression. In this study, we challenge this assertion. Our confocal microscopy analysis and subcellular fractionation assays demonstrate that EX1 does not accumulate in the nucleus. While EX1 appears in nuclear fractions, subsequent thermolysin treatment assays indicate that it adheres to the outer nuclear region rather than localizing inside the nucleus. Furthermore, luciferase complementation imaging and yeast two-hybrid assays reveal that EX1 does not interact with nuclear WRKY18/40. Consequently, our study refines the current model of 1O2 signaling by ruling out the nuclear relocation of intact EX1 as a means of communication between the chloroplast and nucleus.

Genome-wide identification of SAP family genes and characterization of TaSAP6-A1 to improve Cd tolerance in Triticum aestivum L.

Stress Associated Proteins (SAPs) contain A20/AN1 zinc finger domains and, have been proposed to function in various physiological processes such as cold, salinity, drought, heavy metals, damage, and flooding resistance in plants. Here, a total of 131 SAP genes were identified, including T. aestivum (60), T. urartu (10), Ae. Tauschii (16), T. dicoccoides (13), O. sativa (18), and Arabidopsis (14). A phylogenetic analysis revealed that the SAPs are clustered into two subfamilies. The TaSAP genes in the collinear region comprised 34 pairs of duplicated genes formed through segmental duplication events. Overexpressing TaSAP6-A1 in wheat enhanced Cd tolerance, whereas knock-down of this gene increased Cd sensitivity. Yeast two-hybrid (Y2H) and bimolecular fluorescent complementation assays (BiFC) demonstrated interaction between TaSAP6-A1 and phenylalanine ammonia-lyase (TaPAL), the first enzyme in the phenylpropanoid pathway. This study provides a valuable reference for further investigations into the functional and molecular mechanisms of the SAP gene family.

NAC family gene CmNAC34 positively regulates fruit ripening through interacting with CmNAC-NOR in Cucumis melo

Melon (Cucumis melo) is an important economic horticulture crop cultivated worldwide. NAC (NAM, ATAC, and CUC) transcription factors play crucial roles in the transcriptional regulation of various developmental stages in plant growth and fruit development, but little about their gene function is known in melon. Here, we identified 78 CmNAC family genes containing integrated and conserved NAM (no apical meristem) domain in the melon genome by performing genome-wide identification and bioinformatics analysis. Transcriptome data analysis and qRT-PCR results showed that most CmNACs are specifically enriched in the vegetative organ or the reproductive organ in melon. Through genetic transformation, we found that overexpression of CmNAC34 in melons led to the early ripening fruits, suggesting its positive role in promoting fruit maturation. Through performing yeast two-hybrid and bimolecular fluorescence complementation assays, we verified the direct protein interaction between CmNAC34 and CmNAC-NOR. The expression pattern of CmNAC34 and CmNAC-NOR were similar in melon tissues, and subcellular localization also revealed their nuclear protein characteristic. We transformed CmNAC-NOR in melon and found that its overexpression resulted in the early ripening fruits. Then, the yeast one-hybrid and dual luciferase reporter gene assay explored that CmNAC34 protein can bind to the promoters of two Glyoxalase (GLY) genes, which were involved in the abscisic acid signal pathway and associated with the fruit regulation. These findings revealed the molecular characteristics, expression profile, and functional pattern of the NAC family transcription factors genes and provided an insight into the molecular mechanism of CmNAC34 in regulating climacteric fruit ripening.

Mechanism of zju-miR156c-mediated network in regulating witches' broom symptom of Chinese jujube.

Jujube witches' broom, caused by phytoplasma, is a destructive disease of Chinese jujube. Studies have shown that zju-miR156s play an important role in phytoplasma infection in jujube, but the regulatory mechanism between zju-miR156c and witches' broom remains unexplored. In the current study, miRNA-seq and gene expression analysis showed that zju-miR156c was more highly induced in infected jujube plants than the other miRNAs and its target gene was ZjSPL3. In addition, the expression levels of thymidylate kinase gene (TMKJWB) and secreted jujube protein (SJP1JWB) in diseased materials were higher than those in healthy controls. The expression level of zju-miR156c was significantly upregulated, while ZjSPL3 was sharply downregulated and the content of cytokinin (CTK) significantly increased. Overexpression of zju-miR156c in Arabidopsis significantly reduced the expression of AtSPL10 (homologous gene of ZjSPL3) but increased the content of CTK, and the transgenic plants exhibited witches' broom symptoms. In addition, yeast two-hybrid and co-immunoprecipitation assays confirmed that SJP1JWB interacted with ZjERF18. Yeast one-hybrid analysis showed that ZjERF18 could interact with the promoter of zju-MIR156c. In conclusion, our results demonstrated a novel pathogenic module of ZjERF18-zju-miR156c-ZjSPL3-CTK has an important function in the formation of witches' broom caused by SJP1JWB.

Genome-wide identification of the eggplant jasmonate ZIM-domain (JAZ) gene family and functional characterization of SmJAZ10 in modulating chlorophyll synthesis in leaves.

The jasmonate ZIM-domain (JAZ) plays a crucial role in regulating several economic traits in crops. Despite its importance, the characterization of the SmJAZ gene family in eggplant (Solanum melongena L.) has not been documented. In this study, we identified 13 SmJAZ distributed across 9 chromosomes, which were categorized into 5 subgroups based on phylogenetic analysis. Both of them possess TIFY-motif and CCT_2 domains with varying degrees of variation. Promoter cis-element analysis predicted 42 distributed cis-elements that respond to diverse signals. Gene expression analysis demonstrated that SmJAZ exhibited responsiveness to JA, ABA, NaCl, PEG, 4 °C, blue light, and UV-B treatments. Moreover, microRNA interaction predictions identified 150 potential miRNAs, among which ath-miR5021 was found to target 8 SmJAZ mRNAs. Yeast two-hybrid assays demonstrated that most of the SmJAZs were able to interact with SmMYC2 and SmNINJA and could form JAZ-JAZ complexes. Subcellular localization analysis unveiled a diverse array of intranuclear and extranuclear localization signals for SmJAZs. Overexpressing of SmJAZ10 could decrease the chlorophyll content of seedling leaves, and the transcriptome showed that genes related to chlorophyll synthesis, such as SmCHLH, SmPORA, and SmGLK2, underwent down-regulated expression. Overall, these findings serve as a valuable resource for leveraging JA signaling to enhance eggplant quality.

Genome-Wide Identification, Characterization and Expression Patterns of the DBB Transcription Factor Family Genes in Wheat.

Double B-box (DBB) proteins are plant-specific transcription factors (TFs) that play crucial roles in plant growth and stress responses. This study investigated the classification, structure, conserved motifs, chromosomal locations, cis-elements, duplication events, expression levels, and protein interaction network of the DBB TF family genes in common wheat (Triticum aestivum L.). In all, twenty-seven wheat DBB genes (TaDBBs) with two conserved B-box domains were identified and classified into six subgroups based on sequence features. A collinearity analysis of the DBB family genes among wheat, Arabidopsis, and rice revealed some duplicated gene pairs and highly conserved genes in wheat. An expression pattern analysis indicated that wheat TaDBBs were involved in plant growth, responses to drought stress, light/dark, and abscisic acid treatment. A large number of cis-acting regulatory elements related to light response are enriched in the predicted promoter regions of 27 TaDBBs. Furthermore, some of TaDBBs can interact with COP1 or HY5 based on the STRING database prediction and yeast two-hybrid (Y2H) assay, indicating the potential key roles of TaDBBs in the light signaling pathway. Conclusively, our study revealed the potential functions and regulatory mechanisms of TaDBBs in plant growth and development under drought stress, light, and abscisic acid.

Zinc metalloprotease FgM35, which targets the wheat zinc-binding protein TaZnBP, contributes to the virulence of Fusarium graminearum

Metalloproteinases are ubiquitous in organisms. Most metalloproteinases secreted by pathogenic microorganisms are also called virulence factors, because they degrade proteins in the external tissues of the host, thereby reducing the host’s immunity and increasing its susceptibility to disease. Zinc metalloproteinase is one of the most common metalloproteinases. In our report, we studied the biological function of zinc metalloprotease FgM35 in Fusarium graminearum and the pathogen–host interaction during infection. We found that the asexual and sexual reproduction of the deletion mutant ΔFgM35 were affected, as well as the tolerance of F. graminearum to metal stress. In addition, deletion of FgM35 reduced the virulence of F. graminearum. The wheat target TaZnBP was screened using a wheat yeast cDNA library, and the interaction between FgM35 and TaZnBP was verified by HADDOCK molecular docking, yeast two-hybrid, Bi-FC, Luc, and Co-IP assays. The contribution of TaZnBP to plant immunity was also demonstrated. In summary, our work revealed the indispensable role of FgM35 in the reproductive process and the pathogenicity of F. graminearum, and it identified the interaction between FgM35 and TaZnBP as well as the function of TaZnBP. This provides a theoretical basis for further study of the function of metalloproteinases in pathogen–host interactions.

Molecular mechanism of flagellar motor rotation arrest in bacterial zoospores of Actinoplanes missouriensis before germination

Zoospores of the filamentous actinomycete Actinoplanes missouriensis swim vigorously using flagella and stop swimming to initiate germination in response to nutrient exposure. However, the molecular mechanisms underlying swimming cessation remain unknown. A protein (FtgA) of unknown function encoded by a chemotaxis gene cluster (che cluster-1) was found to be required for flagellar rotation arrest; the zoospores of ftgA-knockout mutants kept swimming awkwardly after germination. An ftgA-overexpressing strain exhibited a non-flagellated phenotype. Isolation of a suppressor strain from this strain and further in vivo experiments revealed that the extended N-terminal region of FliN, a component of the C-ring of the flagellar basal body, was involved in the function of FtgA; FliN-P101S canceled the flagellar rotation arrest by FtgA, as well as the negative effect of ftgA-overexpression on flagellation. Furthermore, bacterial two-hybrid assays suggested that FtgA interacted not only with the C-terminal core region of FliN but also with chemotaxis regulatory proteins CheA1 and CheW1-2, which are encoded by che cluster-1. We propose the following working model of motility regulation in A. missouriensis zoospores: the chemotaxis sensory complex initially captures FtgA to allow zoospores to swim and then releases FtgA to stop flagellar rotation (i.e., swimming) in response to external nutrient signals.

Genome-wide analysis of the MADS-box gene family in mango and ectopic expression of MiMADS77 in Arabidopsis results in early flowering

Mango (Mangifera indica L.) is an important tropical fruit, and timely flowering and fruit setting are very important for mango production. The MADS-box gene family is involved in the regulation of flower induction, floral organ specification, and fruit development in plants. The identification and analysis of the MADS-box gene family can lay a foundation for the study of the molecular mechanism of flowering and fruit development in mango. In this study, 119 MiMADS-box genes were identified on the basis of genome and transcriptome data. Phylogenetic analysis revealed that these genes can be divided into two classes. Forty-one type I proteins were further divided into three subfamilies, and seventy-eight type II proteins were further classified into eleven subfamilies. Several pairs of alternative splicing genes were found, especially in the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) subfamily. The MiMADS-box genes were distributed on 18 out of the 20 mango chromosomes. Cis-element analysis revealed many light-, stress-, and hormone-responsive elements in the promoter regions of the mango MiMADS-box genes. Expression pattern analysis revealed that these genes were differentially expressed in multiple tissues in mango. The highly expressed MiMADS77 was subsequently transformed into Arabidopsis, resulting in significant early flowering and abnormal floral organs. Yeast two-hybrid (Y2H) assays revealed that MiMADS77 interacts with several MiMADS-box proteins. In addition, we constructed a preliminary flowering regulatory network of MADS-box genes in mango on the basis of related studies. These results suggest that MiMADS77 genes may be involved in flowering regulation of mango.