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Fusarium verticillioidesis the primary pathogen causing ear rot and stalk rot in corn (Zea mays). It not only affects yields but also produces mycotoxins endangering both human and animal health. Aldehyde dehydrogenase (ALDH) is essential for the oxidation of aldehydes in living organisms, making it a potential target for human drug design. However, there are limited reports on its function in plant pathogenic fungus. In this study, we analyzed the expression levels and gene knockout mutants, revealing that ALDH genes FvALDH-43and FvALDH-96in F. verticillioidesplayed significant roles in pathogenicity and resistance to low-temperature stress by affecting antioxidant capacity. Virtual screening for natural product inhibitors and molecular docking were performed targeting FvALDH-43 and FvALDH-96. Following the biological activity analysis, three natural flavonoid compounds featuring a 2-hydroxyphenol chromene were identified. Among these, Taxifolin exhibited the highest biological activity and low toxicity. Both in vitroand in vivobiological evaluations confirmed that Taxifolin targeted ALDH and inhibited its activity. These findings indicate that aldehyde dehydrogenase may serve as a promising target for the design of novel fungicides.

Northern corn leaf blight caused by Setosphaeria turcicais one of the most important fungal diseases of maize. We previously cloned and analyzed the gene encoding protein kinase C (PKC) in S. turcica, as this Ca2+signaling component is required for growth and pathogenicity of several other plant pathogenic fungi. Here, we assessed the effect of PKCknockout on S. turcicapathogenicity using four quinic-acid-inducible PKCantisense mutants. The conditional mutants enabled us to study the role of PKCin growth, germination, appressorium development, and pathogenicity despite the fact that constitutive PKCknockout is typically lethal in filamentous fungi. The growth rates of the mutants were the same as that of the wild type (WT) on PDA medium but were much lower on PDA medium that contained the inducer 15 mM quinic acid. Conidial germination and appressorium development were almost completely abolished in the induced mutants, and penetration experiments showed that appressoria from the WT could penetrate cellophane, whereas those of the induced mutants could not. Inoculation experiments showed that the mutant strains failed to produce visible lesions on unwounded maize leaves, although they were able to infect wounded leaves, suggesting that they were impaired in the process of host penetration. Characterization of PKCantisense mutants thus highlighted multiple roles of PKC in S. turcicapathogenicity, laying a foundation for understanding and exploiting a key signal transduction pathway in plant pathogenic fungi.

The jasmonic acid (JA) signaling pathway is involved in plant growth, development, and response to abiotic or biotic stresses. MYC2, a bHLH transcription factor, is a regulatory hub in the pathway. The function of ZmMYC7, a putative MYC2 ortholog, in jasmonate-signaled defense responses of maize has not been reported. In this study, we found that ZmMYC7 possesses JID, TAD, bHLH and Zip domains and essential characteristics of transcription factors: a nuclear location and transactivation activity. The ZmMYC7mutants showed markedly increased sensitivity to Fusarium graminearumand Setosphaeria turcica. The expression levels of the defense-associated genes ZmPR1, ZmPR2, ZmPR3, ZmPR5, ZmPR6, and ZmPR7in response to F. graminearuminfection were downregulated in ZmMYC7mutants, while ZmPR4and ZmPR10were up-regulated. ZmMYC7 interacted with members of the ZmJAZ family, including ZmJAZ8, ZmJAZ11, and ZmJAZ12. ZmMYC7 physically interacted with G-box cis-elements in the ZmERF147promoter in vitroand transcriptional activation of ZmERF147by ZmMYC7 was inhibited by ZmJAZ11 and ZmJAZ12. ZmERF147mutants were more susceptible to F. graminearuminfection than inbred line B73 with concomitant down-regulation of all defense-associated ZmPRsexcept ZmPR4. These findings indicate that ZmMYC7 functions in maize resistance to F. graminearumand sheds light on maize defense responses to pathogenic fungi via the JA signaling pathway.

Exploring new herbicide targets based on natural product derivatives is an important research aspect for the generation of innovative pesticides. Ferulic acid ethyl ester (FAEE), a natural product derivative from ferulic acid, has significant herbicidal activity mainly by inhibiting the normal growth of weed seedling roots. However, the FAEE target protein underlying its herbicidal activity has not been identified. In this study, we synthesized an FAEE probe to locate its site of action. We discovered that FAEE entry point was via the root tips. Fourteen major binding proteins were identified using Drug affinity responsive target stability (DARTS) combined with LC-MS/MS, which included 3-ketoacyl-acyl carrier protein synthase I (KAS I) and phenylalanine ammonia-lyase I (PAL I). The KAS I and PAL I proteins/genes expression was changed significantly after exposure to FAEE, as evidenced by combined transcriptomic and proteomic analysis. A molecular docking assay indicated that KAS I and FAEE had a strong binding ability. Combined with previous studies on FAEE mechanism of action, and based on our results, we conclude that FAEE targeting KAS I lead to the blockage of the fatty acid synthesis pathway and result in plant death.

Plant development is highly dependent on energy levels. TARGET OF RAPAMYCIN (TOR) activates the proximal root meristem to promote root development in response to photosynthesis-derived sugars during photomorphogenesis in Arabidopsis thaliana. However, the mechanisms of how root tip homeostasis is maintained to ensure proper root cap structure and gravitropism are unknown. PLETHORA (PLT) transcription factors are pivotal for the root apical meristem (RAM) identity by forming gradients, but how PLT gradients are established and maintained, and their roles in COL development are not well known. We demonstrate that endogenous sucrose induces TOPOISOMERASE1α(TOP1α) expression during the skotomorphogenesis-to-photomorphogenesis transition. TOP1α fine-tunes TORexpression in the root tip columella. TOR maintains columella stem cell identity correlating with reduced quiescent centre cell division in a WUSCHEL RELATED HOMEOBOX5-independent manner. Meanwhile, TOR promotes PLT2expression and phosphorylates and stabilizes PLT2 to maintain its gradient consistent with TORexpression pattern. PLT2 controls cell division and amyloplast formation to regulate columella development and gravitropism. This elaborate mechanism helps maintain root tip homeostasis and gravitropism in response to energy changes during root development.

AbstractAs a valuable and powerful platform technology, yeast surface display (YSD) has been widely used in various biotechnological fields, but little in agricultural applications. Xylanase can be used as an elicitor in the plant–pathogen interaction system and induce corn defence response. In this study, the YSD technology was used to express the xylanase protein family from Setosphaeria turcica, which is a novel strategy for plant protection. First, five genes encoding xylanase were selected from the S. turcicagenome database. The conserved domains of these genes were analyzed, which revealed that these genes were highly conserved. Here, we selected two of the five genes and expressed them by a prokaryotic expression system, but the two xylanase genes were expressed in inclusion bodies and showed no activity. To make them available in the application, we successfully constructed them in a YSD system. The products were detected for enzyme activities and the activities of two xylanases were 16.72 and 19.12 U/mL, respectively. We finally proved that the yeast-displaying xylanase was an efficient strategy to induce corn defence response, as four defence genes in corn, PR-1, PR-4, SODand CATwere induced after treatment with the yeast displaying xylanases.

LysM proteins contain the lysin domain (LysM), bind chitin and are found in various organisms including fungi. In phytopathogenic fungi, certain LysM proteins act as effectors to inhibit host immunity, thus increasing fungal virulence. However, our understanding of the LysM protein family in Setosphaeria turcicais limited. In this study, eight StLysMgenes are identified and designated as StLysM1to StLysM8. The analysis of sequence features indicates that five proteins (StLysM1, StLysM2, StLysM5, StLysM6, and StLysM7) are potential effectors. Phylogenetic analysis suggests that the StLysMs are divided into fungal/bacterial and fungus-specific subclasses. Domain architecture analysis reveals that the five StLysM effectors exclusively harbor the LysM domain, whereas the other three StLysM proteins contain additional functional domains. Sequence conservation analysis shows that the fungal-specific LysM domain sequences share the 8GDxTC12and 29WNP31motifs as well as three highly conserved cysteine residues. Conversely, the LysM domain sequences from the bacterial/fungal branch have few conserved sites. Moreover, expression profiling analysis shows that the StLysM1gene is significantly upregulated during the infection of maize. Yeast secretion assays and transient expression experiments demonstrate that StLysM1 is a secreted protein that can suppress BAX/INF1-induced programmed cell death in Nicotiana benthamiana. Further functional analysis suggests that StLysM1 cannot interact with itself but it can bind chitin. The transient expression of StLysM1inhibits the chitin-triggered plant immune response, increasing susceptibility to the phytopathogenic fungus Botrytis cinereain N. benthamiana. This study reveals that the S. turcicaLySM protein family consists of eight members, highlighting the significance of StLysM1 as a vital effector in regulating plant immunity. The results provide insight into StLysMs and establish a foundation for understanding the roles of StLysM proteins in the pathogenic process of S. turcica.

Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicuscommonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB1) in agricultural settings. However, the specific target of VMA in Fusarium verticillioidesremained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp285, Arg447, Asp452, and Phe665as the binding sites between VMA and FvNth. A ΔFvnthmutant lacking amino acids 250–670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnthdisplayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth. This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB1biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

Fusarium verticillioideshas 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 FvOSHCplays a positive role, establishing it as a novel global regulatory protein involved in the pathogenicity and FB1biosynthesis 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. verticillioidesand the synthesis of FB1was significantly demonstrated. These findings provide valuable insights that can aid in the management of mycotoxin pollution.

Insect chitinase, OfChi-h, from Ostrinia furnacalis, is considered as a promising target for the development of green pesticides. On the basis of the crystal structure of OfChi-h, we successfully obtained a triazolo-quinazolinone scaffold as the novel class of OfChi-h inhibitor via a structure-based virtual screening approach. Rational compound screening enabled us to acquire a potent OfChi-h inhibitor TQ19with a Kivalue of 0.33 μM. Furthermore, the in vivobiological activity of target compounds was assayed. The results showed that compounds TQ8and TQ19could dramatically inhibit the growth and development of Ostrinia nubilalislarvae, and most of the compounds showed higher insecticidal activity than hexaflumuron. This present work reveals that triazolo-quinazolinone derivatives can serve as novel candidates for insect growth regulators.

ABSTRACTLateral roots (LRs) are an important part of plant root systems. In dicots, for example, after plants adapted from aquatic to terrestrial environments, filamentous pseudorhizae evolved to allow nutrient absorption. A typical plant root system comprises a primary root, LRs, root hairs, and a root cap. Classical plant roots exhibit geotropism (the tendency to grow downward into the ground) and can synthesize plant hormones and other essential substances. Root vascular bundles and complex spatial structures enable plants to absorb water and nutrients to meet their nutrient quotas and grow. The primary root carries out most functions during early growth stages but is later overtaken by LRs, underscoring the importance of LR development water and mineral uptake and the soil fixation capacity of the root. LR development is modulated by endogenous plant hormones and external environmental factors, and its underlying mechanisms have been dissected in great detail in Arabidopsis, thanks to its simple root anatomy and the ease of obtaining mutants. This review comprehensively and systematically summarizes past research (largely in Arabidopsis) on LR basic structure, development stages, and molecular mechanisms regulated by different factors, as well as future prospects in LR research, to provide broad background knowledge for root researchers.

ABSTRACTHeterotrimeric G proteins are best known for their role in the transduction of extracellular signals to various downstream effectors. G proteins in higher eukaryotes are intensively studied; however, their roles in foliar pathogens are still elusive. In this study, we cloned the gene Stgg1encoding G protein γ subunit in Setosphaeria turcicaand investigated its function by RNA interference technology. Three independent Stgg1targeted RNAi mutants R3, R5 and R6 with diverse silencing efficiency were generated. Knock-down of Stgg1resulted in a significant reduction in mRNA levels of the genes encoding Gα (Stga1, Stga2, Stga3) but not for Gβ (Stgb1). Stgg1RNAi mutants exhibited significantly elongated hyphal cells with blocked conidium production. In addition, Stgg1RNAi mutants all appeared in lighter colony colour compatible with inhibited secondary metabolites. Further assays demonstrated that Stgg1was required for biosynthesis of melanin and HT-toxin activity. Furthermore, down-regulation of Stgg1largely inhibited the inflection capacity. Thus, we proposed that Stgg1played crucial roles in conidiation, secondary metabolite production and pathogenicity of S. turcicaand is, therefore, an ideal target for drug design against foliar pathogens.

Septins are a conserved group of GTP-binding proteins found in all eukaryotes and are the fourth-most abundant cytoskeletal proteins. Septins of some pathogenic fungi are involved in morphological changes related to infection. Our previous studies have identified four core septins (StSep1-4) in Setosphaeria turcica, the causal agent of northern corn leaf blight, while only StSep4is significantly upregulated during the invasive process. We therefore used forchlorfenuron (FCF), the specific inhibitor of septin, and ΔStSep4knockout mutants to further clarify the role of septins in S. turcicapathogenicity. FCF treatment caused a dose-dependent reduction in S. turcicacolony growth, delayed the formation of infection structures, and reduced the penetration ability. ΔStSep4knockout mutants displayed abnormal mycelium morphology, slow mycelial growth, conidiation deficiency, delayed appressorium development, and weakened pathogenicity. StSep4deletion also broke cell wall integrity, altered chitin distribution, decreased the melanin content, and disrupted normal nuclear localization. A transcriptomic comparison revealed that genes differentially expressed between ΔStSep4and WT were enriched in terms of ribosomes, protein translation, membrane components, and transmembrane transport activities. Our results demonstrate that StSep4is required for morphology and pathogenicity in S. turcica, making it a promising target for the development of novel fungicides.

Here, we investigated the uptake, transport, and subcellular distribution of the pesticides pymetrozine and spirotetramat, and spirotetramat metabolites B-enol, B-glu, B-mono, and B-keto, under hydroponic conditions. Spirotetramat and pymetrozine exhibited high bioconcentrations in lettuce roots, with both having root concentration factor (RCF) values >1 after exposure for 24 h. The translocation of pymetrozine from roots to shoots was higher than that of spirotetramat. Pymetrozine is absorbed in roots mainly via the symplastic pathway and is primarily stored in the soluble fraction of lettuce root and shoot cells. The cell wall and soluble fractions were the major enrichment sites of spirotetramat and its metabolites in root cells. Spirotetramat and B-enol were mainly enriched in the soluble fractions of lettuce shoot cells, whereas B-keto and B-glu accumulated in cell walls and organelles, respectively. Both symplastic and apoplastic pathways were involved in spirotetramat absorption. Pymetrozine and spirotetramat uptake by lettuce roots was passive, with no aquaporin-mediated dissimilation or diffusion. The findings of this study enhance our understanding of the transfer of pymetrozine, spirotetramat, and spirotetramat metabolites from the environment to lettuce, and their subsequent bioaccumulation. This study describes a novel approach for the efficient management of lettuce pest control using spirotetramat and pymetrozine. At the same time, it is of great significance to evaluate the food safety and environmental risks of spirotetramat and its metabolites.

ABSTRACTAnalysis of PCST1expression characteristics and the role of PCST1in response to osmotic stress in Arabidopsis thaliana. The structure of PCST1was analyzed using Bioinformatics method. Real-time PCR, GUS tissue localization and subcellular localization were adopted to analyze the expression pattern of PCST1in Arabidopsis. To validate the transgenic positive strain of PCST1using Real-time PCR, overexpression experiments were performed in wild type. Full-length cDNA was cloned and connected into a binary vector with 35S promoter, and the construction was transformed into wild type. With NaCl and mannitol treatments, the germination rate, green leaves rate, physiological indexes were carried out and counted in Arabidopsis with overexpression of PCST1and T-DNA insertion mutants. The molecular mechanism of PCST1in response to osmotic stress in Arabidopsis was analyzed. Based on the bioinformatic analysis, PCST1 is a hydrophobin with 403 amino acids, and the molecular weight is 45.3236 KDa. It contains only the START (the lipid/sterol – binding StAR – related lipid transfer protein domains) conservative domain. PCST1 possesses phosphatidylcholine binding sites and transmembrane region. Expression pattern analysis showed that expression of PCST1increased with time. The PCST1widely expressed in Arabidopsis, including roots, axils of stem leaves, flowers (sepal, conductive tissue of the petal, thrum, anther and stigmas), and the top and basal parts of the siliquas. It mainly localized in cell membrane. The overexpression of PCST1enhanced the sensitivity to osmotic stress in Arabidopsisbased on the germination rate. While expression of PCST1decreased, and the sensitivity to osmotic stress had no obvious change in Arabidopsis. Its molecular mechanism study showed, that PCST1 response to osmotic stress resistance by regulating the proline, betaine synthesis, as well as the expression of key genes SOS, NCED, CIPK. PCST1 is composed of 403 amino acids. The START conservative domain, a transmembrane structure, the phosphatidyl choline binding sites are contained in PCST1. It is localized in cytoplasmic membrane. The PCST1widely expressed in the root, leaf, flower and siliquas. NaCl and mannitol suppressed the expression of PCST1and PCST1 can negatively control action of Arabidopsisin the osmotic stress. PCST1 regulates the synthetic pathway of proline, betaine and the expression of SOS, NCEDand CIPKin response to the osmotic stress resistance.

ABSTRACTRoot gravitropism is important for anchorage and exploration of soil for water and nutrients. It affects root architecture, which is one of the elements that influence crop yield. The mechanism of primary root gravitropism has been widely studied, but it is still not clear how lateral root gravitropism is regulated. Here, in this study, we found that Topoisomerase I α (TOP1α) repressed lateral root gravitropic growth, which was opposite to the previous report that TOP1α maintains primary root gravitropism, revealing a dual function of TOP1α in root gravitropism regulation. Further investigation showed that Target of Rapamycin (TOR) was suppressed in columella cells of lateral root to inhibit columella cell development, especially amyloplast biosynthesis. Our findings uncovered a new mechanism about lateral root gravitropism regulation, which might provide a theoretical support for improving agricultural production.

ABSTRACTBT4gene was identified to play an important role in Arabidopsisresistance to pstDC3000 in preliminary studies. However, the specific function and molecular mechanism of BT4gene in regulation of Arabidopsisresistance to Botrytis cinereahad not been described to date. In this study, we found that the expression of BT4was induced by wounding and B. cinereainoculation in Arabidopsis. After inoculated with B. cinerea, T-DNA insertion mutants of the BT4gene, bt4, showed significant susceptibility symptoms, whereas no significant symptoms were found in wild-type (WT), the complemented transgenic plants (CE), and the overexpression transgenic plants (OE). After inoculated with B. cinerea, the expression levels of JAR1and PDF1.2genes in bt4mutant were induced; however, the expression levels of these genes in bt4mutant were significantly lower than those in the WT, CE, and OE. These results indicated that the BT4positively regulate the expression of genes in JA/ET signaling pathways. Therefore, the BT4may be involved in the regulation of JA/ET signaling pathways to affect Arabidopsisresistance to B. cinerea.

ABSTRACTBrassinosteroids (BRs) regulate of maize (Zea maysL.) growth, but the underlying molecular mechanism remains unclear. In this study, we used a multi-disciplinary approach to determine how BRs regulate maize morphology and physiology during development. Treatment with the BRs promoted primary root the elongation and growth during germination, and the early development of lateral roots. BRs treatment during the middle growth stage increased the levels of various stress resistance factors, and enhanced resistance to lodging, likely by protecting the plant against stem rot and sheath rot. BRs had no significant effect on plant height during late growth, but it increased leaf angle and photosynthetic efficiency, as well as yield and quality traits. Our findings increase our understanding of the regulatory effects of BR on maize root growth and development and the mechanism by which BR improves disease resistance, which could further the potential for using BR to improve maize yield.

In order to clarify the roles of different protein kinase A (PKA) isoforms during the pathogenesis, a gene coding the catalytic subunit of PKA (PKA-c) of S. turcicawas predicted from the genome database. In addition, the transcriptional profile during the formation of the invasive structure was studied through qPCR. Results showed that the StpkaC2gene harbored an ORF of 1209 bp that encoded the PKA-c with its 402 amino acid residues. Phylogenetic analysis revealed that StpkaC2and StpkaC1belonged to different clusters. Gene expression patterns of StpkaC2during germ tube formation was 2.2 times higher than that at the conidial phase (control), but decreased slightly during appressoria formation and mature stages of infection. During the intrusive filament formation stage, gene expression increased to levels that were 4.2 times higher than the control. Instead, the mRNA levels of StpkaC1were similar at 0, 3, 6 and 12 hpi. However, at 24 hpi, gene expression increased to levels that were 3.2 times higher than that control. Results suggest that StpkaC2is more active than StpkaC1during germ tube and appressoria formation, and mature stages. Conversely, both StpkaC2and StpkaC1are important during the invasive hyphae formation stage.

The uptake, translocation, and subcellular distribution of oxathiapiprolin and famoxadone in tomato plants were investigated using hydroponic experiments. Oxathiapiprolin and famoxadone mainly accumulated in the tomato roots with limited translocation capacity from the roots to the upper part. The root absorption and inhibitor results noted the dominance of the apoplastic and symplastic pathways in the oxathiapiprolin and famoxadone uptake by the tomato roots, respectively. Furthermore, the uptake process for the two fungicides followed passive and aquaporin-dependent transport. Insoluble cell components (cell organelles and walls) were the dominant storage compartments for oxathiapiprolin and famoxadone. In the protoplast, oxathiapiprolin in the soluble fraction had a higher proportion than that of famoxadone. Finally, the uptake and distribution of the two fungicides by the tomato plants was accurately predicted using a partition-limited model. Thus, this study provides an in-depth understanding of the transfer of oxathiapiprolin and famoxadone from the environment to tomato plants.

Chitinase OfChi-h, from the destructive agricultural pest Ostrinia furnacalis, is considered as a promising target for green pest control and management. In this study, structure-based virtual screening and rational molecular optimization led to the synthesis of a series of azo-aminopyrimidine derivatives as a novel class of OfChi-h inhibitors. Among them, the most potent compound 8f, with a benzyl on the amino group at the 4-position of pyrimidine, exhibited a Kivalue of 64.7 nM against OfChi-h. In addition, molecular docking studies were carried out to investigate the basis for the potency of the aminopyrimidines against OfChi-h. Furthermore, the insecticidal activity of the target compounds against Plutella xylostellaand Ostrinia nubilaliswas assessed, and the potent OfChi-h inhibitors 8fand 8ishowed higher insecticidal activity than the control pesticide hexaflumuron. The present work revealed that the azo-aminopyrimidine skeletons characterized by concise chemical structure and high efficiency could be further developed as potential pesticides for the control of lepidopteran pests.