NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize.

Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.

Transcriptomic and Metabolomic Analyses Reveal the Role of Phenylalanine Metabolism in the Maize Response to Stalk Rot Caused by Fusarium proliferatum.

Stalk rot is a prevalent disease of maize (Zea mays L.) that severely affects maize yield and quality worldwide. The ascomycete fungus Fusarium spp. is the most common pathogen of maize stalk rot. At present, the molecular mechanism of Fusarium proliferation during the maize stalk infection that causes maize stalk rot has rarely been reported. In this study, we investigated the response of maize to F. proliferatum infestation by analyzing the phenotypic, transcriptomic, and metabolomic data of inbred lines ZC17 (resistant) and CH72 (susceptible) with different levels of resistance to stalk rot. Physiological and phenotypic results showed that the infection CH72 was significantly more severe than ZC17 after inoculation. Transcriptome analysis showed that after inoculation, the number of differentially expressed genes (DEGs) was higher in CH72 than in ZC17. Nearly half of these DEGs showed the same expression trend in the two inbred lines. Functional annotation and enrichment analyses indicated that the major pathways enriched for DEGs and DEMs included the biosynthesis of plant secondary metabolites, phenylalanine metabolism, biosynthesis of plant hormones, and plant-pathogen interactions. The comprehensive analysis of transcriptome and metabolome data indicated that phenylalanine metabolism and the phenylalanine, tyrosine, and tryptophan biosynthesis pathways played a crucial role in maize resistance to F. proliferatum infection. In addition, a transcription factor (TF) analysis of the DEGs showed that several TF families, including MYB, bHLH, NAC, and WRKY, were significantly activated after inoculation, suggesting that these TFs play important roles in the molecular regulatory network of maize disease resistance. The findings of this study provide valuable insights into the molecular basis of the response of maize to Fusarium proliferatum infection and highlight the importance of combining multiple approaches, such as phenotyping, transcriptomics, and metabolomics, to gain a comprehensive understanding of plant-pathogen interactions.

Genome-wide association study of maize resistance to Pythium aristosporum stalk rot.

Stalk rot, a severe and widespread soil-borne disease in maize, globally reduces yield and quality. Recent documentation reveals that Pythium aristosporum has emerged as one of the dominant causal agents of maize stalk rot. However, a previous study of maize stalk rot disease resistance mechanisms and breeding had mainly focused on other pathogens, neglecting P. aristosporum. To mitigate crop loss, resistance breeding is the most economical and effective strategy against this disease. This study involved characterizing resistance in 295 inbred lines using the drilling inoculation method and genotyping them via sequencing. By combining with population structure, disease resistance phenotype, and genome-wide association study (GWAS), we identified 39 significant single-nucleotide polymorphisms (SNPs) associated with P. aristosporum stalk rot resistance by utilizing six statistical methods. Bioinformatics analysis of these SNPs revealed 69 potential resistance genes, among which Zm00001d051313 was finally evaluated for its roles in host defense response to P. aristosporum infection. Through virus-induced gene silencing (VIGS) verification and physiological index determination, we found that transient silencing of Zm00001d051313 promoted P. aristosporum infection, indicating a positive regulatory role of this gene in maize's antifungal defense mechanism. Therefore, these findings will help advance our current understanding of the underlying mechanisms of maize defense to Pythium stalk rot.

Single-cell RNA sequencing profiles reveal cell type-specific transcriptional regulation networks conditioning fungal invasion in maize roots.

Stalk rot caused by Fusarium verticillioides (Fv) is one of the most destructive diseases in maize production. The defence response of root system to Fv invasion is important for plant growth and development. Dissection of root cell type-specific response to Fv infection and its underlying transcription regulatory networks will aid in understanding the defence mechanism of maize roots to Fv invasion. Here, we reported the transcriptomes of 29 217 single cells derived from root tips of two maize inbred lines inoculated with Fv and mock condition, and identified seven major cell types with 21 transcriptionally distinct cell clusters. Through the weighted gene co-expression network analysis, we identified 12 Fv-responsive regulatory modules from 4049 differentially expressed genes (DEGs) that were activated or repressed by Fv infection in these seven cell types. Using a machining-learning approach, we constructed six cell type-specific immune regulatory networks by integrating Fv-induced DEGs from the cell type-specific transcriptomes, 16 known maize disease-resistant genes, five experimentally validated genes (ZmWOX5b, ZmPIN1a, ZmPAL6, ZmCCoAOMT2, and ZmCOMT), and 42 QTL or QTN predicted genes that are associated with Fv resistance. Taken together, this study provides not only a global view of maize cell fate determination during root development but also insights into the immune regulatory networks in major cell types of maize root tips at single-cell resolution, thus laying the foundation for dissecting molecular mechanisms underlying disease resistance in maize.

Integrative transcriptome and proteome analysis reveals maize responses to Fusarium verticillioides infection inside the stalks.

Fusarium stalk rot caused by Fusarium verticillioides is one of the most devastating diseases of maize that causes significant yield losses and poses potential security concerns for foods worldwide. The underlying mechanisms of maize plants regulating defence against the disease remain poorly understood. Here, integrative proteomic and transcriptomic analyses were employed to identify pathogenesis-related protein genes by comparing differentially expressed proteins (DEPs) and differentially expressed genes (DEGs) in maize stalks after inoculation with F. verticillioides. Functional enrichment analysis showed that DEGs and DEPs were mainly enriched in glutathione metabolism, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis. Fourteen DEGs and DEGs that were highly elevated after inoculation with F. verticillioides were confirmed with parallel reaction monitoring and reverse transcription-quantitative PCR, demonstrating the accountability and reliability of proteomic and transcriptomic data. We also assessed the potential roles of defence-related genes ZmCTA1, ZmWIP1, and ZmLOX2, identified from the multi-omics analysis, during the process of F. verticillioides infection through virus-induced gene silencing. The elevation of stalk rot symptomatic characteristics in the silenced plants revealed their contribution to resistance. We further functionally characterized the roles of ZmLOX2 expression in the defence response of maize plants conditioning fungal invasion via the salicylic acid-dependent pathway. Collectively, this study provides a comprehensive analysis of transcriptome and proteome of maize stalks following F. verticillioides inoculation, and defence-related genes that could inform selection of new genes as targets in breeding strategies.

Development of the maize 5.5K loci panel for genomic prediction through genotyping by target sequencing.

Genotyping platforms are important for genetic research and molecular breeding. In this study, a low-density genotyping platform containing 5.5K SNP markers was successfully developed in maize using genotyping by target sequencing (GBTS) technology with capture-in-solution. Two maize populations (Pop1 and Pop2) were used to validate the GBTS panel for genetic and molecular breeding studies. Pop1 comprised 942 hybrids derived from 250 inbred lines and four testers, and Pop2 contained 540 hybrids which were generated from 123 new-developed inbred lines and eight testers. The genetic analyses showed that the average polymorphic information content and genetic diversity values ranged from 0.27 to 0.38 in both populations using all filtered genotyping data. The mean missing rate was 1.23% across populations. The Structure and UPGMA tree analyses revealed similar genetic divergences (76-89%) in both populations. Genomic prediction analyses showed that the prediction accuracy of reproducing kernel Hilbert space (RKHS) was slightly lower than that of genomic best linear unbiased prediction (GBLUP) and three Bayesian methods for general combining ability of grain yield per plant and three yield-related traits in both populations, whereas RKHS with additive effects showed superior advantages over the other four methods in Pop1. In Pop1, the GBLUP and three Bayesian methods with additive-dominance model improved the prediction accuracies by 4.89-134.52% for the four traits in comparison to the additive model. In Pop2, the inclusion of dominance did not improve the accuracy in most cases. In general, low accuracies (0.33-0.43) were achieved for general combing ability of the four traits in Pop1, whereas moderate-to-high accuracies (0.52-0.65) were observed in Pop2. For hybrid performance prediction, the accuracies were moderate to high (0.51-0.75) for the four traits in both populations using the additive-dominance model. This study suggests a reliable genotyping platform that can be implemented in genomic selection-assisted breeding to accelerate maize new cultivar development and improvement.

Comparative transcriptomic reveals the molecular mechanism of maize hybrid Zhengdan538 in response to water deficit.

Heterosis, known as one of the most successful strategies for increasing grain yield and abiotic/biotic stress tolerance, has been widely exploited in maize breeding. However, the underlying molecular processes are still to be elucidated. The maize hybrid "Zhengdan538" shows high tolerance to drought stress. The transcriptomes of the seedling leaves of its parents, "ZhengA88" and "ZhengT22" and their reciprocal F1 hybrid under well-watered and water deficit conditions, were analyzed by RNA sequencing (RNA-Seq). Transcriptome profiling of the reciprocal hybrid revealed 2994-4692 differentially expressed genes (DEGs) under well-watered and water-deficit conditions, which were identified by comparing with their parents. The reciprocal hybrid was more closely related to the parental line "ZhengT22" than to the parental line "ZhengA88" in terms of gene expression patterns under water-deficit condition. Furthermore, genes showed expression level dominance (ELD), especially the high-parental ELD (Class 3 and 5), accounted for the largest proportion of DEGs between the reciprocal F1 hybrid and their parental lines under water deficit. These ELD genes mainly participated in photosynthesis, energy biosynthesis, and metabolism processes. The results indicated that ELD genes played important roles in hybrid tolerance to water deficit. Moreover, a set of important drought-responsive transcription factors were found to be encoded by the identified ELD genes and are thought to function in improving drought tolerance in maize hybrid plants. Our results provide a better understanding of the molecular mechanism of drought tolerance in hybrid maize.

Transcriptomic Variations and Network Hubs Controlling Seed Size and Weight During Maize Seed Development.

To elucidate the mechanisms underlying seed development in maize, comprehensive RNA-seq analyses were conducted on Zhengdan1002 (ZD1002), Zhengdan958 (ZD958), and their parental lines during seven seed developmental stages. We found that gene expression levels were largely nonadditive in hybrids and that cis-only or trans × cis pattern played a large role in hybrid gene regulation during seed developmental stage. Weighted gene co-expression network (WGCNA) analysis showed that 36 modules were highly correlated (r = -0.90-0.92, p < 0.05) with kernel weight, length, and width during seed development. Forty-five transcription factors and 38 ribosomal protein genes were identified as major hub genes determining seed size/weight. We also described a network hub, Auxin Response Factor 12 of maize (ZmARF12), a member of a family of transcription factor that mediate gene expression in response to auxin, potentially links auxin signal pathways, cell division, and the size of the seeds. The ZmARF12 mutant exhibited larger seed size and higher grain weight. ZmARF12 transcription was negatively associated with cell division during seed development, which was confirmed by evaluating the yield of protoplasts that isolated from the kernels of the mutant and other inbred lines. Transient knock-down of ZmARF12 in maize plants facilitated cell expansion and division, whereas transient silencing of its potential interactor ZmIAA8 impaired cell division. ZmIAA8 expression was repressed in the ZmARF12 over-expressed protoplasts. The mutant phenotype and the genetics studies presented here illustrated evidence that ZmARF12 is a cell division repressor, and potentially determines the final seed size.

Genetic Dissection of Grain Yield of Maize and Yield-Related Traits Through Association Mapping and Genomic Prediction.

High yield is the primary objective of maize breeding. Genomic dissection of grain yield and yield-related traits contribute to understanding the yield formation and improving the yield of maize. In this study, two genome-wide association study (GWAS) methods and genomic prediction were made on an association panel of 309 inbred lines. GWAS analyses revealed 22 significant trait-marker associations for grain yield per plant (GYP) and yield-related traits. Genomic prediction analyses showed that reproducing kernel Hilbert space (RKHS) outperformed the other four models based on GWAS-derived markers for GYP, ear weight, kernel number per ear and row, ear length, and ear diameter, whereas genomic best linear unbiased prediction (GBLUP) showed a slight superiority over other modes in most subsets of the trait-associated marker (TAM) for thousand kernel weight and kernel row number. The prediction accuracy could be improved when significant single-nucleotide polymorphisms were fitted as the fixed effects. Integrating information on population structure into the fixed model did not improve the prediction performance. For GYP, the prediction accuracy of TAMs derived from fixed and random model Circulating Probability Unification (FarmCPU) was comparable to that of the compressed mixed linear model (CMLM). For yield-related traits, CMLM-derived markers provided better accuracies than FarmCPU-derived markers in most scenarios. Compared with all markers, TAMs could effectively improve the prediction accuracies for GYP and yield-related traits. For eight traits, moderate- and high-prediction accuracies were achieved using TAMs. Taken together, genomic prediction incorporating prior information detected by GWAS could be a promising strategy to improve the grain yield of maize.

Physiological and transcriptome analyses for assessing the effects of exogenous uniconazole on drought tolerance in hemp (Cannabis sativa L.).

Uniconazole (S-(+)-uniconazole), a plant growth retardant, exerts key roles in modulating growth and development and increasing abiotic stress tolerance in plants. However, the underlying mechanisms by which uniconazole regulates drought response remain largely unknown. Here, the effects of exogenous uniconazole on drought tolerance in hemp were studied via physiological and transcriptome analyses of the drought-sensitive industrial hemp cultivar Hanma No. 2 grown under drought stress. Exogenous uniconazole treatment increased hemp tolerance to drought-induced damage by enhancing chlorophyll content and photosynthesis capacity, regulating activities of enzymes involved in carbon and nitrogen metabolism, and altering endogenous hormone levels. Expression of genes associated with porphyrin and chlorophyll metabolism, photosynthesis-antenna proteins, photosynthesis, starch and sucrose metabolism, nitrogen metabolism, and plant hormone signal transduction were significantly regulated by uniconazole compared with that by control (distilled water) under drought stress. Numerous genes were differentially expressed to increase chlorophyll content, enhance photosynthesis, regulate carbon-nitrogen metabolism-related enzyme activities, and alter endogenous hormone levels. Thus, uniconazole regulated physiological and molecular characteristics of photosynthesis, carbon-nitrogen metabolism, and plant hormone signal transduction to enhance drought resistance in industrial hemp.

Maize ZmbZIP33 Is Involved in Drought Resistance and Recovery Ability Through an Abscisic Acid-Dependent Signaling Pathway.

Analyzing the transcriptome of maize leaves under drought stress and rewatering conditions revealed that transcription factors were involved in this process, among which ZmbZIP33 of the ABSCISIC ACID-INSENSITIVE 5-like protein 5 family was induced to significantly up-regulated. The functional mechanism of ZmbZIP33 in Abscisic acd (ABA) signaling pathway and its response to drought stress and rewatering has not been studied yet. The present study found that ZmbZIP33 contains a DNA-binding and dimerization domain, has transcriptional activation activity, and is highly homologous to SbABI1,SitbZIP68 and OsABA1. The expression of ZmbZIP33 is strongly up-regulated by drought, high salt, high temperature, and ABA treatments. Overexpression of ZmbZIP33 remarkably increased chlorophyll content and root length after drought stress and rewatering, and, moreover, cause an accumulation of ABA content, thereby improving drought resistance and recovery ability in Arabidopsis. However, silencing the expression of ZmbZIP33 (BMV-ZmbZIP33) remarkably decreased chlorophyll content, ABA content, superoxide dismutase and peroxidase activities, and increased stomatal opening and water loss rate compared with BMV (control). It showed that silencing ZmbZIP33 lead to reduced drought resistance and recovery ability of maize. ABA sensitivity analysis found that 0.5 and 1 µmol/L treatments severely inhibited the root development of overexpression ZmbZIP33 transgenic Arabidopsis. However, the root growth of BMV was greatly inhibited for 1 and 5µmol/L ABA treatments, but not for BMV-ZmbZIP33. Subcellular localization, yeast two-hybrid and BIFC further confirmed that the core components of ABA signaling pathways ZmPYL10 and ZmPP2C7 interacted in nucleus, ZmPP2C7 and ZmSRK2E as well as ZmSRK2E and ZmbZIP33 interacted in the plasma membrane. We also found that expression levels of ZmPYL10 and ZmSRK2E in the BMV-ZmbZIP33 mutant were lower than those of BMV, while ZmPP2C7 was the opposite under drought stress and rewatering. However, expression of ZmPYL10 and ZmSRK2E in normal maize leaves were significantly up-regulated by 3-4 folds after drought and ABA treatments for 24 h, while ZmPP2C7 was down-regulated. The NCED and ZEP encoding key enzymes in ABA biosynthesis are up-regulated in overexpression ZmbZIP33 transgenic line under drought stress and rewatering conditions, but down-regulated in BMV-ZmbZIP33 mutants. Together, these findings demonstrate that ZmbZIP33 played roles in ABA biosynthesis and regulation of drought response and rewatering in Arabidopsis and maize thought an ABA-dependent signaling pathway.

First Report of Maize Stalk Rot Caused by Fusarium kyushuense in China.

During 2017 to 2019, a field survey for maize stalk rot was conducted in 21 counties (districts) across the Guangxi province of China. This disease caused yield losses ranging from 20% to 30%. Maize plants with stalk rot were collected during the late milk stage and pieces of diseased pith tissue were cultured as previously described (Shan et al. 2017). Fungal colonies and mycelia with morphological characteristics of Fusarium species were subcultured onto fresh potato dextrose agar (PDA) and carnation leaf agar (CLA) plates. Based on morphological characteristics and molecular detection by amplification of Fusarium genus-specific primers (Duan et al. 2016), 39 Fusarium isolates were identified. Among them, five isolates from Du'an, Pingguo, Debao, and Daxin had abundant, pale orange to yellow aerial mycelium with deep red pigments when grown on PDA (Fig. 1A; 1B). The average growth rate was 8.0 to 12.0 mm per day at 25°C in the dark. The fungi produced two types of spores on CLA. Microconidia were ovoid to clavate, generally 0- to 3-septate, and 4.6 to 9.4 µm in length (n = 30) (Fig. 1D); Macroconidia were slightly curved with an acute apical cell, mostly 3- to 4- septate, and 19.4 to 38.2 µm in length (n = 30) (Fig. 1C). No chlamydospores were observed. These five isolates were initially identified as Fusarium kyushuense based on morphological features. PCR was performed to amplify three phylogenetic genes (TEF1-α, RPB1, and RPB2) (O'Donnell et al. 1998) and species specific primers kyuR1F/kyuR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3/5-TCCAATGGACTGGGCAGCCAAAACACC-3), kyuR2F/kyuR2R (5-CAGATATACATTTGCCTCGACAC-3/5-TACTTGAGCACGGAGCTTG-3) were used to confirm species identity. The obtained sequences were deposited in GenBank under the accession numbers MT997084, MT997080, MT997081 (TEF1-α); MT550012, MT997085, MT997086 (RPB1); MT550009, MT997089, and MT997090 (RPB2), respectively. Using BLAST, sequences of TEF1-α, RPB1, and RPB2 of the isolates were 99.33% (MH582297.1) to 100% (MG282364.1) similar to those of F. kyushuense strains (Supplementary Table 1). Based on phylogenetic analysis with maximum likelihood methods using tools of the website of CIPRES (http://www.phylo.org), isolates GX27, GX167, and GX204 clustered with F. kyushuense with 100% bootstrap support (Fig. 2). The pathogenicity of the three isolates was tested using young seedlings and adult plants as previously described with modification (Ye et al. 2013; Zhang et al. 2016). The primary roots of three-leaf-old seedlings were inoculated by immersing the roots into a 1 × 106 macroconidia solution, incubating for 6 h at 25°C, and transferring to normal growth conditions (26°C, 16 h light/22°C, 8 h dark). The second or third internode above the soil surface of flowering stage plants grown in a greenhouse was bored with a Bosch electric drill to make a hole (ca. 8 mm in diameter) and inoculated with 0.5 mL of mycelia plug then sealed with petrolatum. The inoculum was created by homogenizing five plates of flourish hyphal mats (approximately 125 mL) with kitchen blender and adjusting to a final volume of 200 mL with sterilized ddH2O. No symptoms were observed in the seedlings or adult plants that were mock-inoculated with PDA plugs. Three days post-inoculation (dpi), roots of the infected seedling turned dark-brown and shrunk and the leaves wilted (Fig. 1E). Typical stalk rot symptoms observed in the inoculated plants were premature wilting of entire plant and hollow and weak stalks, leading to lodging; the longitudinal section of the internodes exhibited obvious dark brown necrosis and reddish discoloration at 14 dpi and 30 dpi, respectively (Fig. 1F). Fusarium kyushuense was re-isolated from the inoculated stalk lesions but not from the control. This is the first record of stalk rot caused by F. kyushuense on maize plants in China. However, F. kyushuense is known to cause maize ear rot in China (Wang et al. 2014) and can produce type A and type B trichothecene mycotoxins in kernels (Aoki and O'Donnell 1998). The occurrence of maize stalk rot and ear rot caused by F. kyushuense should be monitored in China due to the potential risk for crop loss and mycotoxin contamination.

Association Mapping and Transcriptome Analysis Reveal the Genetic Architecture of Maize Kernel Size.

Kernel length, kernel width, and kernel thickness are important traits affecting grain yield and product quality. Here, the genetic architecture of the three kernel size traits was dissected in an association panel of 309 maize inbred lines using four statistical methods. Forty-two significant single nucleotide polymorphisms (SNPs; p < 1.72E-05) and 70 genes for the three traits were identified under five environments. One and eight SNPs were co-detected in two environments and by at least two methods, respectively, and they explained 5.87-9.59% of the phenotypic variation. Comparing the transcriptomes of two inbred lines with contrasting seed size, three and eight genes identified in the association panel showed significantly differential expression between the two genotypes at 15 and 39 days after pollination, respectively. Ten and 17 genes identified by a genome-wide association study were significantly differentially expressed between the two development stages in the two genotypes. Combining environment-/method-stable SNPs and differential expression analysis, ribosomal protein L7, jasmonate-regulated gene 21, serine/threonine-protein kinase RUNKEL, AP2-EREBP-transcription factor 16, and Zm00001d035222 (cell wall protein IFF6-like) were important candidate genes for maize kernel size and development.

First Report of Fusarium thapsinum Causing Maize Stalk Rot in China.

Maize (Zea mays L.) is the most widely grown crop in China, which was planted 41.28 million hectares in 2019 (http://data.stats.gov.cnw/easyquery.htm?cn=C01&zb=A0D0F&sj=2019). Several fungal diseases of maize are reported in which stalk rot has become one of the most destructive diseases in China. The average yield losses affected by the disease are estimated at 10% to 20% (Yu et al. 2016). From 2017 to 2019, a survey was conducted to determine the population diversity of Fusarium species associated with maize diseases in 18 cities across Henan province. Fusarium stalk rot of maize with disease incidence more than 25% was observed in two continuous maize fields at Xuchang city. The diseased stem tissues from junctions in health and disease were chopped into small pieces (3 × 8 mm), superficially disinfected (70% ethyl alcohol for 1 min), placed onto potato dextrose agar (PDA) amended with L-(+)-Lactic-acid (1 g/L), poured in petri plates and incubated at 25°C for 4 days. Mycelia showing morphological characteristic of Fusarium spp. were sub-cultured from single conidium. The pure fungal isolates produced fluffy colonies, white aerial mycelium with yellow pigment in agar. The radial mycelial growth was measured and calculated at an average growth rate 10.9 mm/day at 25°C (Fig. 1A; 1B). Macroconidia produced on carnation leaf agar (CLA) were relatively slender, slightly curved and thick-walled, mostly 3 to 5 marked septa, with a curved and tapering apical cell and poorly developed foot cell, 46.9 ± 5.6 µm × 4.9 ± 0.2 µm (Fig. 1C). Microconidia formed abundantly and were generally oval on CLA, 8.2 ± 0.5 µm × 3.4± 0.1 µm (Fig. 1D). No chlamydospores were observed. Morphological characteristics of the isolates matched the description of Fusarium thapsinum (Leslie and Summerell 2006). To further get the phylogenetic evidence, TEF1-α (translation elongation factor), RPB1 (the largest subunit of RNA polymerase II) and RPB2 (the second largest subunit of RNA polymerase II) were amplified with primer pairs EF1/EF2 (O'Donnell et al. 1998), thapR1F (5'-TTTTCCTCACAAAGGAGCAAATCATG-3')/thapR1R (5'-GTTCACCCAAGATATGGTCGAAAGCC-3'), and thapR2F (5'-ACTCTTTCACATTTGCGCCGAAC-3')/thapR2R (5'-CGGAGCTTTCGTCCAGTGTGAC-3'), and sequenced, respectively. The BLAST search of the sequences of EF1-α, RPB1 and RPB2 shared 99.87% to 100% identity with those of F. thapsinum strains deposited in the GenBank (Supplementary Table 1). Sequences from two isolates (XCCG-3-B-1 and XCCG-3-A-1) were deposited in GenBank (Accession No. MT550014, MT997082 for EF-1α; MT550011, MT997087 for RPB1 and MT550008, MT997091 for RPB2). The phylogenetic relationships based on analysis of the partial sequences showed the representive isolates clustered together with F. thapsinum at 96% bootstrap values (Fig. 2). Combined with the results of morphological characteristics and phylogenetic analysis, the strain designated as Fusarium thapsinum. To complete Koch's postulates, the pathogenicity of the isolates was tested using the silking-stage plants in a greenhouse based on previously described method with modification (Zhang et al. 2016). An 8 mm in diameter wound hole was created at the second or third internode of the plant above the soil surface and injected with 0.5 ml of mycelia plug. The inoculated stalk exhibited internal dark brown necrotic regions and the brown area elongated obviously around the insertion at 14 dpi (days post inoculation). At 30 dpi, the stalks turned soft, hollow and even lodging of the plants for those severe ones, which are similar to those observed on naturally infected maize plants in the field (Fig. 1F). When the roots of the three-leaf-stage seedlings were inoculated with 1×106 macroconidia solution (Ye et al. 2013), the root rot and leaf wilting symptoms were observed (Fig. 1E). While the control plants that were inoculated with only sterile water showed no disease symptoms. The pathogen was re-isolated from the inoculated tissues and the identity was confirmed by the morphological characters. Fusarium thapsinum had been described as causal agent of maize stalk rot in Pakistan (Tahir et al. 2018). To our knowledge, this is the first report of F. thapsinum associated with maize stalk rot in China. The discovery will strengthen the theoretical foundation of maize stalk rot disease management.

A novel pathogenicity determinant hijacks maize catalase 1 to enhance viral multiplication and infection.

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens.

Integrating a genome-wide association study with transcriptomic analysis to detect genes controlling grain drying rate in maize (Zea may, L.).

KEY MESSAGE: Candidate genes on grain drying rate (GDR) were identified, and drying molecular mechanism of grain was explored by integrating genome-wide association with transcriptomic analysis in maize. Grain drying rate (GDR) is a key determinant of grain moisture at harvest. Here, a genome-wide association study (GWAS) of 309 inbred maize lines was used to identify single-nucleotide polymorphisms (SNPs) associated with drying rates of grain, cob and bract. Out of 217,933 SNPs, seven significant SNPs were repeatedly identified in four environments (P < 10-4). Based on genomic position of significant SNPs, six candidate genes were identified, one of which (Zm00001d047468) was verified by transcriptomic data between inbred lines with high and low GDR, indicating stable and reliable correlation with GDR. To further detect more genes correlated with GDR and explore drying molecular mechanism of grain, expression profile of all GWAS-identified genes (4941) detected from different environments, tissues and developmental stage was evaluated by transcriptomic data of six inbred lines with high or low GDR. Results revealed 162 genes exhibit up-regulated expression and another 123 down-regulated in three higher-GDR inbred lines. Based on GO enrichment, 162 up-regulated genes were significantly enriched into grain primary metabolic process, nitrogen compound metabolic process and macromolecule metabolic process (P < 0.05), which indicated grain filling imposes notable influence on GDR before and after physiological maturity. Our results lay foundation in accelerating development of higher-GDR maize germplasm through marker-assisted selection and clarifying genetic mechanism of GDR in maize.

Maize phenylalanine ammonia-lyases contribute to resistance to Sugarcane mosaic virus infection, most likely through positive regulation of salicylic acid accumulation.

Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia-lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA-responsive pathogenesis-related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription-coupled quantitative PCR and RNA-Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up-regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV-infected ZmPAL-silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA-mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV-induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity.

Dual functions of ZmNF-YA3 in photoperiod-dependent flowering and abiotic stress responses in maize.

Nuclear factor-Y (NF-Y) transcription factors are important regulators of several essential biological processes, including embryogenesis, drought resistance, meristem maintenance, and photoperiod-dependent flowering in Arabidopsis. However, the regulatory mechanisms of NF-Ys in maize (Zea mays) are not well understood yet. In this study, we identified an NF-Y transcription factor, ZmNF-YA3. Genome-wide analysis showed that ZmNF-YA3 bound to >6000 sites in the maize genome, 2259 of which are associated with genic sequences. ZmNF-YA3 was found to interact with CONSTANS-like (CO-like) and flowering promoting factor1 (FPF1) through yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Quantitative real-time reverse transcription-PCR (qRT-PCR) combined with yeast one-hybrid assay and EMSA suggested that NF-YA3 could promote early flowering by binding to the FLOWERING LOCUS T-like12 (FT-like12) promoter in maize. Morerover, we also showed that ZmNF-YA3 could improve drought and high-temperature tolerance through binding to the promoter regions of bHLH92, FAMA, and the jasmonic acid activator MYC4, respectively. These results contribute to a comprehensive understanding of the molecular mechanisms and regulatory networks of NF-Y transcription factors in regulating maize flowering time and stress response in maize.

Heterosis-related genes under different planting densities in maize.

Heterosis and increasing planting density have contributed to improving maize grain yield (GY) for several decades. As planting densities increase, the GY per plot also increases, whereas the contribution of heterosis to GY decreases. There are trade-offs between heterosis and planting density, and the transcriptional characterization of heterosis may explain the mechanism involved. In this study, 48 transcriptome libraries were sequenced from four inbred Chinese maize lines and their F1 hybrids. They were planted at densities of 45000 and 67500 plants ha-1. Maternal-effect differentially expressed genes (DEGs) played important roles in processes related to photosynthesis and carbohydrate biosynthesis and metabolism. Paternal-effect DEGs participated in abiotic/biotic stress response and plant hormone production under high planting density. Weighted gene co-expression network analysis revealed that high planting density induced heterosis-related genes regulating abiotic/biotic stress response, plant hormone biosynthesis, and ubiquitin-mediated proteolysis, but repressed other genes regulating energy formation. Under high planting density, maternal genes were mainly enriched in the photosynthesis reaction center, while paternal genes were mostly concentrated in the peripheral antenna system. Four important genes were identified in maize heterosis and high planting density, all with functions in photosynthesis, starch biosynthesis, auxin metabolism, gene silencing, and RNAi.

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis.

The common smut of corn, caused by Ustilago maydis is a troublesome disease of maize. Early and accurate detection of U. maydis is essential for the disease management. In this study, primer set Pep-2 was selected for LAMP (loop-mediated isothermal amplification) from 12 sets of primers targeting three U. maydis effector genes See1, Pit2 and Pep1 according to primer screening. The optimal concentrations of Bst DNA polymerase and Mg2+ as well as inner/outer primer ratio of the LAMP reaction system were screened by combining a single factor experiment and an orthogonal design arrangement. The specificity of this real-time LAMP (RealAmp) assay was confirmed by negative testing for other pathogens. The detection sensitivity of the RealAmp assay was 200 times higher than that of detection through conventional PCR. Results of the RealAmp assay for quantifying the genomic DNA of U. maydis were confirmed by testing with both artificially and naturally infected samples. In addition, the RealAmp reaction could be conducted via an improved tube scanner to implement a "electricity free" assay from template preparation to quantitative detection. The resulting assay could be more convenient for use in the field as a simple, rapid, and effective technique for monitoring U. maydis.