Academic literature on the topic 'Molecular biology|Plant pathology|Biochemistry'

Abstract Basidiomycetes fungi of the family Ustilaginaceae are mainly known as plant pathogens causing smut disease on crops and grasses. However, they are also natural producers of value-added substances like glycolipids, organic acids, polyols, and harbor secretory enzymes with promising hydrolytic activities. These attributes recently evoked increasing interest in their biotechnological exploitation. The corn smut fungus Ustilago maydis is the best characterized member of the Ustilaginaceae. After decades of research in the fields of genetics and plant pathology, a broad method portfolio and detailed knowledge on its biology and biochemistry are available. As a consequence, U. maydis has developed into a versatile model organism not only for fundamental research but also for applied biotechnology. Novel genetic, synthetic biology, and process development approaches have been implemented to engineer yields and product specificity as well as for the expansion of the repertoire of produced substances. Furthermore, research on U. maydis also substantially promoted the interest in other members of the Ustilaginaceae, for which the available tools can be adapted. Here, we review the latest developments in applied research on Ustilaginaceae towards their establishment as future biotech cell factories.

Early in the development of molecular biology, TMV RNA was widely used as a mitochondrial RNA that could be purified easily, and it contributed much to research on protein synthesis. Also, in the early stages of elucidation of the genetic code, artificially produced TMV mutants were widely used and provided the first proof that the genetic code was non–overlapping. In 1982, Goelet et al. determined the complete TMV RNA base sequence of 6395 nucleotides. The four genes (130K, 180K, 30K and coat protein) could then be mapped at precise locations in the TMV genome. Furthermore it had become clear, a little earlier, that genes located internally in the genome were expressed via subgenomic mRNAs. The initiation site for assembly of TMV particles was also determined. However, although TMV contributed so much at the beginning of the development of molecular biology, its influence was replaced by that of Escherichia coli and its phages in the next phase. As recombinant DNA technology developed in the 1980s, RNA virus research became more detached from the frontier of molecular biology. To recover from this setback, a gene–manipulation system was needed for RNA viruses. In 1986, two such systems were developed for TMV, using full–length cDNA clones, by Dawson's group and by Okada's group. Thus, reverse genetics could be used to elucidate the basic functions of all proteins encoded by the TMV genome. Identification of the function of the 30K protein was especially important because it was the first evidence that a plant virus possesses a cell–to–cell movement function. Many other plant viruses have since been found to encode comparable ‘movement proteins’. TMV thus became the first plant virus for which structures and functions were known for all its genes. At the birth of molecular plant pathology, TMV became a leader again. TMV has also played pioneering roles in many other fields. TMV was the first virus for which the amino acid sequence of the coat protein was determined and first virus for which cotranslational disassembly was demonstrated both in vivo and in vitro . It was the first virus for which activation of a resistance gene in a host plant was related to the molecular specificity of a product of a viral gene. Also, in the field of plant biotechnology, TMV vectors are among the most promising. Thus, for the 100 years since Beijerinck's work, TMV research has consistently played a leading role in opening up new areas of study, not only in plant pathology, but also in virology, biochemistry, molecular biology, RNA genetics and biotechnology.

Plants have evolved complex tolerance systems to survive abiotic and biotic stresses. Central to these programmes is a sophisticated conversation of signals between the chloroplast and the nucleus. In this review, we examine the antagonism between abiotic stress tolerance (AST) and immunity: we propose that to generate immunogenic signals, plants must disable AST systems, in particular those that manage reactive oxygen species (ROS), while the pathogen seeks to reactivate or enhance those systems to achieve virulence. By boosting host systems of AST, pathogens trick the plant into suppressing chloroplast immunogenic signals and steer the host into making an inappropriate immune response. Pathogens disrupt chloroplast function, both transcriptionally—by secreting effectors that alter host gene expression by interacting with defence-related kinase cascades, with transcription factors, or with promoters themselves—and post-transcriptionally, by delivering effectors that enter the chloroplast or alter the localization of host proteins to change chloroplast activities. These mechanisms reconfigure the chloroplast proteome and chloroplast-originating immunogenic signals in order to promote infection.

<p> The emergence and spread of <i>Verticillium</i> wilt were observed in cotton plants at the R.R. Foil Plant Science Research Center at Mississippi State during the late summer in 2013 and 2014. Several fungi with different morphology and growth characteristics were isolated from diseased cotton plants. Genomic DNA was extracted from the isolated fungal species and used in molecular typing via PCR amplification and DNA sequencing analysis of the ribosomal internal transcribed spacer (ITS) region. A total of five fungal genera were identified, and <i>Verticillium</i> sp. was the most frequently isolated genus. The isolated <i>Verticillium</i> strains could be <i>Verticillium dahliae, Verticillium longisporum</i> or even <i>Verticillium albo-atrum.</i> A PCR-based genotyping method using <i>VTA2</i> (<i>Verticillium</i> transcription activator) gene specific primers confirmed that the isolated <i>Verticillium</i> strain was <i>Verticillium dahliae,</i> and it caused <i>Verticillium </i> wilt in Mississippi cotton plants. Pathogenicity tests (Koch&rsquo;s postulates) confirmed the earlier qualitative identifications of <i> Verticillium dahliae</i> in the greenhouse.</p>

<p> Understanding the role of phytohormone auxin in defense responses is one of the vital tools for plant breeders to develop maize germplasm lines that exhibit high resistance to <i>Aspergillus flavus</i> and subsequent aflatoxin accumulation. Besides its critical role in different developmental processes throughout the life cycle of plants, auxin is also involved in the network of plant-pathogen interaction as demonstrated in previous studies. However, the actual mechanism for the auxin signaling pathway leading to resistance is unknown. Therefore, the critical gap in the knowledge base is a lack of understanding of the role of auxin signaling in pathogen resistance in maize. Continuation of this gap is an important problem because fungal resistance is a highly quantitative trait and breeding for resistance is a challenge. A complete understanding of the auxin mechanism in resistance could lead the production of corn hybrids with resistance to <i>A. flavus</i> and aflatoxin accumulation. </p><p> The focus of this research was to determine the effect of exogenous auxin on <i>A. flavus</i> growth and production of aflatoxin in growth media. In addition, auxin levels, the amount of aflatoxin, and fungal growth in three resistant (Mp313E, Mp715, and Mp719) and one susceptible (B73) germplasm line were determined. As a result, auxin significantly increased mycelium growth and significantly decreased aflatoxin at a high concentration in potato dextrose broth under the lab conditions. Under the field conditions, auxin levels were low in resistant lines but did not change in response to <i> A. flavus</i> infection. Susceptible line had high auxin levels and auxin levels significantly decreased in response to <i>A. flavus</i> infection. </p>

Subcellular Localization of Tobacco SABP2 under Normal and Stress Conditions Salicylic acid (SA), a phytohormone, plays an important role in plant physiology. SA mediated innate immune pathway is an important pathway for plant immunity against pathogens. Plants resisting pathogen infection synthesize higher levels of Methyl Salicylate (MeSA), which is then converted to SA by the esterase activity of Salicylic Acid Binding Protein 2 (SABP2). The high level of the converted SA leads to enhanced pathogen resistance. The study of subcellular localization of a protein is critical in explaining its potential biochemical functions. SABP2 tagged with eGFP was expressed transiently in Nicotiana benthamiana leaves. The SABP2-eGFP expressing leaves were challenged with bacterial and viral pathogens and observed under confocal microscopy. Fluorescent signals were seen throughout the cell and more concentrated towards the cell periphery. To verify the localization, mCherry fluorescent organelle markers with specific targeting sequences were used. The results indicate that the SABP2 is likely a cytoplasmic protein, and there is no change in its localization upon infection by plant pathogens.

SIP470, a putative tobacco lipid transfer protein, was identified in a yeast two-hybrid screen to interact with SABP2. SABP2 is a critical role in SA-mediated signaling in tobacco and other plants. In vitro studies using purified recombinant SIP470 confirmed that it is a lipid binding protein. In an attempt to determine its role in mediating stress responses, Arabidopsis T-DNA insertion knockout lines lacking SIP470 homolog were used for the analysis. These mutant plants were defective in basal resistance against microbial pathogens. Expression of defense gene PR-1 was also delayed in these mutant plants. Interestingly, these mutant plants were not defective in inducing systemic acquired resistance. Besides biotic stress, these mutant plants also showed increased susceptibility to abiotic stresses. To directly study the role of SIP470 in tobacco plants, transgenic tobacco lines, with reduced levels of SIP470 expression, were generated using RNAi and transgenic lines overexpressing SIP470 were also generated.

The intimate relationship between the higher-order chromatin organisation and the regulation of gene expression is increasingly attracting attention in the scientific community. Thanks to high-resolution microscopy, genome-wide molecular biology tools (3C, ChIP-on-chip), and bioinformatics, detailed structures of chromatin loops, territories, and nuclear domains are gradually emerging. However, to fully reveal a comprehensive map of nuclear organisation, some fundamental questions remain to be answered in order to fit all the pieces of the jigsaw together. The underlying mechanisms, precisely organising the interaction of the different parts of chromatin need to be understood. Previous work in our lab hypothesised and verified the “transcription factory” model for the organisation of mammalian genomes. It is widely assumed that active polymerases track along their templates as they make RNA. However, after allowing engaged polymerases to extend their transcripts in tagged precursors (e.g., Br-U or Br-UTP), and immunolabelling the now-tagged nascent RNA, active transcription units are found to be clustered in nuclei, in small and numerous sites we call “transcription factories”. Previous work suggested the transcription machinery acts both as an enzyme as well as a molecular tie that maintains chromatin loops, and the different classes of polymerases are concentrated in their own dedicated factories. This thesis aims to further characterise transcription factories. Different genes are transcribed by different classes of RNA polymerase (i.e., I, II, or III), and the resulting transcripts are processed differently (e.g., some are capped, others spliced). Do factories specialise in transcribing particular subsets of genes? This thesis developed a method using replicating minichromosomes as probes to examine whether transcription occurs in factories, and whether factories specialise in transcribing particular sets of genes. Plasmids encoding the SV40 origin of replication are transfected into COS-7 cells, where they are assembled into minichromosomes. Using RNA fluorescence in situ hybridisation (FISH), sites where minichromosomes are transcribed are visualised as discrete foci, which specialise in transcribing different groups of genes. Polymerases I, II, and III units have their own dedicated factories, and different polymerase II promoters and the presence of an intron determine the nuclear location of transcription. Using chromosome conformation capture (3C), minichromosomes with similar promoters are found in close proximity. They are also found close to similar endogenous promoters and so are likely to share factories with them. In the second part of this thesis, I used RNA FISH to confirm results obtained by tiling microarrays. Addition of tumour necrosis factor alpha (TNF alpha) to human umbilical vein endothelial cells induces an inflammatory response and the transcription of a selected sub-set of genes. My collaborators used tiling arrays to demonstrate a wave of transcription that swept along selected long genes on stimulation. RNA FISH confirmed these results, and that long introns are co-transcriptionally spliced. Results are consistent with one polymerase being engaged on an allele at any time, and with a major checkpoint that regulates polymerase escape from the first few thousand nucleotides into the long gene.