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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 ZmMYC7 mutants showed markedly increased sensitivity to Fusarium graminearum and Setosphaeria turcica. The expression levels of the defense-associated genes ZmPR1, ZmPR2, ZmPR3, ZmPR5, ZmPR6, and ZmPR7 in response to F. graminearum infection were downregulated in ZmMYC7 mutants, while ZmPR4 and ZmPR10 were 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 ZmERF147 promoter in vitro and transcriptional activation of ZmERF147 by ZmMYC7 was inhibited by ZmJAZ11 and ZmJAZ12. ZmERF147 mutants were more susceptible to F. graminearum infection than inbred line B73 with concomitant down-regulation of all defense-associated ZmPRs except ZmPR4. These findings indicate that ZmMYC7 functions in maize resistance to F. graminearum and sheds light on maize defense responses to pathogenic fungi via the JA signaling pathway.

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

Maize stalk rot caused by Fusarium graminearum can reduce the yield of maize and efficiency of mechanized harvesting. Besides, deoxynivalenol and zearalenone toxins produced by F. graminearum can also affect domestic animals and human health. As chemical fungicides are expensive and exert negative effects on the environment, the use of biological control agents has become attractive in recent years. In the present study, we collected rhizosphere soil with severe stalk rot disease (ZDD), the rhizosphere soil with disease-free near by the ZDD (ZDH), and measured rhizosphere microbial diversity and microbial taxonomic composition by amplicon sequencing targeting either bacteria or fungi. The results showed that Fusarium stalk rot caused by the Fusarium species among which F. graminearum is frequent and can reduce the abundance and alpha diversity of rhizosphere microbial community, and shift the beta diversity of microorganisms. Furthermore, a bacterial strain, Bacillus siamensis GL-02, isolated from ZDD, was found to significantly affect growth of F. graminearum. In vitro and in vivo assays demonstrated that B. siamensis GL-02 had good capability to inhibit F. graminearum. These results revealed that B. siamensis GL-02 could be a potential biocontrol agent for the control of maize stalk rot.

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

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

Abstract Background Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. Methods The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. Results We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. Conclusions AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist’s toolbox.

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

A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea. A novel pathogenicity-related gene BcKMO, which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO-complementing mutant (BCG183/BcKMO) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/BcKMO. Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H2O2, and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1, Pka2, PkaR, Bcg2, Bcg3, bmp1, and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea. Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea.