Genome-wide identification of the Dicer-like family in cotton and analysis of the DCL expression modulation in response to biotic stress in two contrasting commercial cultivars.

BACKGROUND: Dicer-like proteins (DCLs) are essential players in RNA-silencing mechanisms, acting in gene regulation via miRNAs and in antiviral protection in plants and have also been associated to other biotic and abiotic stresses. To the best of our knowledge, despite being identified in some crops, cotton DCLs haven't been characterized until now. In this work, we characterized the DCLs of three cotton species and analyzed their expression profiles during biotic stress. RESULTS: As main results, 11 DCLs in the allotetraploid cotton Gossypium hirsutum, 7 and 6 in the diploid G. arboreum and G. raimondii, were identified, respectively. Among some DCLs duplications observed in these genomes, the presence of an extra DCL3 in the three cotton species were detected, which haven't been found in others eudicots. All the DCL types identified by in silico analysis in the allotetraploid cotton genome were able to generate transcripts, as observed by gene expression analysis in distinct tissues. Based on the importance of DCLs for plant defense against virus, responses of cotton DCLs to virus infection and/or herbivore attack using two commercial cotton cultivars (cv.), one susceptible (FM966) and another resistant (DO) to polerovirus CLRDV infection, were analyzed. Both cvs. Responded differently to virus infection. At the inoculation site, the resistant cv. showed strong induction of DCL2a and b, while the susceptible cv. showed a down-regulation of these genes, wherever DCL4 expression was highly induced. A time course of DCL expression in aerial parts far from inoculation site along infection showed that DCL2b and DCL4 were repressed 24 h after infection in the susceptible cotton. As CLRDV is aphid-transmitted, herbivore attack was also checked. Opposite expression pattern of DCL2a and b and DCL4 was observed for R and S cottons, showing that aphid feeding alone may induce DCL modulation. CONCLUSIONS: Almost all the DCLs of the allotetraploide G. hirsutum cotton were found in their relative diploids. Duplications of DCL2 and DCL3 were found in the three species. All four classes of DCL responded to aphid attack and virus infection in G. hirsutum. DCLs initial responses against the virus itself and/or herbivore attack may be contributing towards virus resistance.

Plant arginyltransferases (ATEs).

Regulation of protein stability and/or degradation of misfolded and damaged proteins are essential cellular processes. A part of this regulation is mediated by the so-called N-end rule proteolytic pathway, which, in concert with the ubiquitin proteasome system (UPS), drives protein degradation depending on the N-terminal amino acid sequence. One important enzyme involved in this process is arginyl-t-RNA transferase, known as ATE. This enzyme acts post-translationally by introducing an arginine residue at the N-terminus of specific protein targets to signal degradation via the UPS. However, the function of ATEs has only recently begun to be revealed. Nonetheless, the few studies to date investigating ATE activity in plants points to the great importance of the ATE/N-end rule pathway in regulating plant signaling. Plant development, seed germination, leaf morphology and responses to gas signaling in plants are among the processes affected by the ATE/N-end rule pathway. In this review, we present some of the known biological functions of plant ATE proteins, highlighting the need for more in-depth studies on this intriguing pathway.

Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates.

BACKGROUND: The RNA silencing pathway is an important anti-viral defense mechanism in plants. As a counter defense, some members of the viral family Luteoviridae are able to evade host immunity by encoding the P0 RNA silencing suppressor protein. Here we explored the functional diversity of P0 proteins among eight cotton leafroll dwarf virus (CLRDV) isolates, a virus associated with a worldwide cotton disease known as cotton blue disease (CBD). METHODS: CLRDV-infected cotton plants of different varieties were collected from five growing fields in Brazil and their P0 sequences compared to three previously obtained isolates. P0's silencing suppression activities were scored based on transient expression experiments in Nicotiana benthamiana leaves. RESULTS: High sequence diversity was observed among CLRDV P0 proteins, indicating that some isolates found in cotton varieties formerly resistant to CLRDV should be regarded as new genotypes within the species. All tested proteins were able to suppress local and systemic silencing, but with significantly variable degrees. All P0 proteins were able to mediate the decay of ARGONAUTE proteins, a key component of the RNA silencing machinery. CONCLUSIONS: The sequence diversity observed in CLRDV P0s is also reflected in their silencing suppression capabilities. However, the strength of local and systemic silencing suppression was not correlated for some proteins.

Complete genome sequences of two new virus isolates associated with cotton blue disease resistance breaking in Brazil.

Since 2006, Brazilian cotton (Gossypium hirsutum) crops planted with cultivars that are resistant to cotton blue disease have developed a new disease termed "atypical" cotton blue disease or atypical vein mosaic disease. Here, we describe the complete genomes of two virus isolates associated with this disease. The new virus isolates, called CLRDV-Acr3 and CLRDV-IMA2, were found to have a high degree of nucleotide and amino acid sequence similarity to previously described isolates of cotton leafroll dwarf virus, the causal agent of cotton blue disease. However, their P0 proteins were 86.1 % identical. These results show that this new disease is caused by a new CLRDV genotype that seems to have acquired the ability to overcome cotton blue disease resistance.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data.

BACKGROUND: Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. METHODS: In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. RESULTS: The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. CONCLUSIONS: SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. AVAILABILITY AND IMPLEMENTATION: SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection.

Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.

The Viral Threat in Cotton: How New and Emerging Technologies Accelerate Virus Identification and Virus Resistance Breeding.

Cotton (Gossypium spp. L., Malvaceae) is the world's largest source of natural fibers. Virus outbreaks are fast and economically devasting regarding cotton. Identifying new viruses is challenging as virus symptoms usually mimic nutrient deficiency, insect damage, and auxin herbicide injury. Traditional viral identification methods are costly and time-consuming. Developing new resistant cotton lines to face viral threats has been slow until the recent use of molecular virology, genomics, new breeding techniques (NBT), remote sensing, and artificial intelligence (AI). This perspective article demonstrates rapid, sensitive, and cheap technologies to identify viral diseases and propose their use for virus resistance breeding.

The Ubiquitin-Proteasome System (UPS) and Viral Infection in Plants.

The ubiquitin-proteasome system (UPS) is crucial in maintaining cellular physiological balance. The UPS performs quality control and degrades proteins that have already fulfilled their regulatory purpose. The UPS is essential for cellular and organic homeostasis, and its functions regulate DNA repair, gene transcription, protein activation, and receptor trafficking. Besides that, the UPS protects cellular immunity and acts on the host's defense system. In order to produce successful infections, viruses frequently need to manipulate the UPS to maintain the proper level of viral proteins and hijack defense mechanisms. This review highlights and updates the mechanisms and strategies used by plant viruses to subvert the defenses of their hosts. Proteins involved in these mechanisms are important clues for biotechnological approaches in viral resistance.

Antibodies for the Coat Protein of Cotton Leafroll Dwarf Virus Detect Commelina sp. as an Intermediary Host for Cotton Blue Disease.

The cotton blue disease, caused by the cotton leafroll dwarf virus (CLRDV), leads to dwarfism, leaf rolling, and production loss in susceptible cotton varieties. To develop an enzyme-linked immunosorbent assay (ELISA) test to detect the virus in cotton and weeds, peptides based on the coat protein were used to produce polyclonal (α-GQE, α-PRN, and α-INK) and monoclonal (α-GQE, α-PRN, and α-NKF) antibodies. All six were tested as capture antibodies, and polyclonal α-GQE and the monocle onal α-NKF were labeled with the enzyme alkaline phosphatase and used as detection antibodies for a double antibody sandwich (DAS) ELISA method, in which p-nitrophenyl phosphate was added and measured by absorbance at 405 nm. The DAS-ELISA sandwich was efficient in discriminating between healthy and diseased plant extracts. The ELISA methodology detected the virus in the weeds Commelina sp., which was confirmed by RT-PCR. The monoclonal antibodies may be used to develop other diagnostic procedures.

Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus.

BACKGROUND: In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae. RESULTS: Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. CONCLUSIONS: This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.

Antiviral activity of glucosylceramides isolated from Fusarium oxysporum against Tobacco mosaic virus infection.

Natural elicitors derived from pathogenic microorganisms represent an ecologic strategy to achieve resistance in plants against diseases. Glucosylceramides (GlcCer) are classified as neutral glycosphingolipids. GlcCer were isolated and purified from Fusarium oxysporum mycelium. F. oxysporum is a plant pathogenic fungus, abundant in soil and causing severe losses in economically important crops such as corn, tobacco, banana, cotton and passion fruit. In this study we evaluate the capacity of GlcCer in inducing resistance in N. tabacum cv Xanthi plants against Tobacco mosaic virus (TMV). Spraying tobacco plants with GlcCer before virus infection reduced the incidence of necrotic lesions caused by TMV. In addition, plants already infected with the virus showed a reduction in hypersensitive response (HR) lesions after GlcCer treatment, suggesting an antiviral effect of GlcCer. Our investigations showed that GlcCer stimulates the early accumulation of H2O2 and superoxide radicals. In addition, the expression of PR-1 (pathogenesis-related 1, with suggested antifungal action), PR-2 (ß-1,3-glucanase), PR-3 (Chitinase), PR-5 (Osmotin), PAL (Phenylalanine ammonia-lyase), LOX (Lipoxygenase) and POX (Peroxidase) genes was highly induced after treatment of tobacco plants with GlcCer and induction levels remained high throughout a period of 6 to 120 hours. Our experiments demonstrate that GlcCer induces resistance in tobacco plants against infection by TMV.

A peptidogalactomannan isolated from Cladosporium herbarum induces defense-related genes in BY-2 tobacco cells.

Cladosporium herbarum is a plant pathogen associated with passion fruit scab and mild diseases in pea and soybean. In this study, a peptidogalactomannan (pGM) of C. herbarum mycelium was isolated and structurally characterized, and its role in plant-fungus interactions was evaluated. C. herbarum pGM is composed of carbohydrates (76%) and contains mannose, galactose and glucose as its main monosaccharides (molar ratio, 52:36:12). Methylation and 13C-nuclear magnetic resonance (13C-NMR) spectroscopy analysis have shown the presence of a main chain containing (1 → 6)-linked α-D-Manp residues, and ß-D-Galf residues are present as (1 → 5)-interlinked side chains. ß-Galactofuranose containing similar structures were characterized by our group in A. fumigatus, A. versicolor, A. flavus and C. resinae. Tobacco BY-2 cells were used as a model system to address the question of the role of C. herbarum pGM in cell viability and induction of the expression of plant defense-related genes. Native and partially acid hydrolyzed pGMs (lacking galactofuranosyl side-chain residues) were incubated with BY-2 cell suspensions at different concentrations. Cell viability drastically decreased after exposure to more than 400 µg ml-1 pGM; however no cell viability effect was observed after exposure to a partially acid hydrolyzed pGM. BY-2 cell contact with pGM strongly induce the expression of plant defense-related genes, such as phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX), as well as the pathogen-related PR-1a, PR-2 and PR-3 genes, suggesting that pGM activates defense responses in tobacco cells. Interestingly, contact with partially hydrolyzed pGM also induced defense-related gene expression at earlier times than native pGM. These results show that the side chains of the (1 → 5)-linked ß-D-galactofuranosyl units from pGM play an important role in the first line fungus-plant interactions mediating plant responses against C. herbarum. In addition, it was observed that pGM and/or C. herbarum conidia are able to induced HR when in contact with tobacco leaves and in vitro plantlets roots, producing necrotic lesions and peroxidase and NO burst, respectively.

microRNAs as reference genes for quantitative PCR in cotton.

Cotton (Gossypium hirsutum) is the most important non-food plant in the world. Studies concerning the fiber quality and plant fitness of cotton at molecular level depend on high sensitive and reproducible gene-expression assays. However, only a few reports have described genes suitable for normalizing gene expression data. In this study, we report for the first time that microRNAs (miRNAs) are reliable reference genes (RGs) for cotton gene expression normalization in quantitative real-time reverse transcription (RT)-PCR. The stability of cotton miRNAs was assayed in root, stem, leaf and flower samples from three different cultivars [FiberMax (FM966), Delta Opal (DO) and Cedro] and under conditions of biotic stress caused by infection with Cotton leafroll dwarf virus (CLRDV). The stability of mRNAs already described as reference genes in cotton was also assessed. The geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to select the best reference genes. In 8 of the 12 sets tested, miRNAs (miR172, 168 and 390) were found to be the best RGs. To validate the best selected RGs, miR159, miR164, miR2118, miR2910, miR3476, GhDCL2 and GhDCL4 expression levels were evaluated under biotic stress conditions, and miR164 and a putative myo-inositol oxigenase gene (GhMIOX) were assessed in leaves and flowers. The RGs selected in this work proved to be excellent reference genes in the two cases studied. Our results support the use of miRNAs as reference genes for miRNA and protein-coding genes.

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing.

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant's vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant's viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant's silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant's anti-viral defense.

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation.

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0(PE), in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0(PE) has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0(PE) destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

Plant arginyltransferases (ATEs)

Abstract Regulation of protein stability and/or degradation of misfolded and damaged proteins are essential cellular processes. A part of this regulation is mediated by the so-called N-end rule proteolytic pathway, which, in concert with the ubiquitin proteasome system (UPS), drives protein degradation depending on the N-terminal amino acid sequence. One important enzyme involved in this process is arginyl-t-RNA transferase, known as ATE. This enzyme acts post-translationally by introducing an arginine residue at the N-terminus of specific protein targets to signal degradation via the UPS. However, the function of ATEs has only recently begun to be revealed. Nonetheless, the few studies to date investigating ATE activity in plants points to the great importance of the ATE/N-end rule pathway in regulating plant signaling. Plant development, seed germination, leaf morphology and responses to gas signaling in plants are among the processes affected by the ATE/N-end rule pathway. In this review, we present some of the known biological functions of plant ATE proteins, highlighting the need for more in-depth studies on this intriguing pathway.

Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast.

Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espírito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV-10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns.