Characterization of horizontal transmission of blueberry latent spherical virus by pollen.

Nicotiana benthamiana plants became infected with blueberry latent spherical virus (BLSV) after pollination with pollen grains produced by BLSV-infected N. benthamiana plants. Interestingly, pollen grains produced by BLSV-infected Vaccinium corymbosum (blueberry), Nicotiana alata, and Petunia × hybrida (petunia) plants also transmitted the virus to healthy N. benthamiana plants after pollination. As seen using aniline blue staining and fluorescence microscopy, pollen grains from BLSV-infected blueberry, N. alata, and petunia plants germinated on stigmas of N. benthamiana, and the pollen tubes penetrated the stigmas in a manner similar to that of N. benthamiana pollen grains on N. benthamiana stigmas. Whole-mount in situ hybridization and chromogenic in situ hybridization analysis showed that infected blueberry and N. benthamiana pollen grains germinated on N. benthamiana stigmas, and virus-containing pollen tubes penetrated the stigmas. Tissue blot hybridization analysis revealed that the initial infection sites were the N. benthamiana stigmas pollinated with infected pollen grains from blueberry and N. benthamiana. In addition, the virus spread from the initial infection sites to the phloem in the stigma and style. Taken together, we suggest that penetrating pollen tubes that harbored the virus results in infection foci in the stigma, and the virus then moves to the vascular tissues in the stigma and style and eventually establishes systemic infection.

LreEF1A4, a Translation Elongation Factor from Lilium regale, Is Pivotal for Cucumber Mosaic Virus and Tobacco Rattle Virus Infections and Tolerance to Salt and Drought.

Eukaryotic translation elongation factors are implicated in protein synthesis across different living organisms, but their biological functions in the pathogenesis of cucumber mosaic virus (CMV) and tobacco rattle virus (TRV) infections are poorly understood. Here, we isolated and characterized a cDNA clone, LreEF1A4, encoding the alpha subunit of elongation factor 1, from a CMV-elicited suppression subtractive hybridization library of Lilium regale. The infection tests using CMV remarkably increased transcript abundance of LreEF1A4; however, it also led to inconsistent expression profiles of three other LreEF1A homologs (LreEF1A1-3). Protein modelling analysis revealed that the amino acid substitutions among four LreEF1As may not affect their enzymatic functions. LreEF1A4 was ectopically overexpressed in petunia (Petunia hybrida), and transgenic plants exhibited delayed leaf and flower senescence, concomitant with increased transcription of photosynthesis-related genes and reduced expression of senescence-associated genes, respectively. A compromised resistance to CMV and TRV infections was found in transgenic petunia plants overexpressing LreEF1A4, whereas its overexpression resulted in an enhanced tolerance to salt and drought stresses. Taken together, our data demonstrate that LreEF1A4 functions as a positive regulator in viral multiplication and plant adaption to high salinity and dehydration.

Disturbance of floral colour pattern by activation of an endogenous pararetrovirus, petunia vein clearing virus, in aged petunia plants.

White areas of star-type bicolour petals of petunia (Petunia hybrida) are caused by post-transcriptional gene silencing (PTGS) of the key enzyme of anthocyanin biosynthesis. We observed blotched flowers and a vein-clearing symptom in aged petunia plants. To determine the cause of blotched flowers, we focused on an endogenous pararetrovirus, petunia vein clearing virus (PVCV), because this virus may have a suppressor of PTGS (VSR). Transcripts and episomal DNAs derived from proviral PVCVs accumulated in aged plants, indicating that PVCV was activated as the host plant aged. Furthermore, DNA methylation of CG and CHG sites in the promoter region of proviral PVCV decreased in aged plants, suggesting that poor maintenance of DNA methylation activates PVCV. In parallel, de novo DNA methylation of CHH sites in its promoter region was also detected. Therefore, both activation and inactivation of PVCV occurred in aged plants. The accumulation of PVCV transcripts and episomal DNAs in blotched regions and the detection of VSR activity support a mechanism in which suppression of PTGS by PVCV causes blotched flowers.

Genetic differences between Korean and American isolates of Petunia vein clearing virus.

Petunia plants are used for urban landscaping in many parts of the world, including South Korea. In this study, we aimed to investigate the occurrence of petunia vein clearing virus (PVCV) infection in petunia plants in Seoul, South Korea. PVCV was detected from 23 of 79 petunia samples collected from Seoul. We obtained the complete genome sequences of the Korean isolates in this study (called PVCV-Kr, Kr2, and Kr3), which were compared with the genome sequence of the USA isolate of the virus (PVCV-USA). The genomic DNA of the three PVCV isolates was found to comprise 7210-7267 nucleotides (nts), which is 4-15 nts longer than the PVCV-USA genome. The genomes of the Kr and Kr2 isolates encode a large polyprotein of 252 kDa (2180 amino acids (aa)). The genome of the Kr3 isolate encodes a large polyprotein of 255 kDa (2203 aa). The polyprotein has six protein domains: a movement protein (MP; 72 aa), a coiled-coil domain (CC; 33 aa), an RNA-binding domain (RB; 18 aa), a protease (PR; 21 aa), a reverse transcriptase (RT; 196 aa), and an RNase H (RH; 121 aa). The large polyprotein and six domains of the three isolates showed 93.9-100.0% sequence homology with those of PVCV-USA. Furthermore, the polymerase polyprotein gene (PR, RT, and RH) of the four PVCV isolates containing the USA isolate grouped with those of Rice tungro bacilliform virus and Soybean chlorotic mottle virus, which belong to the same family (Caulimoviridae). Our findings suggested that the Korean isolates represent a new isolate of PVCV. To our knowledge, this is the first report of PVCV detection in South Korea.

Influence of the terminal left domain on horizontal and vertical transmissions of tomato planta macho viroid and potato spindle tuber viroid through pollen.

Viroids can be transmitted vertically and/or horizontally by pollen. Tomato planta macho viroid (TPMVd) has a high rate of horizontal transmission by pollen, whereas potato spindle tuber viroid (PSTVd) does not. To specify the domain(s) involved in horizontal transmission, four viroid chimeras were created by exchanging the terminal left (TL) and/or pathogenicity (P) domains between PSTVd and TPMVd. PSTVd-based chimeras containing TPMVd-TL and P, or TPMVd-TL alone, displayed a high rate of horizontal transmission. TPMVd-based chimeras containing PSTVd-TL and P lost infectivity, and those containing PSTVd-TL alone displayed a low rate of horizontal transmission. In addition, the vertical transmission rate was also higher in the mutants containing TPMVd-TL than in the others. These findings indicate that the sequences or structures in the TL and P (although the role is limited) domains are important not only for horizontal but also for vertical transmission by pollen.

Characterization of the first tenuivirus naturally infecting dicotyledonous plants.

A mechanically transmissible virus tentatively named "melon chlorotic spot virus" (MeCSV) was isolated in southeastern France from a melon plant showing chlorotic spots and yellowing of the older leaves. Its complete sequence was obtained by Illumina and Sanger sequencing. The genome comprises eight RNAs for a total size of 20,079 nt and is distantly related to Ramu stunt virus and maize yellow stunt virus, two tentative tenuiviruses. MeCSV differs from other tenuiviruses by its number of genomic fragments, by being readily mechanically transmissible, and by infecting only dicotyledonous hosts. MeCSV should thus be considered a member of a tentative new species related to tenuiviruses.

Characterization, phylogeny and recombination analysis of Pedilanthus leaf curl virus-Petunia isolate and its associated betasatellite.

BACKGROUND: Geminiviruses cause major losses to several economically important crops. Pedilanthus leaf curl virus (PeLCV) is a pathogenic geminivirus that appeared in the last decade and is continuously increasing its host range in Pakistan and India. This study reports the identification and characterization of PeLCV-Petunia from ornamental plants in Pakistan, as well as geographical, phylogenetic, and recombination analysis. METHODS: Viral genomes and associated satellites were amplified, cloned, and sequenced from Petunia atkinsiana plants showing typical geminivirus infection symptoms. Virus-satellite complex was analyzed for phylogenetic and recombination pattern. Infectious clones of isolated virus and satellite molecules were constructed using a partial dimer strategy. Infectivity analysis of PeLCV alone and in combination with Digera yellow vein betasatellite (DiYVB) was performed by Agrobacterium infiltration of Nicotiana benthamiana and Petunia atkinsiana plants with infectious clones. RESULTS: PeLCV, in association with DiYVB, was identified as the cause of leaf curl disease on P. atkinsiana plants. Sequence analysis showed that the isolated PeLCV is 96-98% identical to PeLCV from soybean, and DiYVB has 91% identity to a betasatellite identified from rose. Infectivity analysis of PeLCV alone and in combination with DiYVB, performed by Agrobacterium infiltration of infectious clones in N. benthamiana and P. atkinsiana plants, resulted in mild and severe disease symptoms 14 days after infiltration, respectively, demonstrating that these viruses are natural disease-causing agents. Southern blot hybridization indicated successful replication of the virus-betasatellite complex in the infected plants. Phylogenetic analysis suggests that PeLCV originated from Pakistan and later spread to India. Recombination analysis predicted that PeLCV is a donor parent for recombination and evolution of two important begomoviruses, Papaya leaf curl virus (PaLCuV) and Radish leaf curl virus (RaLCuV). The molecular phylogeny of genes encoding coat protein (CP) and replication associated protein (Rep) depict a complex evolutionary pattern of the viruses, with wide diversity in both of the genes. CONCLUSIONS: This study presents PeLCV and DiYVB as a new natural combination resulting in leaf curl disease on P. atkinsiana plants. Phylogenetic analysis, in addition to recent agricultural reports, identify PeLCV as an emerging broad host range Begomovirus that is resident in Pakistan and, more recently, has also spread to India. Recombination analysis showed that PeLCV was involved in a natural recombinational event leading to the evolution of two recombinant begomoviruses, RaLCuV and PaLCuV.

Differences in dynamics of horizontal transmission of Tomato planta macho viroid and Potato spindle tuber viroid after pollination with viroid-infected pollen.

For viroids, pollen transmission is an important transmission pathway to progeny seeds and new hosts. In the current study, we found that Tomato planta macho viroid (TPMVd)-but not Potato spindle tuber viroid (PSTVd)-was horizontally transmitted by pollen from petunia plants. Using tissue-printing hybridization to track the changes in viroid distribution after pollination, we noted that TPMVd was present in petunia stigma, styles, and eventually ovaries, whereas PSTVd was detected in the stigma and upper style but not the ovary. These findings suggest that horizontal transmission of viroids depends on the infection of the lower style and ovary during the elongation of pollen tubes after pollination. Additionally, TPMVd was transmitted horizontally, leading to systematic infection, when we used TPMVd-infected petunia pollen to pollinate the flowers of healthy tomato plants. Fertilization typically does not occur after heterologous pollination and thus likely is not required to accomplish horizontal transmission of viroids.

RETRACTED: WRKY71 and TGA1a physically interact and synergistically regulate the activity of a novel promoter isolated from Petunia vein-clearing virus.

Caulimoviral promoters have become excellent tools for efficient transgene expression in plants. However, the transcriptional framework controlling their systematic regulation is poorly understood. To understand this regulatory mechanism, we extensively studied a novel caulimoviral promoter, PV8 (-163 to +138, 301 bp), isolated from Petunia vein-clearing virus (PVCV). PVCV was found to be Salicylic acid (SA)-inducible and 2.5-3.0 times stronger than the widely used CaMV35S promoter. In silico analysis of the PV8 sequence revealed a unique clustering of two stress-responsive cis-elements, namely, as-11 and W-box1-2, located within a span of 31 bp (-74 to -47) that bound to the TGA1a and WRKY71 plant transcription factors (TFs), respectively. We found that as-1 (TTACG) and W-box (TGAC) elements occupied both TGA1a and WRKY71 on the PV8 backbone. Mutational studies demonstrated that the combinatorial influence of as-1 (-57) and W-box1-2 (-74 and -47) on the PV8 promoter sequence largely modulated its activity. TGA1a and WRKY71 physically interacted and cooperatively enhanced the transcriptional activity of the PV8 promoter. Biotic stress stimuli induced PV8 promoter activity by ~1.5 times. We also established the possible pathogen-elicitor function of AtWRKY71 and NtabWRKY71 TFs. Altogether, this study elucidates the interplay between TFs, biotic stress and caulimoviral promoter function.

Distribution of Tomato planta macho viroid in germinating pollen and transmitting tract.

Vertical and horizontal pollen transmission is important for efficient infection by viroids. Vertical pollen transmission of viroids is attributed to the infection by viroid in the embryo sac through infected pollen. To identify the viroid infection in pollen and pollen tubes elongating through the transmitting tract, we used in situ hybridization to histochemically analyze the distribution of Tomato planta macho viroid (TPMVd) in pollen grains, the stigma, and style of petunia plants. TPMVd was present in the generative nucleus and vegetative nucleus of mature infected pollen grains and germinating pollen grains. During pollen tube growth, TPMVd was present in the vegetative nucleus and two sperm nuclei, which were generated by division of the generative nucleus in the style transmitting tract. These findings indicated that viroid infection in sperm nuclei is responsible for vertical pollen transmission of viroids. TPMVd infection from TPMVd-infected pollen tubes to the transmitting tract was not observed. In addition, TPMVd signals were not confirmed in the stigma and transmitting tract of TPMVd-infected petunia plants, suggesting that viroids may not replicate in these tissues at the stage of mature style. Therefore, TPMVd may leak from the pollen tube somewhere in the ovary, except in the transmitting tract, during the horizontal transmission of TPMVd.

Seed Transmission of Beet Curly Top Virus and Beet Curly Top Iran Virus in a Local Cultivar of Petunia in Iran.

Beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV) are known as the causal agents of curly top disease in beet and several other dicotyledonous plants in Iran. These viruses are transmitted by Circulifer species, and until now, there has been no confirmed report of their seed transmission. A percentage (38.2-78.0%) of the seedlings developed from the seeds of a petunia local cultivar under insect-free conditions showed stunting, interveinal chlorosis, leaf curling, and vein swelling symptoms, and were infected by BCTV when tested by PCR. Presence of BCTV in seed extracts of petunia local cultivar was confirmed by PCR and IC-PCR, followed by sequencing. Agroinoculation of curly top free petunia plants with a BCTV infectious clone resulted in BCTV infection of plants and their developed seeds. These results show the seed infection and transmission of BCTV in a local cultivar of petunia. Similar experiments performed with BCTIV showed that this virus is also seed transmissible in the same cultivar of petunia, although with a lower rate (8.8-18.5%). Seed transmission of curly top viruses may have significant implications in the epidemiology of these viruses.

PhOBF1, a petunia ocs element binding factor, plays an important role in antiviral RNA silencing.

Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway.

A petunia ethylene-responsive element binding factor, PhERF2, plays an important role in antiviral RNA silencing.

Virus-induced RNA silencing is involved in plant antiviral defense and requires key enzyme components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). However, the transcriptional regulation of these critical components is largely unknown. In petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter fragments of phytoene desaturase (PDS) or chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2- RNAi lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching). In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2 Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2, and AGO2 was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wild-type (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi lines were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing.

Molecular characterization of Chilli leaf curl viruses infecting new host plant Petunia hybrida in India.

Petunia hybrida is an important ornamental plant grown in many countries including India. It is a good model plant for the study of genetics and molecular biology. During a survey in 2013-2014, severe leaf curling was observed on most of the P. hybrida grown in the Sikar district, Rajasthan. The infected plants were analyzed for begomovirus infection by rolling circular amplification (RCA) and sequenced. Full length sequences confirmed the association of monopartite begomovirus with betasatellites. Phylogenetic analysis showed the highest percentage of identity with Chilli leaf curl virus (ChLCuV) and therefore considered to be an isolate of ChLCuV. Recombination analysis showed that ChLCuV has broadened its host range by recombination process. To the best our knowledge, this is the first report of natural occurrence of ChLCuV on P. hybrida in India.

Distribution of potato spindle tuber viroid in reproductive organs of petunia during its developmental stages.

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Virus-aided gene expression and silencing using TRV for functional analysis of floral scent-related genes.

Flower scent is a composite character determined by a complex mixture of low-molecular-weight volatile molecules. Despite the importance of floral fragrance, our knowledge on factors regulating these pathways remains sketchy. Virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE) are characterized by a simple inoculation procedure and rapid results as compared to transgenesis, allowing screening and characterization of scent-related genes. Here, we describe methods using TRV as a VIGS/VAGE vector for the characterization of scent-related genes, protein compartmentalization studies, and protein subcellular targeting.

Virus-induced gene silencing using begomovirus satellite molecules.

Virus-induced gene silencing (VIGS) has emerged as a powerful method for studying gene function. VIGS is induced by infecting a plant with a plant virus that has had its genome modified to include a sequence from the host gene to be silenced. DNAß and DNA1 are satellite and single-stranded DNA molecules associated with begomoviruses (family Geminiviridae). We converted DNAß and DNA1 into gene-silencing vectors. The VIGS vectors can induce silencing efficiently in many solanaceous plants. Here, we describe procedures for the use of these two gene-silencing vectors for VIGS in different hosts.

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance.

BACKGROUND: Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance. RESULTS: The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV. CONCLUSION: In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

Virus-induced gene silencing in ornamental plants.

Virus-induced gene silencing (VIGS) provides an attractive tool for high-throughput analysis of the functional effects of gene knockdown. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defense mechanism. The consequences of gene inactivation, even of key metabolic, regulatory, or embryo-lethal genes, can thus be readily analyzed. A number of viral vectors have been developed for VIGS; one of the most frequently employed is based on tobacco rattle virus (TRV) due to its wide host range, efficiency, ease of application, and limited disease symptoms. TRV-based VIGS comprises two vectors. One (RNA2) includes a multiple cloning site into which fragments of target genes can be inserted. We have shown that the TRV/VIGS system can simultaneously silence as many as five independent genes. TRV is a mosaic-type virus, and silencing also occurs in a mosaic pattern. It is therefore desirable to have a reporter that can show where target genes have been silenced. The photobleaching induced by silencing phytoene desaturase (PDS) and the loss of purple pigmentation induced by silencing chalcone synthase (CHS) have successfully been used to indicate the location of coordinate silencing of other target genes. In this chapter, we outline our protocols for the use of VIGS for analysis of gene function, focusing particularly on the use of TRV with petunia and tomato.