FvOshC Is a Key Global Regulatory Target in Fusarium verticillioides for Fumonisin Biosynthesis and Disease Control.

Fusarium verticillioides has a substantial impact on maize production, commonly leading to maize ear rot and the production of fumonisin, a mycotoxin that poses health risks to both humans and animals. Currently, there is a lack of molecular targets for preventing the disease and controlling the toxin. The biological functions of oxysterol-binding proteins (OSBP) in filamentous fungi remain unclear. In this research, 7 oxysterol-binding protein-related proteins were identified in F. verticillioides, and these proteins were obtained through prokaryotic expression and purification. FvOshC was identified as the specific protein that binds to ergosterol through fluorescence titration. Gene knockout complementation techniques confirmed that FvOSHC plays a positive role, establishing it as a novel global regulatory protein involved in the pathogenicity and FB1 biosynthesis in F. verticillioides. Additionally, the interaction between FvOshC and FvSec14 was identified using yeast two-hybrid techniques. Moreover, computer-aided drug design technology was utilized to identify the receptor molecule Xanthatin based on FvOshC. The inhibitory effect of Xanthatin on the growth of F. verticillioides and the synthesis of FB1 was significantly demonstrated. These findings provide valuable insights that can aid in the management of mycotoxin pollution.

Plant immunity suppression by an ß-1,3-glucanase of the maize anthracnose pathogen Colletotrichum graminicola.

BACKGROUND: Many phytopathogens secrete a large number of cell wall degrading enzymes (CWDEs) to decompose host cell walls in order to penetrate the host, obtain nutrients and accelerate colonization. There is a wide variety of CWDEs produced by plant pathogens, including glycoside hydrolases (GHs), which determine the virulence, pathogenicity, and host specificity of phytopathogens. The specific molecular mechanisms by which pathogens suppress host immunity remain obscure. RESULT: In this study, we found that CgEC124 encodes a glycosyl hydrolase with a signal peptide and a conserved Glyco_hydro_cc domain which belongs to glycoside hydrolase 128 family. The expression of CgEC124 was significantly induced in the early stage of Colletotrichum graminicola infection, especially at 12 hpi. Furthermore, CgEC124 positively regulated the pathogenicity, but it did not impact the vegetative growth of mycelia. Ecotopic transient expression of CgEC124 decreased the disease resistance and callose deposition in maize. Moreover, CgEC124 exhibited the ß-1,3-glucanase activity and suppresses glucan-induced ROS burst in maize leaves. CONCLUSIONS: Our results indicate that CgEC124 is required for full virulence of C. graminicola but not for vegetative growth. CgEC124 increases maize susceptibility by inhibiting host reactive oxygen species burst as well as callose deposition. Meanwhile, our data suggests that CgEC124 explores its ß-1,3-glucanase activity to prevent induction of host defenses.

Fractional-order electromagnetic modeling and identification for PMSM servo system.

An accurate electromagnetic model is essential for an optimal controller tuning of the high-performance servo system. This paper proposes a fractional-order electromagnetic model of a permanent magnet synchronous motor (PMSM) servo system and an identification methodology of this model. The reason why the investigated electromagnetic model should be a fractional-order one is addressed with a detailed explanation. The influence of voltage source inverter nonlinearity, which may cause system identification error, is analyzed. An improved inverter nonlinearity model and compensation method are proposed to promote the accuracy of the model parameter identification. Compared with the existing typical electromagnetic models of the PMSM servo system, the current open-loop and closed-loop experiments prove that the proposed fractional-order electromagnetic model with time delay is more accurate for the actual physical system. The effectiveness of the proposed nonlinearity modeling and compensation scheme of the inverter is also verified on an experimental PMSM servo system.

Purine metabolism in plant pathogenic fungi.

In eukaryotic cells, purine metabolism is the way to the production of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and plays key roles in various biological processes. Purine metabolism mainly consists of de novo, salvage, and catabolic pathways, and some components of these pathways have been characterized in some plant pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae and wheat head blight fungus Fusarium graminearum. The enzymatic steps of the de novo pathway are well-conserved in plant pathogenic fungi and play crucial roles in fungal growth and development. Blocking this pathway inhibits the formation of penetration structures and invasive growth, making it essential for plant infection by pathogenic fungi. The salvage pathway is likely indispensable but requires exogenous purines, implying that purine transporters are functional in these fungi. The catabolic pathway balances purine nucleotides and may have a conserved stage-specific role in pathogenic fungi. The significant difference of the catabolic pathway in planta and in vitro lead us to further explore and identify the key genes specifically regulating pathogenicity in purine metabolic pathway. In this review, we summarized recent advances in the studies of purine metabolism, focusing on the regulation of pathogenesis and growth in plant pathogenic fungi.

MicroRNAs involved in the trans-kingdom gene regulation in the interaction of maize kernels and Fusarium verticillioides.

Maize ear rot is a widespread disease and the main pathogen is Fusarium verticillioides. Plant microRNAs (miRNAs) have great effects on disease resistance and it has been reported that maize miRNA participates in defense responses in maize ear rot. However, the trans-kingdom regulation of miRNAs between maize and F. verticillioides remains uncharacterized. In this study, the relationship between miRNA-like RNAs (milRNAs) of F. verticillioides and pathogenicity was investigated, followed by sRNA analysis and degradome sequencing of miRNA profiles and the target genes of maize and F. verticillioides after inoculation. It was found that the milRNA biogenesis positively regulated the pathogenicity of F. verticillioides by knocking out the gene FvDicer2-encoded Dicer-like protein in F. verticillioides. Following inoculation with F. verticillioides, 284 known and 6571 novel miRNAs were obtained in maize, including 28 miRNAs differentially expressed at multiple time points. The target genes of maize differentially expressed miRNAs in F. verticillioides mediated multiple pathways, including autophagy and MAPK signaling pathway. Fifty-one novel F. verticillioides milRNAs were predicted to target 333 genes in maize involved in MAPK signaling pathways, plant hormone signaling transduction and plant-pathogen interaction pathways. Additionally, the miR528b-5p in maize targeted the mRNA of FvTTP which encoded a twice transmembrane protein in F. verticillioides. The FvTTP-knockout mutants displayed decreased pathogenicity and reduced synthesis of fumonisins. Thus, by interfering with the translation of FvTTP, the miR528b-5p inhibited F. verticillioides infection. These findings suggested a novel function of miR528 in resisting F. verticillioides infection. The miRNAs identified in this research and their putative target genes can be used to further elucidate the trans-kingdom functions of microRNAs in plant pathogen interaction.

Activated malate circulation contributes to the manifestation of light-dependent mosaic symptoms.

Mosaic symptoms are commonly observed in virus-infected plants. However, the underlying mechanism by which viruses cause mosaic symptoms as well as the key regulator(s) involved in this process remain unclear. Here, we investigate maize dwarf mosaic disease caused by sugarcane mosaic virus (SCMV). We find that the manifestation of mosaic symptoms in SCMV-infected maize plants requires light illumination and is correlated with mitochondrial reactive oxidative species (mROS) accumulation. The transcriptomic and metabolomic analyses results together with the genetic and cytopathological evidence indicate that malate and malate circulation pathways play essential roles in promoting mosaic symptom development. Specifically, at the pre-symptomatic infection stage or infection front, SCMV infection elevates the enzymatic activity of pyruvate orthophosphate dikinase by decreasing the phosphorylation of threonine527 under light, resulting in malate overproduction and subsequent mROS accumulation. Our findings indicate that activated malate circulation contributes to the manifestation of light-dependent mosaic symptoms via mROS.

Transcriptional differences between major Fusarium pathogens of maize, Fusarium verticillioides and Fusarium graminearum with different optimum growth temperatures.

Fusarium verticillioides and Fusarium graminearum are important pathogens causing disease in maize (Zea mays) worldwide. The distributions of these fungal pathogens vary greatly in different regions and in different years, and are influenced by environmental and climatic conditions. Temperature has significant effects on the growth and mycotoxin production of Fusarium species. In this study, the effects of temperature on the growth and pathogenicity of F. verticillioides and F. graminearum were investigated. F. verticillioides grew fastest and exhibited the strongest pathogenicity to maize stems and grains at 30°C, while F. graminearum grew best at 20°C. Both species produced more toxins at 20°C than at 30°C. To explain the interspecific differences in the relationship of growth and temperature, RNA-seq was used to compare F. verticillioides and F. graminearum cultivated for 4 d at the optimum temperatures of 30°C and 20°C, respectively. Samples of F. verticillioides were also cultivated for 9 d (to maximize toxin production) at 20°C and 30°C and analyzed by RNA-seq to investigate the influence of temperature for different growth stages. The differently expressed genes (DEGs) were identified by comparison of cultures grown for the same amount of time but at different temperatures. GO enrichment analysis showed high enrichment of DEGs in categories of membrane part, catalytic activity, metabolic process, and growth at warmer temperature resulted in more down-regulated DEGs enriched in membrane components in all groups. KEGG analysis revealed enrichment of DEGs related to different temperatures in carbohydrate and amino acid metabolism pathways. For both species, there was decreased expression of many DEGs related to amino acid metabolism when cultivated at warm temperature, such as genes related to beta-alanine metabolism and arginine and proline metabolism. However, changes in genes related to glyoxylate and dicarboxylate metabolism and fatty acid degradation were more related to the growth state. The results showing different responses pattern of these pathways provides a foundation for further investigation of the molecular mechanisms underlying distinct thermal ecological niches of F. verticillioides and F. graminearum.

Real-Time Stylized Humanoid Behavior Control through Interaction and Synchronization.

Restricted by the diversity and complexity of human behaviors, simulating a character to achieve human-level perception and motion control is still an active as well as a challenging area. We present a style-based teleoperation framework with the help of human perceptions and analyses to understand the tasks being handled and the unknown environment to control the character. In this framework, the motion optimization and body controller with center-of-mass and root virtual control (CR-VC) method are designed to achieve motion synchronization and style mimicking while maintaining the balance of the character. The motion optimization synthesizes the human high-level style features with the balance strategy to create a feasible, stylized, and stable pose for the character. The CR-VC method including the model-based torque compensation synchronizes the motion rhythm of the human and character. Without any inverse dynamics knowledge or offline preprocessing, our framework is generalized to various scenarios and robust to human behavior changes in real-time. We demonstrate the effectiveness of this framework through the teleoperation experiments with different tasks, motion styles, and operators. This study is a step toward building a human-robot interaction that uses humans to help characters understand and achieve the tasks.

Novel factors contributing to fungal pathogenicity at early stages of Setosphaeria turcica infection.

The fungal pathogen Setosphaeria turcica causes leaf blight on maize, which leads to considerable crop losses. However, how S. turcica establishes sustained systemic infection is largely unknown. Here, we report several novel factors contributing to S. turcica pathogenicity, identified using a genomic and transcriptional screen at different stages of S. turcica appressorium development. We identified two cytoskeleton regulators, SLM1 and SLM2, that are crucial for hypha and appressorium development. The SLM1 and SLM2 transcripts accumulated during germling stage but their levels were notably reduced at the appressorium stage. Deletion of SLM2 dramatically affected cell morphology, penetration ability, and pathogenicity. We also identified three different types of S. turcica glycosyl hydrolases that are critical for plant cell wall degradation. Their transcripts accumulated during the appressorium infection stage induced by cellophane and maize leaf. Most importantly, we characterized a novel and specific S. turcica effector, appressorium-coupled effector 1 (StACE1), whose expression is coupled to appressorium formation in S. turcica. This protein is required for maize infection and induces cell death on expression in Nicotiana benthamiana. These observations suggest that the phytopathogen S. turcica is primed in advance with multiple strategies for maize infection, which are coupled to appressorium formation at the early infection stages.

The zinc finger protein StMR1 affects the pathogenicity and melanin synthesis of Setosphaeria turcica and directly regulates the expression of DHN melanin synthesis pathway genes.

The infection and colonization of pathogenic fungi are often regulated by transcription factors. In our previous study, the zinc finger protein-encoding gene StMR1 was found to be highly expressed during the infection process of Setosphaeria turcica, the pathogen causing northern corn leaf blight. Evolutionary tree analysis showed that this gene was associated with regulatory factors of melanin synthesis. However, the regulatory mechanism of melanin synthesis and its effect on pathogenicity remain unclear. In this study, the function of StMR1 was analyzed by gene knockout. When the expression level of StMR1 in the mutants was significantly reduced, the colony color became lighter, the mycelia were curved and transparent, and the mutant showed a significant loss of pathogenicity. In addition, compared with wild-type, the accumulation of melanin decreased significantly in ΔStmr1. RNA-seq analysis revealed 1,981 differentially expressed genes between the wild-type and knockout mutant, among which 39 genes were involved in melanin metabolism. qPCR revealed that the expression levels of six key genes in the melanin synthesis pathway were significantly reduced. ChIP-PCR and yeast one-hybrid assays confirmed that StMR1 directly binds to the promoters of St3HNR, St4HNR, StPKS, and StLAC2 in the DHN melanin synthesis pathway and regulates gene expression. The C2H2-type zinc fingers and Zn(Ⅱ)2Cys6 binuclear cluster in StMR1 were important for the binding to targets.

Mechanosensation triggers enhanced heavy metal ion uptake by non-glandular trichomes.

The trichomes of Arabidopsis thaliana serve as accumulation sites for heavy metals such as Cd2+, and thereby both help plants cope with heavy metal stress and detoxify the soil. These trichomes are also believed to prime plant defenses against insect herbivores in response to mechanical stimulation. Because Cd2+ in such trichomes may be beneficial for plant defenses, we hypothesized that mechanical stimulation would enhance sequestration of Cd2+ in trichomes. We quantified the distribution and concentration of Cd2+ in leaves of A. thaliana, of the glabrous mutant gl1-1 of A. thaliana, and Brassica rapa L. subsp. pekinensis (Lour.) Hanelt (Chinese cabbage) and examined how these changed following mechanical stimulation of the trichomes or leaves. Light brushing or exposure to caterpillars of Spodoptera exigua led trichomes of both A. thaliana and Chinese cabbage to accumulate Cd2+ complexes more rapidly and to a higher concentration than trichomes in unstimulated controls. Comparison to responses in leaves of gl1-1 mutants suggested that this acceleration and enhancement of Cd2+ storage requires signaling through trichomes. In wild type A. thaliana, Cd2+ was found exclusively in trichomes, whereas in gl1-1 mutants, Cd2+ was found mainly in the - mesophyll cells. Results suggest a mechanobiological pathway for improving heavy metal detoxification of soils through the action of hyperaccumulator plant leaves containing non-glandular trichomes.

Sugarcane mosaic virus orchestrates the lactate fermentation pathway to support its successful infection.

Viruses often establish their own infection by altering host metabolism. How viruses co-opt plant metabolism to support their successful infection remains an open question. Here, we used untargeted metabolomics to reveal that lactate accumulates immediately before and after robust sugarcane mosaic virus (SCMV) infection. Induction of lactate-involved anaerobic glycolysis is beneficial to SCMV infection. The enzyme activity and transcriptional levels of lactate dehydrogenase (LDH) were up-regulated by SCMV infection, and LDH is essential for robust SCMV infection. Moreover, LDH relocates in viral replicase complexes (VRCs) by interacting with SCMV-encoded 6K2 protein, a key protein responsible for inducing VRCs. Additionally, lactate could promote SCMV infection by suppressing plant defense responses. Taken together, we have revealed a viral strategy to manipulate host metabolism to support replication compartment but also depress the defense response during the process of infection.

A Genome Resource of Setosphaeria turcica, Causal Agent of Northern Leaf Blight of Maize.

The heterothallic ascomycete Setosphaeria turcica (anamorph Exserohilum turcicum) causes northern corn leaf blight, which results in devastating yield losses and a reduction in feed value. Although genome sequences of two model strains of the pathogen are available (https://mycocosm.jgi.doe.gov/mycocosm/home), previous drafts were assembled using short read technologies, making evolutionary and genetic linkage inferences difficult. Here, race 23N of S. turcica strain Et28A was sequenced again using Illumina HiSeq and PacBio Sequel technologies, and assembled to approximately 43,480,261 bp on 30 scaffolds. In all, 13,183 protein-coding genes were predicted, 13,142 of them were well annotated. This S. turcica genome resource is important for understanding the genetics behind pathogen evolution and infection mechanisms.

Setosphaeria turcica ATR turns off appressorium-mediated maize infection and triggers melanin-involved self-protection in response to genotoxic stress.

Eukaryotic organisms activate conserved signalling networks to maintain genomic stability in response to DNA genotoxic stresses. However, the coordination of this response pathway in fungal pathogens remains largely unknown. In the present study, we investigated the mechanism by which the northern corn leaf blight pathogen Setosphaeria turcica controls maize infection and activates self-protection pathways in response to DNA genotoxic insults. Appressorium-mediated maize infection by S. turcica was blocked by the S-phase checkpoint. This repression was dependent on the checkpoint central kinase Ataxia Telangiectasia and Rad3 related (ATR), as inhibition of ATR activity or knockdown of the ATR gene recovered appressorium formation in the presence of genotoxic reagents. ATR promoted melanin biosynthesis in S. turcica as a defence response to stress. The melanin biosynthesis genes StPKS and StLac2 were induced by the ATR-mediated S-phase checkpoint. The responses to DNA genotoxic stress were conserved in a wide range of phytopathogenic fungi, including Cochliobolus heterostrophus, Cochliobolus carbonum, Alternaria solani, and Alternaria kikuchiana, which are known causal agents for plant diseases. We propose that in response to genotoxic stress, phytopathogenic fungi including S. turcica activate an ATR-dependent pathway to suppress appressorium-mediated infection and induce melanin-related self-protection in addition to conserved responses in eukaryotes.

Heterologous expression of Stlac2, a laccase isozyme of Setosphearia turcica, and the ability of decolorization of malachite green.

The active laccases of ascomycetous fungus Setosphaeria turcica were identified by Native-PAGE and ESI-MS/MS, and one of these isozymes Stlac2 was heterologous expressed to investigate the decolorization of malachite green. Setosphaeria turcica produced three active laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 °C. The activity of recombinant Stlac2 was inhibited by 10 mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10 mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. CONCLUSIONS: Generally, recombinant protein of fungal laccase Stlac2 was active without glycosylation and decolorize malachite green efficiently, which has potential industrial applications.

The Relationship Analysis on Corn Stalk Rot and Ear Rot According to Fusarium Species and Fumonisin Contamination in Kernels.

Fusarium diseases, including corn root rot, sheath rot, stalk rot, and ear rot are frequently occurring in maize producing areas of China. Fusarium stalk rot and ear rot are the most serious diseases and often occur at the same time, but it is unclear whether there is a correlation between Fusarium composition and disease occurrence. This study was conducted to clarify the relationship between the two diseases. A total of 49 corn stalk rot samples were collected from 15 regions of eight provinces in China from 2016 to 2018. The pathogens were isolated and identified separately from stalks, ear stems, and kernels. The contents of the fumonisins (FB1 and FB2) were detected in kernels. The results showed that the main Fusarium species were found in corn kernels, ear stems and stalks at the same time. The results showed that 1201 strains of Fusarium verticillioides, 668 strains of Fusarium oxysporum, 574 strains of Fusarium graminearum species complex (FGSC), 318 strains of Fusarium equiseti, 95 strains of Fusarium proliferatum, and 40 strains of Fusarium subglutinans were isolated from 1470 corn kernels, 245 ear stems, and 1225 stalks randomly selected from 49 samples. The contamination rate of fumonisins in the 49 samples was 57.1% with an average content of 1.9 µg/g, of which four samples exhibited higher levels as set by the European Commission (4.0 µg/g). These results provide a certain association between stalk rot and ear rot and lay a foundation to study the relationships among Fusarium maize diseases.

Expression, purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli.

Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET-30a expression plasmid. The recombinant laccase was purified and visualized on SDS-PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg-1 , the optimum temperature and pH value were 60 °C and 4.0 respectively, measured by oxidation of 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). Purified laccase activity under different metal ions and an inhibitor were tested, revealing that laccase activity increased by approximately 434.8% with Fe3+ , and 217.4% with Cu2+ at 10 mmol · L-1 concentrations, Mn2+ increased the laccase activity only at 5 mmol · L-1 , while Na+ increased activity at 1 mmol · L-1 but inhibited activity at 5 and 10 mmol · L-1 . SDS increased laccase activity at 1 mmol · L-1 , and inhibited activity at 5 and 10 mmol · L-1 .

The StLAC2 gene is required for cell wall integrity, DHN-melanin synthesis and the pathogenicity of Setosphaeria turcica.

Laccases are blue multicopper oxidases, play important roles in various biological processes. These processes include fungal dihydroxynaphthalene (DHN)-melanin biosynthesis and pathogenicity, cellular growth, morphogenesis, and differentiation. This study investigated functions of the laccase gene StLAC2 in Setosphaeria turcica. The Δlac2 mutant colony color was distinct from that of the S. turcica wild-type (WT) isolate, and the mutants exhibited defective conidial formation. In contrast to the WT, the mutants exhibited a lighter color on the 2, 2-azino-di-[3-ethylbenzo-thia-zolin-sulphonate] (ABTS) plates, and the intracellular laccase activity was lower. Notably, StLAC2 gene loss correlated with decreased DHN-melanin biosynthesis and affected the integrity of the cell wall, where the StLAC2 gene mutants showed thinner, more transparent walls with a higher number of mitochondria than the WT. The Δlac2 mutants also lost their pathogenicity in maize. The results indicated that the StLAC2 gene involved in cell wall integrity, melanin biosynthesis and appressorial and conidial formation.

StPBS2, a MAPK kinase gene, is involved in determining hyphal morphology, cell wall development, hypertonic stress reaction as well as the production of secondary metabolites in Northern Corn Leaf Blight pathogen Setosphaeria turcica.

Mitogen activated protein kinase kinase (MAPKK) is a crucial component in the MAPK signaling pathway. However, the functions of MAPKKs in foliar pathogens remain poorly understood. In the current study, a MAPKK gene designated as StPBS2 was cloned from Setosphaeria turcica and the functions of this gene were investigated by RNAi technology. Four independent StPBS2 gene silence transformants with different efficiencies were confirmed by real time PCR. Compared to the wild type strain (WT), these transformants showed decreased colony growth, shortened hyphae cell length, broadened cell width and an obvious reduction in conidium yield. Moreover, the cell wall of the transformants was thicker and they were also more sensitive to substances that interfere with cell wall biosynthesis than WT. Additionally, the transformants displayed higher sensitivity to hypertonic stress than WT and the sensitivity was associated with the level of silencing of StPBS2. They were also resistant to the fungicides iprodione, procymidone and fludioxonil, to which WT almost completely sensitive. The transformants produced more red secondary metabolites than WT and the production was enhanced with increasing silencing level and increased glucose content in PDA medium. Our results suggest that StPBS2 is involved in morphogenesis, condiogenesis, cell wall development, hypertonic stress reaction and resistance to fungicides, as well as in the biosynthesis of secondary metabolites in S. turcica.