Flavones are a major group of flavonoids with diverse functions and are extensively distributed in land plants. There are two different classes of FLAVONE SYNTHASE (FNS) enzymes that catalyze the conversion of the flavanones into flavones. The FNSI class comprises soluble Fe2+/2-oxoglutarate-dependent dioxygenases, and FNSII enzymes are oxygen- and NADPH-dependent cytochrome P450 membrane-bound monooxygenases. Here, we describe the identification and characterization of FNSI enzymes from maize (Zea mays) and Arabidopsis (Arabidopsis thaliana). In maize, ZmFNSI-1 is expressed at significantly higher levels in silks and pericarps expressing the 3-deoxy flavonoid R2R3-MYB regulator P1, suggesting that ZmFNSI-1 could be the main enzyme for the synthesis of flavone O-glycosides. We also show here that DOWNY MILDEW RESISTANT6 (AtDMR6), the Arabidopsis homologous enzyme to ZmFNSI-1, has FNSI activity. While dmr6 mutants show loss of susceptibility to Pseudomonas syringae, transgenic dmr6 plants expressing ZmFNSI-1 show similar susceptibility to wild-type plants, demonstrating that ZmFNSI-1 can complement the mutant phenotype. AtDMR6 expression analysis showed a tissue- and developmental stage-dependent pattern, with high expression in cauline and senescing leaves. Finally, we show that Arabidopsis cauline and senescing leaves accumulate apigenin, demonstrating that Arabidopsis plants have an FNSI activity involved in the biosynthesis of flavones. The results presented here also suggest cross talk between the flavone and salicylic acid pathways in Arabidopsis; in this way, pathogens would induce flavones to decrease salicylic acid and, hence, increase susceptibility.
This week’s Research in Focus is reprinted from an In Brief published by Science Editor Nancy Hoffman in The Plant Cell, which summarizes an article newly published by Gerttula et al. This study examines the formation of tension wood, a specialized tissue that forms on the upper side of a fallen woody angiosperm stem that exerts force to vertically reorient the stem. The authors develop a model that leads from gravity perception through hormonal and transcriptional responses to the anatomical changes associated with this unusual and important type of wood.
This study was aimed at investigating the toxicological effects of GO on beneficial Bacillus soil microbes. Five bacterial isolates screened from the rhizosphere of a common pulse-growing agricultural field were identified as Bacillus marisflavi, Bacillus cereus, Bacillus subtilis, Bacillus megaterium, and Bacillus mycoides. To study the effect of GO under in vitro conditions, GO was prepared and characterized by various analytical techniques. Our results suggest that GO decreases cell viability in a concentration- and time-dependent manner by regulating biochemical changes and demonstrate that GO nanoparticles can negatively impact beneficial bacterial communities in the soil.
After performing de novo transcript assembly of >1 billion RNA-Sequencing reads obtained from 22 samples of different Norway spruce (Picea abies) tissues that were not surface sterilized, we found that assembled sequences captured a mix of plant, lichen, and fungal transcripts. The latter were likely expressed by endophytic and epiphytic symbionts, indicating that these organisms were present, alive, and metabolically active. Here, we show that these serendipitously sequenced transcripts need not be considered merely as contamination, as is common, but that they provide insight into the plant’s phyllosphere. Notably, we could classify these transcripts as originating predominantly from Dothideomycetes and Leotiomycetes species, with functional annotation of gene families indicating active growth and metabolism, with particular regards to glucose intake and processing, as well as gene regulation.
Quantitative trait loci (QTL) play important roles in controlling rice blast disease. In the present study, 10 field isolates of the races IA1, IB1, IB17, and IC1 of US rice blast fungusMagnaporthe oryzae collected in 1996 and 2009 were used to identify blast resistance QTL with a recombinant inbred line (RIL) population consisting of 227 F7 individuals derived from the cross of rice (Oryza sativa L.) cultivars Lemont and Jasmine 85. Jasmine 85 is an indicacultivar that is moderately resistant, and Lemont is a tropical japonica cultivar susceptible to rice blast in greenhouse inoculation. Disease reactions of the parents and RILs were evaluated under greenhouse conditions. A total of six resistance QTL, qBLR8, qBLR10-1,qBLR10-2, qBLR10-3, qBLR12-1, and qBLR12-2, were identified on chromosomes 8, 10, and 12, respectively. Phenotypic variation, conditioned by these six resistance QTL, ranged from 5.37 to 39.18%. Among them, qBLR12-1 and qBLR12-2 provided the strongest resistance to the newest isolates of the most virulent race IA1 of M. oryzae. Three of these resistance QTL have been identified using different blast isolates in a previous study.qBLR10-1, qBLR10-2, and qBLR10-3 have not been previously found in this cross. These confirmed and new resistance QTL will be useful for the development of rice cultivars with improved effective resistance to rice blast via a marker-assisted selection (MAS) approach.
Next-generation sequencing technologies allow an almost exhaustive survey of the transcriptome, even in species with no available genome sequence. To produce a Unigene set representing most of the expressed genes of pea, 20 cDNA libraries produced from various plant tissues harvested at various developmental stages from plants grown under contrasting nitrogen conditions were sequenced. Around one billion reads and 100 Gb of sequence were de novo assembled. Following several steps of redundancy reduction, 46 099 contigs with N50 length of 1667 nt were identified. These constitute the ‘Caméor’ Unigene set. The high depth of sequencing allowed identification of rare transcripts and detected expression for approximately 80% of contigs in each library. The Unigene set is now available online (http://bios.dijon.inra.fr/FATAL/cgi/pscam.cgi), allowing (i) searches for pea orthologs of candidate genes based on gene sequences from other species, or based on annotation, (ii) determination of transcript expression patterns using various metrics, (iii) identification of uncharacterized genes with interesting patterns of expression, and (iv) comparison of gene ontology pathways between tissues. This resource has allowed identification of the pea orthologs of major nodulation genes characterized in recent years in model species, as a major step towards deciphering unresolved pea nodulation phenotypes. In addition to a remarkable conservation of the early transcriptome nodulation apparatus between pea and Medicago truncatula, some specific features were highlighted. The resource provides a reference for the pea exome, and will facilitate transcriptome and proteome approaches as well as SNP discovery in pea.
Transcription Activator-Like (TAL) effectors are type III-delivered transcription factors that enhance virulence of plant pathogenic Xanthomonas species through the activation of host susceptibility (S) genes. TAL effectors recognize their DNA target(s) via a partially degenerate code whereby modular repeats in the TAL effector bind to nucleotide sequences in the host promoter. While this knowledge has greatly facilitated our power to identify new (S) genes, it can also be easily used to screen plant genomes for variations in TAL effectors target sequences and predict for loss-of-function gene candidates in silico. In a proof-of-principle experiment, we screened a germplasm of 169 rice accessions for polymorphism in the promoter of the major bacterial blight susceptibility (S) gene OsSWEET14 which encodes a sugar transporter targeted by numerous strains of X. oryzae pv. oryzae. We identified a single allele with a deletion of 18-bp overlapping with the binding sites targeted by several TAL effectors known to activate the gene. We show that this allele, which we call xa41(t), confers resistance against half of the tested Xoo strains representative of various geographic origins and genetic lineages, highlighting the selective pressure for the pathogen to accommodate with OsSWEET14 polymorphism and reciprocally the apparent limited possibilities for the host to create variability at this particular S gene. Analysis of xa41(t) conservation across the Oryza genus enabled to raise scenarios as to its evolutionary history prior and during domestication. Our findings demonstrate that resistance through TAL effector-dependent loss of S genes expression can be greatly fostered upon knowledge-based molecular screening of large collection of host plants.
Mathilde Hutin, François Sabot, Alain Ghesquière, Ralf Koebnik and Boris Szurek
Receptor-like cytoplasmic kinases (RLCKs) are a subset of plant receptor-like kinases lacking both extracellular and transmembrane domains. Some of the 46 members in the Arabidopsis RLCK subfamily VII have been linked to plant innate immunity, however most remain uncharacterized. Thus, multiple subfamily VII members are expected to be involved in plant immune signaling. Here, we investigate the role of PBL13, a subfamily VII RLCK with unique domain architecture. Unlike other characterized RLCKs, PBL13 T-DNA insertion lines exhibit enhanced disease resistance after inoculation with virulent Pseudomonas syringae. The pbl13-2 knockout also exhibits elevated basal level expression of the PR1 defense marker gene, enhanced ROS burst in response to perception of bacterial microbial patterns, and accelerated flagellin-induced activation of MAP kinases. Recombinant PBL13 is an active kinase and its primary autophosphorylated sites map to a 15 amino acid repeat motif unique to PBL13. Complementation of pbl13-2 with PBL13-3xFLAG converts the enhanced resistance and elevated ROS phenotypes back to wild type levels. In contrast, kinase dead PBL13K111A-3xFLAG was unable to rescue pbl13-2 disease phenotypes. Consistent with the enhanced ROS burst in the pbl13-2 knockout, PBL13 is able to associate with the NADPH oxidase RBOHD by split-luciferase complementation assay, and this association is disrupted by flagellin treatment. We conclude that the PBL13 kinase negatively regulates plant innate immunity to pathogenic bacteria and can associate with RBOHD prior to pathogen perception. These data are consistent with the hypothesis that PBL13 acts to prevent inappropriate activation of defense responses in the absence of pathogen challenge.
N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour.
Programmed cell death (PCD) is crucial in plant organogenesis and survival. In this review the involvement of mitochondria and chloroplasts in PCD execution is critically assessed. Recent findings support a central role for mitochondria in PCD, with newly identified components of the mitochondrial electron transport chain (mETC), FOF1 ATP synthase, cardiolipins, and ATPase AtOM66. While chloroplasts received less attention, their contribution to PCD is well supported, suggesting that they possibly contribute by producing reactive oxygen species (ROS) in the presence of light or even contribute through cytochrome f release. Finally we discuss two working models where mitochondria and chloroplasts could cooperatively execute PCD: mitochondria initiate the commitment steps and recruit chloroplasts for swift execution or, alternatively, mitochondria and chloroplasts could operate in parallel. Trends
Mitochondrial involvement in plant PCD is only partly similar to animal PCD systems. The presence of chloroplasts provides plants with unique possibilities to remove unwanted cells, especially in the light. Alternative models of how mitochondria and chloroplasts may cooperate during plant PCD are proposed.
ROS appear to be a common thread in many plant PCD systems but can originate from various subcellular sources, including mitochondria, chloroplasts, and plasma membrane NADPH oxidases. Their relative contributions remain unclear.
Energy-organelle membrane permeabilization is likely to play a role in plant PCD execution, but how this is achieved remains controversial.
Small molecules and metabolites such as ATP, Ca2+, and NO are likely to affect plant PCD initiation and execution.
Diphenylamine derivatives have been reported with good fungicidal, insecticidal, acaricidal, rodenticidal and/or herbicidal activities. To find new lead compound of this kind, a series of novel diphenylamine derivatives were designed and synthesized by the approach of Intermediate Derivatization Methods. All compounds were identified by 1H NMR and elemental analysis. Bioassays demonstrated that some compounds substituted at 2,4,6-positions or 2,4,5-positions of phenyl ring B exhibited excellent fungicidal activities. The optimal compounds P30 and P33 showed 80% and 85% control respectively against cucumber downy mildew at 12.5 mg L−1, both 100% control against rice blast at 0.3 mg L−1 and both 100% control against cucumber gray mold at 0.9 mg L−1. The relationship between structure and fungicidal activities was discussed as well.
Publication date: December 2015 Source:Plant Physiology and Biochemistry, Volume 97 Author(s): Nikola Ptáčková, Jitka Klempová, Michal Obořil, Sylvie Nedělová, Jan Lochman, Tomáš Kašparovský Cryptogein, a protein from oomycete...
Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B1 (FB1). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB1, whereas LOH1-overexpressing plants showed no increase in FB1 resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB1. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance for improved plant performance.
Magnaporthaceae is a family of ascomycetes that includes three fungi of great economic importance: Magnaporthe oryzae, Gaeumannomyces graminis var. tritici, and Magnaporthe poae. These three fungi cause widespread disease and loss in cereal and grass crops including rice blast disease (M. oryzae), take-all disease in wheat and other grasses (G. graminis), and summer patch disease in turf grasses (M. poae). Here, we present the finished genome sequence for M. oryzae and draft sequences for M. poae and G. graminis var. tritici. We used multiple technologies to sequence and annotate the genomes of M. oryzae, M. poae, and G. graminis var. tritici. The M. oryzae genome is now finished to 7 chromosomes while M. poae and G. graminis var. tritici are sequenced to 40.0X and 25.0X coverage respectively. Gene models were developed using multiple computational techniques and further supported by RNAseq data. In addition, we performed preliminary analysis of genome architecture and repetitive element DNA.
Understanding the genetic basis of pathogen susceptibility in various crop plants is crucial to increasing the stability of food, feed, and fuel production. Varietal differences in defence responses provide insights into the mechanisms of resistance and are a key resource for plant breeders. To explore the role of salicylic acid in the regulation of defence in cacao, we demonstrated that SA treatment decreased susceptibility to a pod rot pathogen, Phytophthora tropicalis in two genotypes, Scavina 6 and Imperial College Selection 1, which differ in their resistance to several agriculturally important pathogens. Transient overexpression of TcNPR1, a major transcriptional regulator of the SA-dependent plant immune system, also increased pathogen tolerance in cacao leaves. To explore further the genetic basis of resistance in cacao, we used microarrays to measure gene expression profiles after salicylic acid (SA) treatment in these two cacao genotypes. The two genotypes displayed distinct transcriptional responses to SA. Unexpectedly, the expression profile of the susceptible genotype ICS1 included a larger number of pathogenesis-related genes that were induced by SA at 24h after treatment, whereas genes encoding many chloroplast and mitochondrial proteins implicated in reactive oxygen species production were up-regulated in the resistant genotype, Sca6. Sca6 accumulated significantly more superoxide at 24h after treatment of leaves with SA. These experiments revealed critical insights regarding the molecular differences between cacao varieties, which will allow a better understanding of defence mechanisms to help guide breeding programmes.
Healthy soils are the foundation of the food system. Our soils are the basis for agriculture and the medium in which nearly all food-producing plants grow. Healthy soils produce healthy crops that in turn nourish people and animals. Indeed, soil quality is directly linked to food quality and quantity.
With a global population that is projected to exceed 9 billion by 2050, compounded by competition for land and water resources and the impact of climate change, our current and future food security hinges on our ability to increase yields and food quality using the soils that are already under production today.
Photosynthetic picoeukaryotes (PPEs) are important components of the marine picophytoplankton community playing a critical role in CO2 fixation but also as bacterivores, particularly in the oligotrophic gyres. Despite an increased interest in these organisms and an improved understanding of the genetic diversity of this group we still know little of the environmental factors controlling the abundance of these organisms. Here, we investigated the quantitative importance of eukaryotic parasites in the free-living fraction as well as in associations with PPEs along a transect in the South Atlantic. Using TSA-FISH (Tyramide Signal Amplification - Fluorescence in situ hybridization) we provide quantitative evidence of the occurrence of free-living fungi in open ocean marine systems, while Perkinsozoa and Syndiniales parasites were not abundant in these waters. Using flow cytometric cell sorting of different PPE populations followed by a dual-labelled TSA-FISH approach we also demonstrate fungal associations, potentially parasitic, occurring with both pico-Prymnesiophyceae and pico-Chrysophyceae. These data highlights the necessity for further work investigating the specific role of marine fungi as parasites of phytoplankton to improve understanding of carbon flow in marine ecosystems.
The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.
Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances.
Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified CTP1 (chloroplast-targeted protein 1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in a N-terminal signal-dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells.
Even though vast amounts of genome-wide gene expression data have become available in plants, it remains a challenge to effectively mine this information for the discovery of genes and gene networks, for instance those that control agronomically important traits. These networks reflect potential interactions among genes and, therefore, can lead to a systematic understanding of the molecular mechanisms underlying targeted biological processes. We discuss methods to analyze gene networks using gene expression data, specifically focusing on four common statistical approaches used to reconstruct networks: correlation, feature selection in supervised learning, probabilistic graphical model, and meta-prediction. In addition, we discuss the effective use of these methods for acquiring an in-depth understanding of biological systems in plants. Trends
Gene networks are valuable for gene function discovery and candidate gene prioritization.
Statistical methods for gene network reconstruction using transcriptome data have been vastly improved.
Methods to evaluate the quality of a constructed network are available and improve the ability of the user to use the networks to inform research.
Meta-prediction methods that combine multiple statistical models are generally more robust and accurate for gene network reconstruction.
The use of gene networks to functionally annotate genes is a boon for genomes for which little functional information exists.
Volatile compounds and extrafloral nectar are common defenses of wild plants; however, in crops they bear an as-yet underused potential for biological control of pests and diseases. Odor emission and nectar secretion are multigene traits in wild plants, and thus form difficult targets for breeding. Furthermore, domestication has changed the capacity of crops to express these traits. We propose that breeding crops for an enhanced capacity for tritrophic interactions and volatile-mediated direct resistance to herbivores and pathogens can contribute to environmentally-friendly and sustainable agriculture. Natural plant volatiles with antifungal or repellent properties can serve as direct resistance agents. In addition, volatiles mediating tritrophic interactions can be combined with nectar-based food rewards for carnivores to boost indirect plant defense. Trends
Hundreds of studies convincingly demonstrate functioning indirect defenses in wild plants, but breeding approaches have never considered the underlying traits (e.g., food rewards or shelter for carnivores, and volatiles that mediate information-based interactions) as desirable targets.
We argue that induced plant volatiles, owing to their multiple roles as signals, repellents, and antimicrobial compounds, bear an as-yet underused potential for biological control, and that future breeding efforts should enhance the capacity of crops to engage in tritrophic interactions.
We also present ecological and evolutionary considerations that can explain why the constitutive release of volatile compounds that have evolved as inducible defenses is not likely to work, and why extrafloral nectar is likely to represent a better food reward for carnivores than floral nectar.
In this study, we have used untargeted global metabolomic analysis to determine and compare the chemical nature of the metabolites altered during the infection of tomato plants (cv. Ailsa Craig) with Botrytis cinerea (Bot) or Pseudomonas syringae pv. tomato DC3000 (Pst), pathogens that have different invasion mechanisms and lifestyles. We also obtained the metabolome of tomato plants primed using the natural resistance inducer hexanoic acid and then infected with these pathogens. By contrasting the metabolomic profiles of infected, primed, and primed + infected plants, we determined not only the processes or components related directly to plant defense responses, but also inferred the metabolic mechanisms by which pathogen resistance is primed. The data show that basal resistance and hexanoic acid-induced resistance to Bot and Pst are associated with a marked metabolic reprogramming. This includes significant changes in amino acids, sugars and free fatty acids, and in primary and secondary metabolism. Comparison of the metabolic profiles of the infections indicated clear differences, reflecting the fact that the plant's chemical responses are highly adapted to specific attackers. The data also indicate involvement of signaling molecules, including pipecolic and azelaic acids, in response to Pst and, interestingly, to Bot. The compound 1-methyltryptophan was shown to be associated with the tomato–Pst and tomato–Bot interactions as well as with hexanoic acid-induced resistance. Root application of this Trp-derived metabolite also demonstrated its ability to protect tomato plants against both pathogens
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