Epigenetics in the plant-virus interaction.

Plants have developed diverse molecular mechanisms to resist viruses. RNA silencing plays a dominant role in antiviral defense. Recent studies have correlated plant antiviral silencing to epigenetic modification in genomic DNA and protein by remodeling the expression levels of coding genes. The plant host methylation level is reprogrammed in response to viral challenge. Genomes of some viruses have been implicated in the epigenetic modification via small RNA-mediated transcriptional gene silencing and post-transcriptional gene silencing. These mechanisms can be primed prior to a virus attack through methylation changes for antiviral defense. This review highlights the findings concerning the methylation changes in plant-virus interactions and demonstrates a possible direction to improve the understanding of plant host methylation regulation in response to viral infection.

Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-wide Sequencing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated Arabidopsis plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%-97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals.

Blue Native/SDS-PAGE and iTRAQ-Based Chloroplasts Proteomics Analysis of Nicotiana tabacum Leaves Infected with M Strain of Cucumber Mosaic Virus Reveals Several Proteins Involved in Chlorosis Symptoms.

Virus infection in plants involves necrosis, chlorosis, and mosaic. The M strain of cucumber mosaic virus (M-CMV) has six distinct symptoms: vein clearing, mosaic, chlorosis, partial green recovery, complete green recovery, and secondary mosaic. Chlorosis indicates the loss of chlorophyll which is highly abundant in plant leaves and plays essential roles in photosynthesis. Blue native/SDS-PAGE combined with mass spectrum was performed to detect the location of virus, and proteomic analysis of chloroplast isolated from virus-infected plants was performed to quantify the changes of individual proteins in order to gain a global view of the total chloroplast protein dynamics during the virus infection. Among the 438 proteins quantified, 33 showed a more than twofold change in abundance, of which 22 are involved in the light-dependent reactions and five in the Calvin cycle. The dynamic change of these proteins indicates that light-dependent reactions are down-accumulated, and the Calvin cycle was up-accumulated during virus infection. In addition to the proteins involved in photosynthesis, tubulin was up-accumulated in virus-infected plant, which might contribute to the autophagic process during plant infection. In conclusion, this extensive proteomic investigation on intact chloroplasts of virus-infected tobacco leaves provided some important novel information on chlorosis mechanisms induced by virus infection.

The cucumber mosaic virus movement protein suppresses PAMP-triggered immune responses in Arabidopsis and tobacco.

Cucumber Mosaic Virus (CMV) has a small RNA genome that encodes a limited number of proteins, but can infect many plant species, including Arabidopsis thaliana and Nicotiana benthamiana. Virus proteins thus have multiple means of conferring their pathogenicity during the infection process. However, the pathogenic mechanism of CMV remains unclear. Here we discovered that the expression of the CMV movement protein (MP) in A. thaliana and N. benthamiana can suppress reactive oxygen species (ROS) production triggered by multiple pathogen-associated molecular patterns (PAMPs), such as bacteria-derived peptide flg22, elf18, and fungal-derived chitin. Transgenic Arabidopsis plants expressing the MP were compromised in flg22-induced immune activation and were more susceptible to Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC- strain infection. Further analysis revealed that flg22-induced resistance gene expression was also compromised in MP transgenic plants. The CMV MP protein was previously reported to function in cell-to-cell movement processes, and our findings offer a new molecular mechanism for the CMV MP protein in suppression of host PAMP-triggered immune responses.

Probing Adsorption Behaviors of BSA onto Chiral Surfaces of Nanoparticles.

Chiral properties of nanoscale materials are of importance as they dominate interactions with proteins in physiological environments; however, they have rarely been investigated. In this study, a systematic investigation is conducted for the adsorption behaviors of bovine serum albumin (BSA) onto the chiral surfaces of gold nanoparticles (AuNPs), involving multiple techniques and molecular dynamic (MD) simulation. The adsorption of BSA onto both L- and D-chiral surfaces of AuNPs shows discernible differences involving thermodynamics, adsorption orientation, exposed charges, and affinity. As a powerful supplement, MD simulation provides a molecular-level understanding of protein adsorption onto nanochiral surfaces. Salt bridge interaction is proposed as a major driving force at protein-nanochiral interface interaction. The spatial distribution features of functional groups (COO- , NH3+ , and CH3 ) of chiral molecules on the nanosurface play a key role in the formation and location of salt bridges, which determine the BSA adsorption orientation and binding strength to chiral surfaces. Sequentially, BSA corona coated on nanochiral surfaces affects their uptake by cells. The results enhance the understanding of protein corona, which are important for biological effects of nanochirality in living organisms.

Characterization of siRNAs derived from cucumber mosaic virus in infected tobacco plants.

This study characterized the viral small interfering RNAs (vsiRNAs) from Nicotiana tabacum cv. Samsun infected with a cucumber mosaic virus (CMV) 2b-deficient mutant. Most vsiRNAs were 21 -22 nucleotides in length and the 5'-terminal ends were dominated by A and U, respectively. The observed vsiRNAs were heterogeneously distributed throughout the CMV genome; however, most of the vsiRNAs were derived from sense strands, as opposed to antisense strands. These results demonstrate the conserved and specific function of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins in tobacco. Finally, it was revealed that vsiRNAs target abundant host genes, indicating complex roles for CMV vsiRNAs during the development of symptoms.

Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for the final evaluation. After the second evaluation, the final amplification curves and melting curves have been achieved.

Chlorophyll fluorescence lifetime imaging provides new insight into the chlorosis induced by plant virus infection.

KEY MESSAGE: Leaf chlorosis induced by plant virus infection has a short fluorescence lifetime, which reflects damaged photosynthetic complexes and degraded chloroplasts. Plant viruses often induce chlorosis and necrosis, which are intimately related to photosynthetic functions. Chlorophyll fluorescence lifetime measurement is a valuable noninvasive tool for analyzing photosynthetic processes and is a sensitive indicator of the environment surrounding the fluorescent molecules. In this study, our central goal was to explore the effect of viral infection on photosynthesis by employing chlorophyll fluorescence lifetime imaging (FLIM), steady-state fluorescence, non-photochemical quenching (NPQ), transmission electron microscopy (TEM), and pigment analysis. The data indicated that the chlorophyll fluorescence lifetime of chlorotic leaves was significantly shorter than that of healthy control leaves, and the fitted short lifetime component of chlorophyll fluorescence of chlorotic leaves was dominant. This dominant short lifetime component may result from damage to the structure of thylakoid, which was confirmed by TEM. The NPQ value of chlorotic leaves was slightly higher than that of healthy green leaves, which can be explained by increased neoxanthin, lutein and violaxanthin content relative to chlorophyll a. The difference in NPQ is slight, but FLIM can provide simple and direct characterization of PSII structure and photosynthetic function. Therefore, this technique shows great potential as a simple and rapid method for studying mechanisms of plant virus infection.

The detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy.

The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H2 O2 ) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H2 O2 and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H2 O2 in leaves. In this study, the M strain of Cucumber mosaic virus (M-CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock-inoculated leaves, H2 O2 accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H2 O2 accumulation was always higher than in healthy leaves. Subcellular H2 O2 localization observed using CLSM showed that H2 O2 in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H2 O2 was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H2 O2 and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.

Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads.

BACKGROUND: Adapter trimming is a prerequisite step for analyzing next-generation sequencing (NGS) data when the reads are longer than the target DNA/RNA fragments. Although typically used in small RNA sequencing, adapter trimming is also used widely in other applications, such as genome DNA sequencing and transcriptome RNA/cDNA sequencing, where fragments shorter than a read are sometimes obtained because of the limitations of NGS protocols. For the newly emerged Nextera long mate-pair (LMP) protocol, junction adapters are located in the middle of all properly constructed fragments; hence, adapter trimming is essential to gain the correct paired reads. However, our investigations have shown that few adapter trimming tools meet both efficiency and accuracy requirements simultaneously. The performances of these tools can be even worse for paired-end and/or mate-pair sequencing. RESULTS: To improve the efficiency of adapter trimming, we devised a novel algorithm, the bit-masked k-difference matching algorithm, which has O(kn) expected time with O(m) space, where k is the maximum number of differences allowed, n is the read length, and m is the adapter length. This algorithm makes it possible to fully enumerate all candidates that meet a specified threshold, e.g. error ratio, within a short period of time. To improve the accuracy of this algorithm, we designed a simple and easy-to-explain statistical scoring scheme to evaluate candidates in the pattern matching step. We also devised scoring schemes to fully exploit the paired-end/mate-pair information when it is applicable. All these features have been implemented in an industry-standard tool named Skewer (https://sourceforge.net/projects/skewer). Experiments on simulated data, real data of small RNA sequencing, paired-end RNA sequencing, and Nextera LMP sequencing showed that Skewer outperforms all other similar tools that have the same utility. Further, Skewer is considerably faster than other tools that have comparative accuracies; namely, one times faster for single-end sequencing, more than 12 times faster for paired-end sequencing, and 49% faster for LMP sequencing. CONCLUSIONS: Skewer achieved as yet unmatched accuracies for adapter trimming with low time bound.

Quantitative determination of chondroitin sulfate with various molecular weights in raw materials by pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

A simple and reliable HPLC method was developed for quantification of chondroitin sulfate (CS). The procedure is based on precolumn hydrolysis of CS to liberate galactosamine and subsequent derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Hydrolysis and derivatization conditions were optimized. A linear correlation coefficient of 0.9999 was calculated within the range of 10-1500 µg/mL from the standard curve. The method produces good precision and good accuracy (100.75 % recovery). An advantage over other common methods is its ability to quantify CS of all molecular weights and structures, as evidenced by the determination of CS fractions with narrow molecular weight distributions obtained through depolymerization by different methods, while enzymatic HPLC was proven to be infeasible. Extraction recoveries of CS from monosaccharide mixed samples were > 93 %. The reliability was also validated by a small difference (-1.95 % to 4.12 %) relative to enzymatic HPLC results in analysing representative CS samples of different animal origins and suppliers.

Polymerase chain reaction-based methods for the rapid identification of Amanita exitialis.

Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to develop an efficient, accurate, and user-friendly PCR-based method for identifying A. exitialis that could facilitate the prevention, diagnosis, and treatment of associated food poisoning. A. exitialis-specific primers and probes were designed based on the internal transcribed spacer region variations of 27 mushroom species. Specificity was confirmed using conventional and real-time PCR for 23 non-target mushroom species, including morphologically similar and closely related species. Compared to conventional PCR, real-time PCR was more sensitive (detectable DNA concentration: 1.36 × 10-2 ng/µL vs. 1.36 × 10-3) and efficient (analysis time: 1 h vs. 40 min). Furthermore, the real-time PCR results could be immediately visualized using amplification curve analysis. The results present two robust PCR-based methods for A. exitialis identification that can facilitate food safety.

Rapid and sensitive detection of maize chlorotic mottle virus using surface plasmon resonance-based biosensor.

We report a biosensor based on surface plasmon resonance (SPR) for the selective detection of maize chlorotic mottle virus (MCMV). 11-Mercaptoundecanoic acid was applied on a gold surface to form a self-assembled monolayer, and a layer of anti-MCMV antibody was crosslinked on the surface for specific recognition of MCMV. The effects of coupling reaction time and antibody concentration on detection sensitivity were studied. The coverage mass change is a function of the concentration of MCMV with a dynamic range from 1 to 1000 ppb. The detection limit is approximately 1 ppb, which is approximately two orders of magnitude higher than that of the existing enzyme-linked immunosorbent assay (ELISA) method. The developed SPR sensor showed highly specific recognition for both purified MCMV and crude extracts from real-world samples.

Molecular weight determination of low molecular weight hyaluronic acid and chondroitin sulfate by gel permeation chromatography.

Low molecular weight (LWM) hyaluronic acid (HA) and chondroitin sulfate (CS) have a wide range of applications. To determine their molecular weight (MW), we developed a gel permeation chromatography (GPC) method, which is calibrated based on serrated peaks in the chromatograms. MW calibrants were obtained from the enzymolysis of HA and CS using hyaluronidase. The identical structure of calibrants and samples ensured the soundness of the method. The highest confidence MWs were up to 14,454 and 14,605 for HA and CS, respectively, and the standard curves showed very high correlation coefficients. Thanks to the changeless relationship between MW and its contribution to the GPC integral, the second calibration curves could be derived via one GPC column, also embodied correlation coefficients of >0.9999. The discrepancies of MW values were minuscule, and the measurement of a sample could be conducted in <30 min. The accuracy of the method was verified using LWM heparins, and the measured Mw values showed a 1.2 %-2.0 % error relative to pharmacopeia results. The MW results obtained for LWM-HA and LWM-CS samples were also consistent with the results obtained by multiangle laser light scattering. The method was also verified be able to measure the very low MWs.

The effects of the E3 ubiquitin-protein ligase UBR7 of Frankliniella occidentalis on the ability of insects to acquire and transmit TSWV.

The interactions between plant viruses and insect vectors are very complex. In recent years, RNA sequencing data have been used to elucidate critical genes of Tomato spotted wilt ortho-tospovirus (TSWV) and Frankliniella occidentalis (F. occidentalis). However, very little is known about the essential genes involved in thrips acquisition and transmission of TSWV. Based on transcriptome data of F. occidentalis infected with TSWV, we verified the complete sequence of the E3 ubiquitin-protein ligase UBR7 gene (UBR7), which is closely related to virus transmission. Additionally, we found that UBR7 belongs to the E3 ubiquitin-protein ligase family that is highly expressed in adulthood in F. occidentalis. UBR7 could interfere with virus replication and thus affect the transmission efficiency of F. occidentalis. With low URB7 expression, TSWV transmission efficiency decreased, while TSWV acquisition efficiency was unaffected. Moreover, the direct interaction between UBR7 and the nucleocapsid (N) protein of TSWV was investigated through surface plasmon resonance and GST pull-down. In conclusion, we found that UBR7 is a crucial protein for TSWV transmission by F. occidentalis, as it directly interacts with TSWV N. This study provides a new direction for developing green pesticides targeting E3 ubiquitin to control TSWV and F. occidentalis.

Integrated flow cytometric and proteomics analyses reveal the regulatory network underlying sugarcane protoplast responses to fusion.

BACKGROUND: Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion. METHOD: We employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion. RESULTS: Flow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17-1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion. PURPOSE AND SIGNIFICANCE: Overall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.

Rapid on-site detection of Acidovorax citrulli by cross-priming amplification.

Cross-priming amplification (CPA) for Acidovorax citrulli detection was evaluated in this study. The sensitivity of CPA assay for pure bacterial culture was 3.7 × 10(3) CFU/ml. Bacteria on naturally infected watermelon seeds were detected using CPA assay, suggesting this method is suitable for A. citrulli on-site detection from watermelon seeds.

Metabolite profiles of rice cultivars containing bacterial blight-resistant genes are distinctive from susceptible rice.

The metabolic changes of bacterial blight-resistant line C418/Xa23 generated by molecular marker-assisted selection (n= 12), transgenic variety C418-Xa21 generated by using the Agrobacterium-mediated system (n= 12), and progenitor cultivar C418 (n= 12) were monitored using gas chromatography/mass spectrometry. The validation, discrimination, and establishment of correlative relationships between metabolite signals were performed by cluster analysis, principal component analysis, and partial least squares-discriminant analysis. Significant and unintended changes were observed in 154 components in C418/Xa23 and 48 components in C418-Xa21 compared with C418 (P< 0.05, Fold change > 2.0). The most significant decreases detected (P< 0.001) in both C418/Xa23 and C418-Xa21 were in three amino acids: glycine, tyrosine, and alanine, and four identified metabolites: malic acid, ferulic acid, succinic acid, and glycerol. Linoleic acid was increased specifically in C418/Xa23 which was derived from traditional breeding. This line, possessing a distinctive metabolite profile as a positive control, shows more differences vs. the parental than the transgenic line. Only succinic acid that falls outside the boundaries of natural variability between the two non-transgenic varieties C418 and C418/Xa23 should be further investigated with respect to safety or nutritional impact.

Complex small RNA-mediated regulatory networks between viruses/viroids/satellites and host plants.

Host plants deploy the small RNA (sRNA)-directed RNA silencing pathway to resist invasion by acellular microorganisms (viruses/viroids/satellites), and, in turn, this pathway is exploited by pathogenic agents to create an environment conducive to infection. Previous known sRNA-RNA systems consist of host endogenous microRNAs (miRNAs) mediating the regulation of host mRNAs and virus/viroid/satellite-derived small interfering RNAs (vsiRNAs) targeting their genomic RNAs. However, more in-depth explorations have substantially expanded the understanding of the complexity of sRNA-RNA regulatory networks. Here, we review some recently discovered sRNA-mediated regulatory systems. Specifically, in addition to virus-encoded proteins acting as virulence factors, vsiRNAs can serve as important pathogenic determinants targeting host mRNAs and noncoding RNAs to promote virus/viroid/satellite infection and trigger symptoms that may be side effects of infection. Additionally, virus-activated but host-derived siRNAs (vasiRNAs) regulate endogenous plant gene expression related to virus resistance or pathogenicity. The inhibitory effect of miRNAs on plant endogenous mRNAs and viral RNAs (vRNAs) has also been identified. Furthermore, siRNA-based interregulation occurring between viruses and their parasite satellite RNAs (satRNAs) enables coexisting virus-satRNA-plant homoeostasis. Thus, the underlying mechanisms of plant-virus/viroid/satellite competition and symbiosis are largely obscured by these diverse sRNA-RNA combinations. Guided by the intricate regulatory network-based principle at the RNA level, practically applicable and feasible strategies have been developed for the management of plant viruses/viroids/satellites for which effective control measures are lacking.

Chromosome-level genome assembly of Bactrocera dorsalis reveals its adaptation and invasion mechanisms.

Bactrocera dorsalis is an invasive polyphagous pest causing considerable ecological and economic damage worldwide. We report a high-quality chromosome-level genome assembly and combine various transcriptome data to explore the molecular mechanisms of its rapid adaptation to new environments. The expansions of the DDE transposase superfamily and key gene families related to environmental adaptation and enrichment of the expanded and unique gene families in metabolism and defence response pathways explain its environmental adaptability. The relatively high but not significantly different expression of heat-shock proteins, regardless of the environmental conditions, suggests an intrinsic mechanism underlying its adaptation to high temperatures. The mitogen-activated protein kinase pathway plays a key role in adaptation to new environments. The prevalence of duplicated genes in its genome explains the diversity in the B. dorsalis complex. These findings provide insights into the genetic basis of the invasiveness and diversity of B. dorsalis, explaining its rapid adaptation and expansion.