Comparative physiological and transcriptome analysis provide insights into the inhibitory effect of 6-pentyl-2H-pyran-2-one on Clarireedia jacksonii.

Clarireedia spp. is a destructive phytopathogenic fungus that causes turf dollar spot of bent-grass, leading to widespread lawn death. In this study, we explored the antifungal capability of 6-pentyl-2H-pyran-2-one (6PP), a natural metabolite volatilized by microorganisms, which plays an important role in the biological control of turfgrass dollar spot. However, the mechanisms by which 6PP inhibits Clarireedia jacksonii remain unknown. In the present study, C. jacksonii mycelial growth was inhibited by the 6PP treatment and the 6PP treatment damaged cell membrane integrity, causing an increase in relative conduc-tivity. Furthermore, physiological and biochemistry assay showed that 6PP treatment can enhance reactive oxygen species (ROS) levels, malondialdehyde (MDA) content obviously increased with 6PP exposure, increased alchohol dehydrogenase (ADH) and depleted acetalde-hyde dehydrogenase (ALDH), and activated the activities of many antioxidant enzymes in C. jacksonii. Gen Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that some genes in C. jacksonii after 6PP treatment related to integrity of the cell wall and membrane, and oxidative stress were significantly downregulated. It is worth mentioning that the fatty acid degradation pathway is significantly upregulated, with an increase in ATP content and ATP synthase activity, which may promote fungal cell apoptosis. Moreover, we found that the expression of ABC transporters, and glutathione metabolism encoding genes were increased to respond to external stimuli. Taken together, these findings revealed the potential antifungal mechanism of 6PP against Clarireedia spp., which also provides a theoretical basis for the commercial utilization of 6PP as a green pesticide in the future.

New Insights into the Mechanism of Trichoderma virens-Induced Developmental Effects on Agrostis stolonifera Disease Resistance against Dollar Spot Infection.

Trichoderma is internationally recognized as a biocontrol fungus for its broad-spectrum antimicrobial activity. Intriguingly, the crosstalk mechanism between the plant and Trichoderma is dynamic, depending on the Trichoderma strains and the plant species. In our previous study, the Trichoderma virens 192-45 strain showed better pathogen inhibition through the secretive non-volatile and volatile substrates. Therefore, we studied transcriptional and metabolic responses altered in creeping bentgrass (Agrostis stolonifera L.) with T. virens colonization prior to a challenge with Clarireedia homoeocarpa. This fungal pathogen causes dollar spot on various turfgrasses. When the pathogen is deficient, the importance of T. virens to the enhancement of plant growth can be seen in hormonal production and microbe signaling, such as indole-3-acrylic acid. Therefore, these substrates secreted by T. virens and induced genes related to plant growth can be the 'pre-defense' for ensuing pathogen attacks. During C. homoeocarpa infection, the Trichoderma-plant interaction activates defense responses through the SA- and/or JA-dependent pathway, induced by T. virens and its respective exudates, such as oleic, citric, and stearic acid. Thus, we will anticipate a combination of genetic engineering and exogenous application targeting these genes and metabolites, which could make creeping bentgrass more resistant to dollar spot and other pathogens.

Genome-Wide Identification and Analysis of the NF-Y Transcription Factor Family Reveal Its Potential Roles in Salt Stress in Alfalfa (Medicago sativa L.).

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that plays an important role in various biological processes in plants, such as flowering regulation, drought resistance, and salt stress. However, few in-depth studies investigated the alfalfa NF-Y gene family. In this study, in total, 60 MsNF-Y genes, including 9 MsNF-YAs, 26 MsNF-YBs, and 25 MsNF-YCs, were identified in the alfalfa genome. The genomic locations, gene structures, protein molecular weights, conserved domains, phylogenetic relationships, and gene expression patterns in different tissues and under different stresses (cold stress, drought stress, and salt stress) of these NF-Y genes were analyzed. The illustration of the conserved domains and specific domains of the different subfamilies of the MsNF-Y genes implicates the conservation and diversity of their functions in alfalfa growth, development, and stress resistance. The gene expression analysis showed that 48 MsNF-Y genes (7 MsNF-YAs, 22 MsNF-YBs, and 19 MsNF-YCs) were expressed in all tissues at different expression levels, indicating that these genes have tissue expression specificity and different biological functions. In total, seven, seven, six, and eight MsNF-Y genes responded to cold stress, the ABA treatment, drought stress, and salt stress in alfalfa, respectively. According to the WGCNA, molecular regulatory networks related to salt stress were constructed for MsNF-YB2, MsNF-YB5, MsNF-YB7, MsNF-YB15, MsNF-YC5, and MsNF-YC6. This study could provide valuable information for further elucidating the biological functions of MsNF-Ys and improving salt tolerance and other abiotic stress resistance in alfalfa.

Zoysia japonica Chlorophyll b Reductase Gene NOL Participates in Chlorophyll Degradation and Photosynthesis.

The degradation of chlorophyll is of great significance to plant growth. The chlorophyll b reductase NOL (NYC1-like) is in charge of catalyzing the degradation of chlorophyll b and maintaining the stability of the photosystem. However, the molecular mechanisms of NOL-mediated chlorophyll degradation, senescence, and photosynthesis and its functions in other metabolic pathways remain unclear, especially in warm-season turfgrass. In this study, ZjNOL was cloned from Zoysia japonica. It is highly expressed in senescent leaves. Subcellular localization investigation showed ZjNOL is localized in the chloroplast and the bimolecular fluorescence complementation (BiFC) results proved ZjNOL interacts with ZjNYC1 in vivo. ZjNOL promoted the accumulation of abscisic acid (ABA) and carbohydrates, and the increase of SAG14 at the transcriptional level. ZjNOL simultaneously led to the excessive accumulation of reactive oxygen species (ROS), the activation of antioxidant enzymes, and the generation of oxidative stress, which in turn accelerated senescence. Chlorophyll fluorescence assay (JIP-test) analysis showed that ZjNOL inhibited photosynthetic efficiency mainly through damage to the oxygen-evolving complex. In total, these results suggest that ZjNOL promotes chlorophyll degradation and senescence and negatively affects the integrity and functionality of the photosystem. It could be a valuable candidate gene for genome editing to cultivate Z. japonica germplasm with prolonged green period and improved photosynthesis efficiency.

Integrated Analysis of Coding and Non-coding RNAs Reveals the Molecular Mechanism Underlying Salt Stress Response in Medicago truncatula.

Salt stress is among the most severe abiotic stresses in plants worldwide. Medicago truncatula is a model plant for legumes and analysis of its response to salt stress is helpful for providing valuable insights into breeding. However, few studies have focused on illustrating the whole-transcriptome molecular mechanism underlying salt stress response in Medicago truncatula. Herein, we sampled the leaves of Medicago truncatula treated with water or NaCl and analyzed the characteristics of its coding and non-coding RNAs. We identified a total of 4,693 differentially expressed mRNAs (DEmRNAs), 505 DElncRNAs, 21 DEcircRNAs, and 55 DEmiRNAs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that their functions were mostly associated with metabolic processes. We classified the lncRNAs and circRNAs into different types and analyzed their genomic distributions. Furthermore, we predicted the interactions between different RNAs based on the competing endogenous RNA (ceRNA) theory and identified multiple correlation networks, including 27 DEmiRNAs, 43 DEmRNAs, 19 lncRNAs, and 5 DEcircRNAs. In addition, we comprehensively analyzed the candidate DEmRNAs and ceRNAs and found that they were involved in Ca+ signaling, starch and sucrose biosynthesis, phenylpropanoid and lignin metabolism, auxin and jasmonate biosynthesis, and transduction pathways. Our integrated analyses in salt stress response in Medicago truncatula revealed multiple differentially expressed coding and non-coding RNAs, including mRNAs, lncRNAs, circRNAs, and miRNAs, and identified multiple DEmRNA and ceRNA interaction pairs that function in many pathways, providing insights into salt stress response in leguminous plants.

Single-molecule long-read sequencing analysis improves genome annotation and sheds new light on the transcripts and splice isoforms of Zoysia japonica.

BACKGROUND: Zoysia japonica is an important warm-season turfgrass used worldwide. Although the draft genome sequence and a vast amount of next-generation sequencing data have been published, the current genome annotation and complete mRNA structure remain incomplete. Therefore, to analyze the full-length transcriptome of Z. japonica, we used the PacBio single-molecule long-read sequencing method in this study. RESULTS: First, we generated 37,056 high-confidence non-redundant transcripts from 16,005 gene loci. Next, 32,948 novel transcripts, 913 novel gene loci, 8035 transcription factors, 89 long non-coding RNAs, and 254 fusion transcripts were identified. Furthermore, 15,675 alternative splicing events and 5325 alternative polyadenylation sites were detected. In addition, using bioinformatics analysis, the underlying transcriptional mechanism of senescence was explored based on the revised reference transcriptome. CONCLUSION: This study provides a full-length reference transcriptome of Z. japonica using PacBio single-molecule long-read sequencing for the first time. These results contribute to our knowledge of the transcriptome and improve the knowledge of the reference genome of Z. japonica. This will also facilitate genetic engineering projects using Z. japonica.

Chlorophyll Metabolism and Gene Expression in Response to Submergence Stress and Subsequent Recovery in Perennial Ryegrass Accessions Differing in Growth Habits.

Submergence-induced leaf senescence may alter chlorophyll metabolism. The objective of this study was to characterize chlorophyll biosynthesis and degradation in contrasting perennial ryegrass (Lolium perenne) in response to submergence stress and recovery. The light-green and fast-growing accession PI238938 and the darker-green and slow-growing cultivar BrightStar SLT were exposed to 0, 6 h, 1-, 3-, and 7-d of submergence stress and 1- and 5- d of de-submergence, respectively. Plant growth of PI238938 were more severely inhibited by submergence stress and recovery. Both accessions showed increased leaf malondialdehyde under stress and recovery, but reduced chlorophyll (Chl) concentrations were observed at 3- and 7-d of stress and at recovery. The reduction in Chl was more severe in BrightStar SLT at 7 d of stress. The concentration of 5-aminolevulenic acid was unaffected by stress but increased at 1d of recovery. Activities of 5-aminolevulinic acid dehydratase (ALAD) involved in Chl biosynthesis remained unchanged under stress and recovery, while the activities of Chl degrading enzymes chlorophyllase (CHL) and pheophytinase (PPH) increased at 3 d or 7 d of stress, and returned to the control level after recovery in both accessions. The downregulation of Chl-biosynthetic genes CHLI, POR, and CHLP and the upregulation of Chl-degrading genes CLH, PPH, and SGR were observed in both accessions under most of the stress periods. BrightStar SLT exhibited much lower expressions of the Chl-biosynthetic genes PBGD, CHS, and CHID under stress, while PI238938 had remarkably higher expressions of genes involved in Chl breakdown including CLH, PPH, PAO, RCCR, and SGR, and the expressions of these genes remained at a higher level at 1 d of recovery. The results indicated that submergence-induced leaf senescence and declines in Chl were associated with downregulation of more Chl-biosynthetic genes in slow-growing genotype and upregulation of more Chl-degrading genes in fast-growing genotype of perennial ryegrass.

Altered Promoter and G-Box Binding Factor for 1-Deoxy-d-Xylulose-5-Phosphate Synthase Gene Grown from Poa pratensis Seeds after Spaceflight.

In plant cells, the nucleus DNA is considered the primary site of injury by the space environment, which could generate genetic alteration. As the part of genomic mutation, genetic variation in the promoter region could regulate gene expression. In the study, it is observed that there is a deletion in the upstream regulatory region of the 1-deoxy-d-xylulose-5-phosphate synthase 1 gene (PpDXS1) of Poa pratensis dwarf mutant and the PpDXS1 transcript abundance is lower in the dwarf mutant. It is indicated that the deletion in the promoter region between wild type and dwarf mutant could be responsible for the regulation of PpDXS1 gene expression. The PpDXS1 promoter of dwarf mutant shows a lower activity as determined by dual luciferase assay in Poa pratensis protoplast, as well as the GUS activity is lower in transgenic Poa pratensis plant. To further investigate the effect of the deletion in the promoter region on PpDXS1 transcript accumulation, the transient assay and yeast one-hybrid experiment demonstrate that the deletion comprises a motif which is a target of G-box binding factor (GBF1), and the motif correlates with an increase in transactivation by GBF1 protein. Taken together, these results indicate that the deletion in the promoter of PpDXS1 isolated from dwarf mutant is sufficient to account for the decrease in PpDXS1 transcript level and GBF1 can regulate the PpDXS1 gene expression, and subsequently affect accumulation of various isoprenoids throughout the plant.

Correction: De Novo Transcriptome Analysis for Kentucky Bluegrass Dwarf Mutants Induced by Space Mutation.

[This corrects the article DOI: 10.1371/journal.pone.0151768.].

De Novo Transcriptome Analysis for Kentucky Bluegrass Dwarf Mutants Induced by Space Mutation.

Kentucky bluegrass (Poa pratensis L.) is a major cool-season turfgrass requiring frequent mowing. Utilization of cultivars with slow growth is a promising method to decrease mowing frequency. In this study, two dwarf mutant selections of Kentucky bluegrass (A12 and A16) induced by space mutation were analyzed for the differentially expressed genes compared with the wild type (WT) by the high-throughput RNA-Seq technology. 253,909 unigenes were obtained by de novo assembly. 24.20% of the unigenes had a significant level of amino acid sequence identity to Brachypodium distachyon proteins, followed by Hordeum vulgare with 18.72% among the non-redundant (NR) Blastx top hits. Assembled unigenes were associated with 32 pathways using KEGG orthology terms and their respective KEGG maps. Between WT and A16 libraries, 4,203 differentially expressed genes (DEGs) were identified, whereas there were 883 DEGs between WT and A12 libraries. Further investigation revealed that the DEG pathways were mainly involved in terpenoid biosynthesis and plant hormone metabolism, which might account for the differences of plant height and leaf blade color between dwarf mutant and WT plants. Our study presents the first comprehensive transcriptomic data and gene function analysis of Poa pratensis L., providing a valuable resource for future studies in plant dwarfing breeding and comparative genome analysis for Pooideae plants.

Corrigendum: De novo assembly of the Japanese lawngrass (Zoysia japonica Steud.) root transcriptome and identification of candidate unigenes related to early responses under salt stress.

[This corrects the article on p. 610 in vol. 6, PMID: 26347751.].

De novo assembly of the Japanese lawngrass (Zoysia japonica Steud.) root transcriptome and identification of candidate unigenes related to early responses under salt stress.

Japanese lawngrass (Zoysia japonica Steud.) is an important warm-season turfgrass that is able to survive in a range of soils, from infertile sands to clays, and to grow well under saline conditions. However, little is known about the molecular mechanisms involved in its resistance to salt stress. Here, we used high-throughput RNA sequencing (RNA-seq) to investigate the changes in gene expression of Zoysia grass at high NaCl concentrations. We first constructed two sequencing libraries, including control and NaCl-treated samples, and sequenced them using the Illumina HiSeq™ 2000 platform. Approximately 157.20 million paired-end reads with a total length of 68.68 Mb were obtained. Subsequently, 100,800 unigenes with an N50 length of 1104 bp were assembled using Trinity, among which 70,127 unigenes were functionally annotated (E ≤ 10(-5)) in the non-redundant protein (NR) database. Furthermore, three public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, and Clusters of Orthologous Groups (COGs), were used for gene function analysis and enrichment. The annotated genes included 46 Gene Ontology (GO) terms, 120 KEGG pathways, and 25 COGs. Compared with the control, 6035 genes were significantly different (false discovery rate ≤0.01, |log2Ratio|≥1) in the NaCl-treated samples. These genes were enriched in 10 KEGG pathways and 58 GO terms, and subjected to 25 COG categories. Using high-throughput next-generation sequencing, we built a database as a global transcript resource for Z. japonica Steud. roots. The results of this study will advance our understanding of the early salt response in Japanese lawngrass roots.

[Research progress of interaction between Toxoplasma gondii rhoptry proteins and host cells].

Rhoptry proteins are the major virulence factors of Toxoplasma gondii. They locate in different parts of the host cells, and can affect the membrane, cytoskeleton structure and active factors of the host cells, so as to block the cell intrinsic defense mechanisms of the host, and let T. gondii invade, parasitize and proliferate in the host successfully. The function and ac- tion mode of rhoptry proteins reflect the pathogenic mechanism of T. gondii, which holds great significance to looking for toxoplasmosis drug targets and developing molecule vaccines. This paper reviews the research progess of the interaction between rhoptry proteins of T. gondii and host cells.

First-principles investigations of Ti-substituted hydroxyapatite electronic structure.

The electronic structure of Ti-substituted hydroxyapatite is investigated using density functional theory within a periodic slab model. Two sorption mechanisms have been considered: i.e., Ti(4+) and Ti(OH)(2)(2+) as the likely species to exchange with Ca(2+). Ti(4+) has a small ionic radius compared to Ca(2+) and can dope into both distinct sites, showing no site preference; however, when two H were removed from the OH channel to obtain charge compensation, preferential site II substitution appears, accompanied with a large O shift forming a strong Ti-O bond. The species Ti(OH)(2)(2+) displays a strong site preference: substitution by Ti(OH)(2)(2+) on the hydroxyl channel (site II) is exothermic and favored strongly over the Ca column (site I). Ti(OH)(2)(2+) substitution for Ca(2+) induces a large geometry relaxation and distortion, especially within the OH channel and Ca(2+) column, with a considerable shift of Ti compared to the Ca sites in pure HA. These results are consistent with the experimental observation that material synthesis with high Ti doping (atomic ratio > 0.1) shows irregular particles formation with reduced crystallinity. The calculated cell shape and volume relaxations indicate that the volume and cell parameters both expand in all the substituted HA models. The site preference and volume expansion differences found are attributed to the metal ion shift caused in meeting the requirement of strong Ti-O coordination in site I and site II polyhedra.