Genomic Structure of Three Phenotypically Different Isolates of Peach Latent Mosaic Viroid: Implications of the Existence of Constraints Limiting the Heterogeneity of Viroid Quasispecies

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.     

Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (-) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.     

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation

Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3'-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short insertions/deletions at their 3' termini failed to cross-link. Because U81 and C289 are conserved in PLMVd variants and because the initiation site of PLMVd minus-strand RNA maps at a short double-stranded motif containing C289, the UV-photo-cross-linkable element of tertiary structure may be functionally significant. A second cross-linked species similar in size and sequence to the monomeric circular PLMVd form, observed in some PLMVd variants, probably derives from UV-induced ligation of the two termini resulting from self-cleavage.     

Deep-Sequencing of the Peach Latent Mosaic Viroid Reveals New Aspects of Population Heterogeneity

Viroids are small circular single-stranded infectious RNAs characterized by a relatively high mutation level. Knowledge of their sequence heterogeneity remains largely elusive and previous studies, using Sanger sequencing, were based on a limited number of sequences. In an attempt to address sequence heterogeneity from a population dynamics perspective, a GF305-indicator peach tree was infected with a single variant of the Avsunviroidae family member Peach latent mosaic viroid (PLMVd). Six months post-inoculation, full-length circular conformers of PLMVd were isolated and deep-sequenced. We devised an original approach to the bioinformatics refinement of our sequence libraries involving important phenotypic data, based on the systematic analysis of hammerhead self-cleavage activity. Two distinct libraries yielded a total of 3,939 different PLMVd variants. Sequence variants exhibiting up to ∼17% of mutations relative to the inoculated viroid were retrieved, clearly illustrating the high level of divergence dynamics within a unique population. While we initially assumed that most positions of the viroid sequence would mutate, we were surprised to discover that ∼50% of positions remained perfectly conserved, including several small stretches as well as a small motif reminiscent of a GNRA tetraloop which are the result of various selective pressures. Using a hierarchical clustering algorithm, the different variants harvested were subdivided into 7 clusters. We found that most sequences contained an average of 4.6 to 6.4 mutations compared to the variant used to initially inoculate the plant. Interestingly, it was possible to reconstitute and compare the sequence evolution of each of these clusters. In doing so, we identified several key mutations. This study provides a reliable pipeline for the treatment of viroid deep-sequencing. It also sheds new light on the extent of sequence variation that a viroid population can sustain, and which may give rise to a quasispecies.     

Deep Sequencing of the Small RNAs Derived from Two Symptomatic Variants of a Chloroplastic Viroid: Implications for Their Genesis and for Pathogenesis

Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21–24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (−) RNAs presenting a biased distribution of their 5′ nucleotide, and adopting a hotspot profile along the genomic (+) and (−) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12–14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Comparative transmission of, and varietal reaction to, three isolates of rice tungro virus disease

Comparative transmission by leafhoppers of three tungro isolates obtained from the Philippines, India and Malaysia, and of an infectious clone of the Philippine isolate of rice tungro bacilliform virus (RTBV) by agroinoculation, was conducted on 12 rice cultivars. The symptoms, including height of inoculated plants were recorded and the efficiency of RTBV and rice tungro spherical virus (RTSV) transmission was determined by enzyme-linked immunosorbent assay. In most cases, the reduction of height and leaf symptoms of plants infected with RTBV and/or RTSV by the three isolates were similar in any given cultivar. On cultivar ASD 7 , the Malaysian isolate showed more severe yellow orange leaf discolouration symptoms than the Indian isolate which in turn had more severe leaf discolouration than the Philippine isolate. On the other hand, cultivars ASD 7 and Ptb 18 produced the most severe yellow orange leaf discolouration when agroinoculated with an infectious RTBV clone of the Philippine isolate. There was some variation in the transmission profile of the two tungro viruses among the three isolates. However, there was no one clear set of characteristics by which one could use cultivars to distinguish isolates. The amount of viral DNA in agroinfected plants of cultivars Utri merah, Balimau putih, Utri Rajapan and ARC 11554 was low, while the amount was high in cultivars TN1, ASD7, Ptb 18 and TKM 6. There was high correlation between the amount of viral coat protein by ELISA and viral nucleic acid by DNA hybridisation on 10 agroinoculated rice cultivars; this might indicate that similar proportions of the total RTBV DNA are encapsidated in each cultivar.     

Molecular variability analyses of <Emphasis Type="Italic">Apple chlorotic leaf spot virus</Emphasis> capsid protein

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91–100% and 70–98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.     

Strains of Zucchini Yellow Mosaic Virus in Muskmelon (Cucumis melo L.)

According to the reaction of muskmelon line PI 414723, 22 natural isolates of Zucchini Yellow Mosaic Virus (ZYMV) were grouped into two pathotypes. When inoculated by isolates belonging to pathotype 0, most of the PI 414723 plants (over 70%) remained symptomless while few plants developed systemic chloronecrotic spotting and more rarely yellowing, stunting, mosaic and leaf deformations. When contaminated by pathotype 1 all PI 414723 plants developed systemic chloronecrotic spotting. Two variants were obtained from representative strains of these two pathotypes, able to induce yellowing, stunting, mosaic and leaf deformation on all inoculated PI 414723 plants. These variants could not be differentiated from their originating strains either by host range, serology or aphid transmission properties. Therefore they are regarded as belonging to a third group called pathotype 2. No relation was observed between the type of symptom developed on PI 414723 and the ability to induce a rapid wilting reaction of melon cv. Doublon (pathotype F).     

Infectibility and sensitivity of UK raspberry, blackberry and hybrid berry cultivars to Rubus viruses

Six blackberry or hybrid berry cultivars and 19 raspberry cultivars were assessed for their infectibility with, and sensitivity to, graft inoculation with 10 distinct viruses found infecting Rubus in the UK. Cultivars were grafted with each of, two isolates of the pollen borne raspberry bushy dwarf virus (RBDV), five aphid borne viruses: black raspberry necrosis, raspberry leaf mottle (RLMV), raspberry leaf spot (RLSV), rubus yellow net and raspberry vein chlorosis (RVCV); and isolates of the nematode transmitted nepoviruses, arabis mosaic, raspberry ringspot, strawberry latent ringspot and tomato black ring. All tested cultivars were infectible with a resistance breaking isolate of RBDV but only about half of that number with the Scottish type isolate of the virus. The raspberry cvs Autumn Bliss, and occasionally Glen Garry and Glen Prosen, developed leaf yellowing symptoms following infection with RBDV, but none of the other infected cultivars showed obvious leaf symptoms when kept in a heated glasshouse during the growing season. All tested cultivars were infectible with each of the four viruses transmitted in nature by the aphid, Amphorophora idaei. Most were infected symptomlessly, but seven cultivars developed severe leaf spotting symptoms due to infection with RLMV or RLSV. All but one of the raspberry cultivars were infectible with RVCV, which is transmitted in nature by the aphid Aphis idaei, and almost all infected plants developed leaf symptoms; only one of the hybrid berry or blackberry cultivars tested was infected with RVCV. In tests with the four nepoviruses, all tested cultivars, except Tummelberry, were infectible with at least one or more of these viruses. However, cultivars responded differently to challenge inoculation with different isolates of individual nepoviruses. Several cultivars developed chlorotic leaf mottling following infection with some nepovirus isolates. The implications of these results for virus control are discussed in the light of the changing pattern of virus and virus vector incidence in the UK.     

Biological and Molecular Characterization of Three Isolates of Tomato aspermy virus Infecting Chrysanthemums in India

Severe mosaic, chlorotic ringspots and flower deformation were observed during the winter of November 2006–February 2007 on chrysanthemums ( Chrysanthemum morifolium ) at three locations in India: Lucknow (UP), Dhanbad (MP) and Kolkata (WB). Tomato aspermy virus (TAV) was detected in affected plants by ELISA and by RT-PCR using TAV specific primers. These TAV isolates were mechanically transmitted to test plant species and also by aphids ( Aphis gossypii ) to Lycopersicon esculentum . The complete RNA 3 of each TAV isolate was cloned and sequenced and determined to be 2386 nucleotides (nt) long, and to encode two open reading frames (ORFs): the movement protein (MP) of 741 nt and the coat protein (CP) of 657 nt translating in to 246 and 218 amino acid (aa), respectively. When RNA 3 sequences of the Indian isolates were multiple aligned with seven other strains of TAV occurring worldwide, Indian isolates shared 98–99% identities among themselves and with the KC, V, P, B, I and C strains of TAV. In phylogenetic analysis, the Lucknow and Kolkata isolates of TAV clustered together and showed a close relationship with the KC-TAV strain from South Korea, whereas the Dhanbad isolate formed an independent cluster and showed closeness with the V-TAV strains from Spain and Australia. Recombination events were also observed in the CP region of the Dhanbad isolate, supporting its diverse behaviour. This is the first report of the complete RNA 3 sequence of these three Indian TAV isolates.     

Serological and molecular characteristics of pea seed borne mosaic virus-PSbMV from lentil (Lens culinaris Medik L.) in Iran

Abstract

During 2016 growing season, samples with leaf yellowing and mosaic like symptoms were collected from main lentil (Lens culinaris Medik L.) fields. Specific ELISA positive PSbMV samples were selected for RT-PCR. Using specific pair of primer towards CP gene region of the virus, PCR product of approx.235?bp was amplified. Based on the nucleotide sequences of the CP gene PSbMV isolates were classified in two major groups. In which, isolates in group I divided into two subgroups of Iranian isolates (B2) and (G1), along with Czech Republic, Australia, China, Greece, Pakistan and Egypt were placed in the subgroup-I. Isolate (V18) from Iran placed independently in group II. In the grouping based on the amino acid sequencing the isolates divided into two phylogenetic groups. Iranian isolates along with an isolate from Australia categorized in group-II, however world isolates were all clustered in group-I.     

黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究

利用RT_PCR的方法,获得了黄瓜花叶病毒卫星RNA XJs1的全长侵染性cDNA克隆pMSC20。序列分析显示,XJs1全长384nt(GenBank登录号:DQ070748),比较XJs1与具有代表性的CMV卫星RNA的序列结构表明,在XJs1核苷酸序列的325nt~350nt间,具有典型的坏死型卫星RNA保守序列。通过体外转录,将XJs1与不含卫星RNA的辅助病毒分离物CMV_AH组合接种普通烟和心叶烟并进行检测。初步研究结果表明,XJs1为一致弱卫星RNA。