Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic ...translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5'-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.
Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.
Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism. We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were ...identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3, 15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.
Clusters of functionally related genes are a general feature of prokaryotic gene organization but are much less prevalent in eukaryotes. The discovery that genes for certain types of metabolic ...pathways are clustered in filamentous fungi is relatively recent. Only 8 years ago in a discussion of the proline utilization pathway genes, Hull et al. (1989) commented that "The organization and regulation of the genes involved in L-proline catabolism in the ascomycete fungus Aspergillus nidulans are of particular interest because, rather unusually for functionally related eukaryotic genes, they are all clustered." This view was supported by studies of metabolic pathway gene linkage relationships from a variety of eukaryotes. Although genetic evidence was available suggesting that some metabolic pathway genes were closely linked in A. nidulans, recognition of gene clustering as an important feature of fungal metabolic pathways had to await the molecular characterization of specific pathways. Fungal gene clusters can be broadly defined as the close linkage of two or more genes that participate in a common metabolic or developmental pathway. Fungi possess numerous pathways for what can be described as "dispensable" metabolic functions, and research over the last 5 years has shown that the genes for these dispensable pathways are often organized in gene clusters. The term dispensable metabolic pathways is used here to describe pathways that either are not required for growth or are only required for growth under a limited range of conditions. Dispensable metabolic pathways are typically expressed under suboptimal growth conditions and most likely function to enhance fungal survival in response to nutrient deprivation or competing organisms. In this review, two major types of dispensable metabolic pathways will be discussed, catabolic pathways for the utilization of low-molecular-weight nutrients such as proline or quinate (nutrient utilization pathways) and biosynthetic pathways for low-molecular-weight compounds which include antibiotics and mycotoxins (natural product pathways). Nutrient utilization pathways increase the metabolic versatility of filamentous fungi, enabling them to utilize a variety of complex compounds as alternative sources of nutrients. Expression of the appropriate catabolic pathways can be critically important for survival under limiting nutrient growth conditions. However, many of these nutrients are not commonly encountered by fungi, so there is little benefit derived from expressing pathway genes constitutively. To ensure that the required catabolic pathways are expressed appropriately in response to changing nutritional conditions while simultaneously limiting the loss of cellular resources due to unnecessary pathway gene expression, fungi have developed complex regulatory systems.
Several species of the genus Fusarium and related fungi produce trichothecenes which are sesquiterpenoid epoxides that act as potent inhibitors of eukaryotic protein synthesis. Interest in the ...trichothecenes is due primarily to their widespread contamination of agricultural commodities and their adverse effects on human and animal health. In this review, we describe the trichothecene biosynthetic pathway in Fusarium species and discuss genetic evidence that several trichothecene biosynthetic genes are organized in a gene cluster. Trichothecenes are highly toxic to a wide range of eukaryotes, but their specific function, if any, in the survival of the fungi that produce them is not obvious. Trichothecene gene disruption experiments indicate that production of trichothecenes can enhance the severity of disease caused by Fusarium species on some plant hosts. Understanding the regulation and function of trichothecene biosynthesis may aid in development of new strategies for controlling their production in food and feed products.
The internal ribosome entry sites (IRES), IRESCP, 148CR and IRESMP,75CR, precede the coat protein (CP) and movement protein (MP) genes of crucifer-infecting tobamovirus (crTMV), respectively. In the ...present work, we analyzed the activity of these elements in transgenic plants and other organisms. Comparison of the relative activities of the crTMV IRES elements and the IRES from an animal virus-encephalomyocarditis virus-in plant, yeast, and HeLa cells identified the 148-nt IRESCP,148CR as the strongest element that also displayed IRES activity across all kingdoms. Deletion analysis suggested that the polypurine (A)-rich sequences (PARSs) contained in IRESCP,148CR are responsible for these features. On the basis of those findings, we designed artificial PARS-containing elements and showed that they, too, promote internal translation from dicistronic transcripts in vitro, in tobacco protoplasts and in HeLa cells. The maximum IRES activity was obtained from multiple copies of either (A)4G(A)2(G)2 or G(A)2-5 as contained in IRESCP, 148CR. Remarkably, even homopolymeric poly(A) was moderately active, whereas a poly(G) homopolymer was not active. Furthermore, a database search for existing PARS sequences in 5′-untranslated regions (5′UTR) of genes in tobacco genome allowed the easy identification of a number of IRES candidates, in particular in the 5′UTR of the gene encoding Nicotiana tabacum heat-shock factor 1 (NtHSF1). Consistent with our prediction, the 5′UTR of NtHSF1 turned out to be an IRES element active in vitro, in plant protoplasts and HeLa cells. We predict that PARS elements, when found in other mRNAs, will show a similar activity.
The use of Unmanned Aerial Vehicles (UAVs) to monitor large colonies of seabirds avoids challenges associated with conventional methods, but manual image processing is expensive. Development of ...semi-automated analytical methods rely on high image spatial resolution, which requires a trade-off between securing low area coverage and high spatial resolution flying at low altitude vs high area coverage but low spatial resolution flying at higher altitudes. Increasing individual bird detection probabilities requires maximizing contrast between target and background, which can be enhanced using thermal sensors. We applied a semi-automatic analytical method to multispectral UAV derived imagery to count a mixed breeding colony of Herring Gulls (Larus argentatus) and Lesser Blackbacked Gulls (L. fuscus). We trained the computer to detect different image classes by their spectral signature in several orthomosaics obtained from UAV flights at different altitudes using different cameras. Highest agreement with the manual counts was achieved by low flying (20 m) using the highest camera resolution (97.7 ± 1.1% for the Herring Gulls, omission error 2.6%, commission error 0.5%; 94.8 ± 1.8% for Lesser Black-backed Gulls, omission error 6.5%, commission error 1.6%). Method precision varied between trials, confirming the importance of low altitude flying with high quality cameras, and a 40% reduction in detection noise from adding a thermal sensor.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Trichothecene biosynthetic pathway genes are localized within a gene cluster in Fusarium sporotrichioides and require the zinc-finger containing protein, TRI6, for expression. We show here that TRI6 ...is able to bind within the promoter regions of nine different pathway genes and that TRI6 binding is involved in pathway gene activation. TRI6 binding occurs at three distinct sites in the TRI5 promoter, all of which contain the sequence TNAGGCCT. DNA fragments from the promoter regions of six other pathway genes containing this sequence are also substrates for TRI6 binding. Specific nucleotide changes in the TNAGGCCT sequence dramatically reduced TRI6 binding. Analysis of TRI6 binding within the TRI3 and TRI11 promoters and the TRI4–TRI6 intergenic region which do not contain the TNAGGCCT motif suggests that the minimum sequence required for TRI6 binding is YNAGGCC. Two potential TRI6 binding sites, T4A and T4B, were identified within the intergenic region for the divergently transcribed TRI4 and TRI6 genes. Alteration or deletion of the T4A site resulted in the loss of nearly all in vitro TRI6 binding and was correlated with the loss of promoter activity in vivo as measured by the expression of mutant TRI4p/GUS fusions. This establishes a physiological role for TRI6 binding and demonstrates that TRI6 is directly involved in the regulation of pathway gene expression. To determine if a predicted Cys2His2 zinc-finger motif at the C-terminus of TRI6 is involved in DNA binding, a C187A mutant was constructed in TRI6 using site-directed mutagenesis. The C187A mutant did not bind promoter DNA fragments, supporting the role of C187 in DNA binding. In addition, a TRI6 homologue in the distantly related macrocyclic trichothecene pathway of Myrothecium roridum (MRTRI6) was also shown to bind to the same TRI5 and TRI4 promoter fragments bound by TRI6. Together, these data confirm our previous proposal that TRI6 is an activator of trichothecene pathway gene expression and that DNA binding employs the C-terminal region of TRI6 containing three predicted Cys2His2 zinc fingers.
Application-Aware Networking is a promising approach to provide good application quality to users in scenarios with limited network resources, like today's access networks. With SDN, a particularly ...interesting method to enable flowbased traffic management in networks has become available. In this work we take a look at how a specific application, i.e., YouTube Streaming, can benefit from such an SDN-based Application-Aware Network. We implement and investigate an approach based on Deep Packet Inspection (DPI) and one based on direct information input from the application in an OpenFlow testbed in order to show, how these different types of application information can be exploited to enhance the Quality of Experience (QoE). Furthermore, we determine the overhead caused by each of the presented approaches.
In plant pararetroviruses, pregenomic RNA serves both as a template for replication through reverse transcription and a polysictronic mRNA. This RNA has a complex leader sequence preceding the first ...large ORF. The leader contains multiple short ORFs and strong secondary structure, both inhibiting ribosome scanning. Translation on this RNA is initiated by shunting, in which scanning ribosomes bypass a large portion of the leader with the inhibitory secondary structure and short ORFs. In Cauliflower mosaic virus (CaMV), the ribosome shunting mechanism involves translation of the 5'-proximal short ORF terminating in front of the secondary structure that appears to force ribosomes to take off and resume scanning at a landing site downstream of the structure. Using two plant protoplast systems and shunt-competent wheat-germ extracts, we demonstrate that in Rice tungro bacilliform virus (RTBV) shunting also depends on the first short ORF followed by strong secondary structure. Swapping of the conserved shunt elements between CaMV and RTBV revealed the importance of nucleotide composition of the landing sequence for efficient shunting. The results suggest that the mechanism of ribosome shunting is evolutionary conserved in plant pararetroviruses.
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