First report of Plum bark necrosis stem pitting-associated virus infecting grapevine in China.

BACKGROUND: Virus disease is one of the main diseases in grapevine, and there has been no report on Plum bark necrosis and stem pitting-associated virus infecting grapevine in China. OBJECTIVE: The leaf samples of grapevine cultivar 'Cabernet Gernischt' were collected from Shandong province, which the leaves suffered from viral-like symptoms with spotting and crinkle. METHODS: Small RNA-seq combined with reverse transcription PCR (RT-PCR) were performed to detect the potential viruses in these field samples. Phylogenetic tree was constructed using the neighbor joining method in MEGA 5.1 CONCLUSIONS: This is the first report of PBNSPaV infecting grapevine in China, contributing to a better understanding of the epidemiology and host range distribution of this pathogen.

Revisiting the Role of Transcription Factors in Coordinating the Defense Response Against Citrus Bark Cracking Viroid Infection in Commercial Hop (Humulus Lupulus L.).

Transcription factors (TFs) play a major role in controlling gene expression by intricately regulating diverse biological processes such as growth and development, the response to external stimuli and the activation of defense responses. The systematic identification and classification of TF genes are essential to gain insight into their evolutionary history, biological roles, and regulatory networks. In this study, we performed a global mining and characterization of hop TFs and their involvement in Citrus bark cracking viroid CBCVd infection by employing a digital gene expression analysis. Our systematic analysis resulted in the identification of a total of 3,818 putative hop TFs that were classified into 99 families based on their conserved domains. A phylogenetic analysis classified the hop TFs into several subgroups based on a phylogenetic comparison with reference TF proteins from Arabidopsis thaliana providing glimpses of their evolutionary history. Members of the same subfamily and subgroup shared conserved motif compositions. The putative functions of the CBCVd-responsive hop TFs were predicted using their orthologous counterparts in A. thaliana. The analysis of the expression profiling of the CBCVd-responsive hop TFs revealed a massive differential modulation, and the expression of the selected TFs was validated using qRT-PCR. Together, the comprehensive integrated analysis in this study provides better insights into the TF regulatory networks associated with CBCVd infections in the hop, and also offers candidate TF genes for improving the resistance in hop against viroids.

A sensitive nested multiplex RT-PCR assay for the simultaneous detection of three common viruses infecting pear plants.

A highly sensitive nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assay was developed for the simultaneous detection of Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) infecting pear trees. In the assay, a set of three forward primers specific to each of the three viruses and a universal reverse primer was used as external primers in the first-round PCR, which was followed by a second-round PCR developed previously. The nmRT-PCR assay was 104 times more sensitive than conventional mRT-PCR assay in detecting the three viruses in in vitro pear plantlets. This assay was subsequently used to detect these viruses in leaf and bark samples of cultivated and wild pear trees from orchards and demonstrated to be highly sensitive and reliable. This is the first report describing a use of nmRT-PCR for the sensitive and simultaneous detection of the three viruses infecting pear plants. The assay would be useful for the certification of pear planting materials and surveillance of nursery stocks.

Double-Stranded RNA-Enriched Preparations to Identify Viroids by Next-Generation Sequencing.

Approaches based on next-generation sequencing (NGS) coupled with bioinformatics tools have been developed for detecting viruses and viroids infecting herbaceous and woody plants. Here we describe a protocol to extract nucleic acids from citrus bark and enrich them in double-stranded RNAs. These preparations can be efficiently used for generating cDNA libraries that, after pair-end sequencing and bioinformatics analyses, allow efficient identification of the viroids infecting the source plant.

Rootstock-scion interaction affecting citrus response to CTV infection: a proteomic view.

Citrus tristeza virus (CTV) is the causal agent of various diseases with dramatic effects on citrus crops worldwide. Most Citrus species, grown on their own roots, are symptomless hosts for many CTV isolates. However, depending on different scion-rootstock combination, CTV infection should result in distinct syndromes, being 'tristeza' the more severe one, leading to a complete decline of the susceptible plants in a few weeks. Transcriptomic analyses revealed several genes involved either in defense response, or systemic acquired resistance, as well as transcription factors and components of the phosphorylation cascades, to be differentially regulated during CTV infection in Citrus aurantifolia species. To date little is known about the molecular mechanism of this host-pathogen interaction, and about the rootstock effect on citrus response to CTV infection. In this work, the response to CTV infection has been investigated in tolerant and susceptible scion-rootstock combinations by two-dimensional gel electrophoresis (2DE). A total of 125 protein spots have been found to be differently accumulated and/or phosphorylated between the two rootstock combinations. Downregulation in tolerant plants upon CTV infection was detected for proteins involved in reactive oxygen species (ROS) scavenging and defense response, suggesting a probable acclimation response able to minimize the systemic effects of virus infection. Some of these proteins resulted to be modulated also in absence of virus infection, revealing a rootstock effect on scion proteome modulation. Moreover, the phospho-modulation of proteins involved in ROS scavenging and defense response, further supports their involvement either in scion-rootstock crosstalk or in the establishment of tolerance/susceptibility to CTV infection.

Characterization of a virus infecting Citrus volkameriana with citrus leprosis-like symptoms.

A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves but not in healthy controls. Double-stranded (ds) RNAs ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay; however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease.

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus.

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

Efficient methods for sample processing and cDNA synthesis by RT-PCR for the detection of grapevine viruses and viroids.

Template preparation is important in reverse-transcription polymerase chain reaction (RT-PCR)-based detection methods. A TissueLyser with tungsten carbide beads was used for simultaneous processing of up to 48 samples under the same conditions in the development of a simple and rapid procedure to prepare a plant extract for RT reaction. A sandpaper method was also developed by which wood tissue of dormant cuttings could be macerated easily to process with minimal time and effort. It was also demonstrated that the combination use of random primers and oligo dT primer in an RT reaction was efficient for simultaneous cDNA synthesis of viral and viroid RNAs in plant extracts. These template preparation methods were used for the amplification of Grapevine leafroll-associated virus-1,-2, and -3; Grapevine virus A and B; Grapevine rupestris stem pitting-associated virus; Grapevine fleck virus; and Grapevine fanleaf virus. All these viruses tested in this study were reliably detected up to a 10(3)-fold or higher dilution of the original extract. Besides, Hop stunt viroid and Grapevine yellow speckle viroid 1 were well amplified in the same manner as the template preparation and following PCR for virus detection. These methods would contribute to cost-effective testing of a large number of samples through the year and help to detect viral pathogens in grapevine.