Identification, full-length genome sequencing, and field survey of citrus vein enation virus in Italy

Citrus vein enation virus (CVEV) was described in Spain and then it has been reported in several citrus growing areas of Asia, America and Australia. Here, the occurrence of CVEV in Italy has been documented for the first time. The full genome sequence of a CVEV Italian isolate (14Q) was determined by high-throughput sequencing and the presence of the virus was confirmed by RT-PCR and graft-transmission to indicator plants, from which the virus was recovered six-months post-inoculation. Phylogenetic analysis based on the full-length genome of CVEV isolates from different countries showed that they are phylogenetically related to each other based on their geographic origin, rather than on their host and that the Italian isolate is more closely related to the Spanish isolate than to the other ones. A field survey revealed the presence of CVEV in some areas of Campania region (southern Italy), prevalently infecting lemon trees. In the frame of this survey, kumquat was identified for the first time as a host of CVEV. No symptoms were observed in the field so far. The infection of asymptomatic hosts and the transmission by aphid species present in Italy increase the risk that the virus could further spread

Identification and characterization of citrus concave gum-associated virus infecting citrus and apple trees by serological, molecular and high-throughput sequencing approaches

Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined

Close similarities between Cherry chlorotic rusty spot disease from Italy and Cherry leaf scorch from Spain

Cherry chlorotic rusty spot (CCRS), a disease affecting sweet and sour cherry in Southern Italy was regularly found associated with an unidentified fungus and with a complex pattern of viral-like double-stranded RNAs as well as with two small circular RNAs (cherry small circular RNAs, cscRNAs). Further studies revealed that i) the ds-RNAs correspond to the genome of different mycoviruses belonging to the genera Chrysovirus, Partitivirus and Totivirus and ii) the two viroid-like RNAs consist of two groups of variants with similar sequences but differing in size (394–415 and 372–377 nt for cscRNA1 and cscRNA2, respectively). Here we report that the dsRNAs of Chrysovirus and Partitivirus have been detected by RT-PCR analysis with CCRS specific primers in nucleic acid preparations from cherry leaves affected by cherry leaf scorch (CLS) in Spain, a disease whose etiological agent is the ascomycetes Apiognomonia erythrostoma, order Diaporthales. Moreover, Northern-blot hybridization assays showed that a viroid-like RNA comigrating and sharing high sequence similarity with the cscRNA1 previously reported in Italy, accumulate in leaves from CLS affected trees in Spain. These data, together with other evidence showing similar symptoms, disease cycle and fungal fructifications in CCRS and CLS affected trees, suggest a close relationship between the two cherry disorders.Keywords: dsRNAs, cscRNAs, Apiognomonia erythrostoma, Diaporthale

Genetic variability of the coat protein gene of isolates of Citrus variegation virus from Campania (southern Italy)

Six new sequences of the coat protein (CP) gene of Citrus variegation virus (CVV) isolates, some producing crinkly leaf symptoms and some asymptomatic, and collected in Campania (southern Italy), are presented. Comparison with previously reported CP gene sequences of isolates from various locations worldwide confirmed a high degree of coat protein gene conservation in Campania (lowest similarity among all CVV sequences 92.4%). No relationship was found between amino-acid substitutions and host species or symptoms. Phylogenetic analysis proved that CP sequences from Campania isolates clustered in a new group when compared to those reported in the data bank

A novel self-cleaving viroid-like RNA identified in RNA preparations from a citrus tree is not directly associated with the plant

Viroid and viroid-like satellite RNAs are infectious, circular, non-protein coding RNAs reported in plants only so far. Some viroids (family Avsunviroidae) and viroid-like satellite RNAs share self-cleaving activity mediated by hammerhead ribozymes (HHRzs) endowed in both RNA polarity strands. Using a homology-independent method based on the search for conserved structural motifs of HHRzs in reads and contigs from high-throughput sequenced RNAseq libraries, we identified a novel small (550 nt) viroid-like RNA in a library from a Citrus reticulata tree. Such a viroid-like RNA contains a HHRz in both polarity strands. Northern blot hybridization assays showed that circular forms of both polarity strands of this RNA (tentatively named citrus transiently-associated hammerhead viroid-like RNA1 (CtaHVd-LR1)) exist, supporting its replication through a symmetric pathway of the rolling circle mechanism. CtaHVd-LR1 adopts a rod-like conformation and has the typical features of quasispecies. Its HHRzs were shown to be active during transcription and in the absence of any protein. CtaHVd-LR1 was not graft-transmissible, and after its first identification, it was not found again in the original citrus source when repeatedly searched in the following years, suggesting that it was actually not directly associated with the plant. Therefore, the possibility that this novel self-cleaving viroid-like RNA is actually associated with another organism (e.g., a fungus), in turn, transiently associated with citrus plants, is propose

Sound Event Detection with Binary Neural Networks on Tightly Power-Constrained IoT Devices

Sound event detection (SED) is a hot topic in consumer and smart city applications. Existing approaches based on Deep Neural Networks are very effective, but highly demanding in terms of memory, power, and throughput when targeting ultra-low power always-on devices. Latency, availability, cost, and privacy requirements are pushing recent IoT systems to process the data on the node, close to the sensor, with a very limited energy supply, and tight constraints on the memory size and processing capabilities precluding to run state-of-the-art DNNs. In this paper, we explore the combination of extreme quantization to a small-footprint binary neural network (BNN) with the highly energy-efficient, RISC-V-based (8+1)-core GAP8 microcontroller. Starting from an existing CNN for SED whose footprint (815 kB) exceeds the 512 kB of memory available on our platform, we retrain the network using binary filters and activations to match these memory constraints. (Fully) binary neural networks come with a natural drop in accuracy of 12-18% on the challenging ImageNet object recognition challenge compared to their equivalent full-precision baselines. This BNN reaches a 77.9% accuracy, just 7% lower than the full-precision version, with 58 kB (7.2 times less) for the weights and 262 kB (2.4 times less) memory in total. With our BNN implementation, we reach a peak throughput of 4.6 GMAC/s and 1.5 GMAC/s over the full network, including preprocessing with Mel bins, which corresponds to an efficiency of 67.1 GMAC/s/W and 31.3 GMAC/s/W, respectively. Compared to the performance of an ARM Cortex-M4 implementation, our system has a 10.3 times faster execution time and a 51.1 times higher energy-efficiency.Comment: 6 pages conferenc

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from S. cerevisiae, S. pombe, and the β- and γ- isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate actin dendritic networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 SNP variants in the follicle stimulating hormone receptor (fshr) consistent with an XX / XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. Fshr displayed differential gene expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 non-synonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.info:eu-repo/semantics/acceptedVersio

Expression of Odorant Receptor Family, Type 2 OR in the Aquatic Olfactory Cavity of Amphibian Frog Xenopus tropicalis

Recent genome wide in silico analyses discovered a new family (type 2 or family H) of odorant receptors (ORs) in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN), it remains unknown if type 2 ORs (OR2) function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions