Transcriptomic and Functional Analyses Reveal the Different Roles of Vitamins C, E, and K in Regulating Viral Infections in Maize.

Maize lethal necrosis (MLN), one of the most important maize viral diseases, is caused by maize chlorotic mottle virus (MCMV) infection in combination with a potyvirid, such as sugarcane mosaic virus (SCMV). However, the resistance mechanism of maize to MLN remains largely unknown. In this study, we obtained isoform expression profiles of maize after SCMV and MCMV single and synergistic infection (S + M) via comparative analysis of SMRT- and Illumina-based RNA sequencing. A total of 15,508, 7567, and 2378 differentially expressed isoforms (DEIs) were identified in S + M, MCMV, and SCMV libraries, which were primarily involved in photosynthesis, reactive oxygen species (ROS) scavenging, and some pathways related to disease resistance. The results of virus-induced gene silencing (VIGS) assays revealed that silencing of a vitamin C biosynthesis-related gene, ZmGalDH or ZmAPX1, promoted viral infections, while silencing ZmTAT or ZmNQO1, the gene involved in vitamin E or K biosynthesis, inhibited MCMV and S + M infections, likely by regulating the expressions of pathogenesis-related (PR) genes. Moreover, the relationship between viral infections and expression of the above four genes in ten maize inbred lines was determined. We further demonstrated that the exogenous application of vitamin C could effectively suppress viral infections, while vitamins E and K promoted MCMV infection. These findings provide novel insights into the gene regulatory networks of maize in response to MLN, and the roles of vitamins C, E, and K in conditioning viral infections in maize.

Field application of nanoliposomes delivered quercetin by inhibiting specific hsp70 gene expression against plant virus disease.

BACKGROUND: The annual economic loss caused by plant viruses exceeds 10 billion dollars due to the lack of ideal control measures. Quercetin is a flavonol compound that exerts a control effect on plant virus diseases, but its poor solubility and stability limit the control efficiency. Fortunately, the development of nanopesticides has led to new ideas. RESULTS: In this study, 117 nm quercetin nanoliposomes with excellent stability were prepared from biomaterials, and few surfactants and stabilizers were added to optimize the formula. Nbhsp70er-1 and Nbhsp70c-A were found to be the target genes of quercetin, through abiotic and biotic stress, and the nanoliposomes improved the inhibitory effect at the gene and protein levels by 33.6 and 42%, respectively. Finally, the results of field experiment showed that the control efficiency was 38% higher than that of the conventional quercetin formulation and higher than those of other antiviral agents. CONCLUSION: This research innovatively reports the combination of biological antiviral agents and nanotechnology to control plant virus diseases, and it significantly improved the control efficiency and reduced the use of traditional chemical pesticides.

Identification and functional characterization of NbMLP28, a novel MLP-like protein 28 enhancing Potato virus Y resistance in Nicotiana benthamiana.

BACKGROUND: Major latex proteins (MLPs) belong to the MLP subfamily in Bet v 1 protein family and respond to both biotic and abiotic stresses, which play critical roles in plant disease resistance. As the type species of widely distributed and economically devastating Potyvirus, Potato virus Y (PVY) is one of the major constraints to important crop plants including tobacco (Nicotiana benthamiana) worldwide. Despite the great losses owing to PVY infection in tobacco, there is no previous study investigating the potential role of MLPs in developing resistance to viral infection. RESULTS: In this study, for the first time we have identified and functionally analyzed the MLP-like protein 28 from N. benthamiana, denoted as NbMLP28 and investigated its role in conferring resistance to N. benthamiana against PVY infection. NbMLP28 was localized to the plasmalemma and nucleus, with the highest level in the root. NbMLP28 gene was hypothesized to be triggered by PVY infection and was highly expressed in jasmonic acid (JA) signaling pathway. Further validation was achieved through silencing of NbMLP28 through virus-induced gene silencing (VIGS) that rendered N. benthamiana plants more vulnerable to PVY infection, contrary to overexpression that enhanced resistance. CONCLUSIONS: Taken together, this is the first study describing the role of NbMLP28 in tobacco against PVY infection and provide a pivotal point towards obtaining pathogen-resistant tobacco varieties through constructing new candidate genes of MLP subfamily.

RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph.

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

CLC-Nt1 affects Potato Virus Y infection via regulation of endoplasmic reticulum luminal Ph.

Chloride channel (CLC) proteins are important anion transporters conserved in organisms ranging from bacteria and yeast to plants and animals. According to sequence comparison, some plant CLCs are predicted to function as Cl- /H+ antiporters, but not Cl- channels. However, no direct evidence was provided to verify the role of these plant CLCs in regulating the pH of the intracellular compartment. We identified tobacco CLC-Nt1 interacting with the Potato virus Y (PVY) 6K2 protein. To investigate its physiological function, homologous genes of CLC-Nt1 in Nicotiana benthamiana were knocked out using the CRISPR/Cas9 system. Complementation experiments were subsequently performed by expression of wild-type or point-mutated CLC-Nt1 in knockout mutants. The data presented herein demonstrate that CLC-Nt1 is localized at endoplasmic reticulum (ER). Using a pH-sensitive fluorescent protein (pHluorin), we found that loss of CLC-Nt1 function resulted in a decreased ER luminal pH. Secreted GFP (secGFP) was retained mostly in ER in knockout mutants, indicating that CLC-Nt1 is also involved in protein secretion. PVY infection induced a rise in ER luminal pH, which was dependent on functional CLC-Nt1. By contrast, loss of CLC-Nt1 function inhibited PVY intracellular replication and systemic infection. We propose that PVY alters ER luminal pH for infection in a CLC-Nt1-dependent manner.

Molecular characterization and pathogenicity of a novel monopartite geminivirus infecting tobacco in China.

The occurrence of geminiviruses causes significant economic losses in many economically important crops. In this study, a novel geminivirus isolated from tobacco in Sichuan province of China, named tomato leaf curl Chuxiong virus (TLCCxV), was characterized by small RNA-based deep sequencing. The full-length of TLCCxV genome was determined to be 2744 nucleotides (nt) encoding six open reading frames. Phylogenetic and genome-wide pairwise identity analysis revealed that TLCCxV shared less than 91% identities with reported geminiviruses. A TLCCxV infectious clone was constructed and successfully infected Nicotiana benthamiana, N. tabacum, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants. Furthermore, expression of the V2, C1 and C4 proteins through a potato virus X vector caused severe chlorosis or necrosis symptom in N. benthamiana. Taken together, we identified a new geminivirus in tobacco plants, and found that V2, C1 and C4 contribute to symptom development.

ZmmiR398b negatively regulates maize resistance to sugarcane mosaic virus infection by targeting ZmCSD2/4/9.

MicroRNAs (miRNAs) are widely involved in various biological processes of plants and contribute to plant resistance against various pathogens. In this study, upon sugarcane mosaic virus (SCMV) infection, the accumulation of maize (Zea mays) miR398b (ZmmiR398b) was significantly reduced in resistant inbred line Chang7-2, while it was increased in susceptible inbred line Mo17. Degradome sequencing analysis coupled with transient co-expression assays revealed that ZmmiR398b can target Cu/Zn-superoxidase dismutase2 (ZmCSD2), ZmCSD4, and ZmCSD9 in vivo, of which the expression levels were all upregulated by SCMV infection in Chang7-2 and Mo17. Moreover, overexpressing ZmmiR398b (OE398b) exhibited increased susceptibility to SCMV infection, probably by increasing reactive oxygen species (ROS) accumulation, which were consistent with ZmCSD2/4/9-silenced maize plants. By contrast, silencing ZmmiR398b (STTM398b) through short tandem target mimic (STTM) technology enhanced maize resistance to SCMV infection and decreased ROS levels. Interestingly, copper (Cu)-gradient hydroponic experiments demonstrated that Cu deficiency promoted SCMV infection while Cu sufficiency inhibited SCMV infection by regulating accumulations of ZmmiR398b and ZmCSD2/4/9 in maize. These results revealed that manipulating the ZmmiR398b-ZmCSD2/4/9-ROS module provides a prospective strategy for developing SCMV-tolerant maize varieties.

N6-Methyladenosine RNA Modification Regulates Maize Resistance to Maize Chlorotic Mottle Virus Infection.

Maize chlorotic mottle virus (MCMV) is one of the main viruses causing significant losses in maize. N6-methyladenosine (m6A) RNA modification has been proven to play important regulatory roles in plant development and stress response. In this study, we found that MCMV infection significantly up-regulated the m6A level in maize, and methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were performed to investigate the distribution of m6A modified peaks and gene expression patterns in MCMV-infected maize plants. The results showed that 1325 differentially methylated genes (DMGs) and 47 differentially methylated and expressed genes (DMEGs) were identified and analyzed. Moreover, the results of virus-induced gene silencing (VIGS) assays showed that ZmECT18 and ZmGST31 were required for MCMV infection, while silencing of ZmMTC, ZmSCI1 or ZmTIP1 significantly promoted MCMV infection in maize. Our findings provided novel insights into the regulatory roles of m6A modification in maize response to MCMV infection.

Whole-transcriptome characterization and functional analysis of lncRNA-miRNA-mRNA regulatory networks responsive to sugarcane mosaic virus in maize resistant and susceptible inbred lines.

Sugarcane mosaic virus (SCMV) is one of the most important pathogens causing maize dwarf mosaic disease, which seriously affects the yield and quality of maize. Currently, the molecular mechanism of non-coding RNAs (ncRNAs) responding to SCMV infection in maize is still uncovered. In this study, a total of 112 differentially expressed (DE)-long non-coding RNAs (lncRNAs), 24 DE-microRNAs (miRNAs), and 1822 DE-messenger RNAs (mRNAs), and 363 DE-lncRNAs, 230 DE-miRNAs, and 4376 DE-mRNAs were identified in maize resistant (Chang7-2) and susceptible (Mo17) inbred lines in response to SCMV infection through whole-transcriptome RNA sequencing, respectively. Moreover, 4874 mRNAs potentially targeted by 635 miRNAs were obtained by degradome sequencing. Subsequently, several crucial SCMV-responsive lncRNA-miRNA-mRNA networks were established, of which the expression levels of lncRNA10865-miR166j-3p-HDZ25/69 (class III homeodomain-leucine zipper 25/69) module, and lncRNA14234-miR394a-5p-SPL11 (squamosal promoter-binding protein-like 11) module were further verified. Additionally, silencing lncRNA10865 increased the accumulations of SCMV and miR166j-3p, while silencing lncRNA14234 decreased the accumulations of SCMV and SPL11 targeted by miR394a-5p. This study revealed the interactions of lncRNAs, miRNAs and mRNAs in maize resistant and susceptible materials, providing novel clues to reveal the mechanism of maize in resistance to SCMV from the perspective of competing endogenous RNA (ceRNA) regulatory networks.

Characterization of small interfering RNAs derived from pepper mild mottle virus in infected pepper plants by high-throughput sequencing.

Pepper mild mottle virus (PMMoV) infects pepper plants and induces severe yield losses in China. However, the molecular interaction between PMMoV and pepper plants is largely unknown. RNA silencing is a eukaryotically conserved mechanism against viruses mediated by virus-derived small interfering RNAs (vsiRNAs) in plants. In this study, the profiles of vsiRNAs from PMMoV in infected pepper plants were obtained by high-throughput sequencing. The results showed that vsiRNAs were predominantly 21 and 22 nucleotides (nts) in length, and had a U bias at the 5'-terminal. The single-nucleotide resolution maps revealed that vsiRNAs were heterogeneously distributed throughout PMMoV genomic RNAs and hotspots of sense and antisense strands were mainly located in the RdRp and CP coding regions. The host transcripts targeted by vsiRNAs were predicted and they are mainly involved in physiological pathways related to stress response, cell regulation, and metabolism process. In addition, PMMoV infection induced significant up-regulation of CaAGO1a/1b/2, CaDCL2 and CaRDR1 gene transcripts in pepper plants, which are important components involved in antiviral RNA silencing pathway. Taken together, our results suggest the possible roles of vsiRNAs in PMMoV-pepper interactions.

Recombinase Polymerase Amplification Assay for Simultaneous Detection of Maize Chlorotic Mottle Virus and Sugarcane Mosaic Virus in Maize.

Maize chlorotic mottle virus (MCMV) can cause maize lethal necrosis (MLN) when coinfected with potyvirids, such as sugarcane mosaic virus (SCMV), maize dwarf mosaic virus, or wheat streak mosaic virus. MLN is often caused by coinfection of MCMV and SCMV, which has been reported in China and several countries of Africa. In this study, a recombinase polymerase amplification (RPA) assay was established for simultaneous detection of MCMV and SCMV in maize. The RPA assay can be completed within 30 min at 38 °C. The primers for the RPA assay were specific since no crossreaction was detected with other selected viruses that infected maize in China. The detection limit of the RPA method was 102 copies µL-1, which was about 10-fold more sensitive than that of the conventional PCR method. Moreover, the RPA assay can be successfully applied to detect maize samples collected in the field. These results demonstrated that the established RPA assay is a rapid and efficient method to conduct simultaneous detection of MCMV and SCMV, which provides an alternative technology for MLN diagnosis.

Purification and Characterization of a Secretory Alkaline Metalloprotease with Highly Potent Antiviral Activity from Serratia marcescens Strain S3.

In this study we report a secretory protein that was purified from Serratia marcescens strain S3 isolated from soil from the tobacco rhizosphere. Subsequent mass spectrometry and annotation characterized the protein as secretory alkaline metalloprotease (SAMP). SAMP plays a crucial role in inhibiting Tobacco mosaic virus (TMV). Transmission electron microscopy (TEM), dynamic light scattering (DLS), confocal microscopy, and microscale thermophoresis (MST) were employed to investigate the anti-TMV mechanism of SAMP. Our results demonstrated that SAMP, as a hydrolytic metal protease, combined and hydrolyzed TMV coat proteins to destroy the virus particles. This study is the first to investigate the antiviral effects of a S. marcescens metalloprotease, and our finding suggests that S. marcescens-S3 may be agronomically useful as a disease-controlling factor active against Tobacco mosaic virus.

RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph / Análisis RNA-seq de la respuesta del oomiceto fitopatógeno Phytophthora parasitica al fungicida dimetomorf

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

Phytophthora parasitica es un importante oomiceto que origina enfermedades en una variedad de plantas; el fungicida dimetomorf es específico contra oomicetos. El objetivo de este estudio fue utilizar la tecnología de RNA-seq para descubrir rápidamente el mecanismo por el que el dimetomorf actúa en el tratamiento de P. parasitica. Descubrimos que la expresión de 832 genes se modificaba significativamente tras el tratamiento con dimetomorf, incluyendo 365 genes que son sobrerregulados y 467 genes que son subrregulados. El análisis de enriquecimiento de ontología de genes (GO), análisis de enriquecimiento de las vías y pruebas de verificación permitieron extraer las conclusiones siguientes: 1) el tratamiento de P. parasitica con dimetomorf origina cambios en los niveles de expresión de los genes relacionados con la pared celular y su síntesis; 2) el tratamiento con dimetomorf origina una reducción de la permeabilidad de la membrana celular, así como cambios en la expresión de ciertas proteínas relacionadas con el transporte, y 3) el tratamiento con dimetomorf incrementó las especies reactivas del oxígeno y redujo la expresión de los genes relacionados con el control del estrés oxidativo.