Virus Genes

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Gene expression and population polymorphism of maize Iranian mosaic virus in Zea mays, and intracellular localization and interactions of viral N, P, and M proteins in Nicotiana benthamiana

  • Abozar Ghorbani
  • Keramatollah Izadpanah
  • Ralf G. Dietzgen


Maize Iranian mosaic virus (MIMV; Mononegavirales, Rhabdoviridae, Nucleorhabdovirus) infects maize and several other poaceous plants. MIMV encodes six proteins, i.e., nucleocapsid protein (N), polymerase cofactor phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G), and large RNA-dependent RNA polymerase (L). In the present study, MIMV gene expression and genetic polymorphism of an MIMV population in maize were determined. N, P, P3, and M protein genes were more highly expressed than the 5′ terminal G and L genes. Twelve single nucleotide polymorphisms were identified across the genome within a MIMV population in maize from RNA-Seq read data pooled from three infected plants indicating genomic variations of potential importance to evolution of the virus. MIMV N, P, and M proteins that are known to be involved in rhabdovirus replication and transcription were characterized as to their intracellular localization and interactions. N protein accumulated exclusively in the nucleus and interacted with itself and with P protein. P protein accumulated in both the nucleus and cell periphery and interacted with itself, N and M proteins in the nucleus. M protein was localized in the cell periphery and on endomembranes, and interacted with P protein in the nucleus. MIMV proteins show a distinctive combination of intracellular localizations and interactions.


Maize rhabdovirus Protein–protein interactions Population polymorphism Viral gene expression 


Plant rhabdoviruses with unsegmented genomes are taxonomically classified into the two genera Nucleorhabdovirus and Cytorhabdovirus [1]. Members of the family Rhabdoviridae infect mammals, fish, plants, and arthropods [2]. The genomes of all rhabdoviruses encode a core set of five canonical structural proteins, i.e., nucleocapsid protein (N), polymerase cofactor phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L) that is a RNA-dependent RNA polymerase. These genes are organized in the conserved order 3ʹ–N–P–M–G–L–5ʹ and are separated by conserved intergenic regions, while the coding region is flanked by non-coding regulatory 3′ leader and 5′ trailer regions [3, 4]. Plant rhabdovirus genomes encode one or more additional accessory proteins between N-P, P-M, or G-L genes [5].

Maize Iranian mosaic virus (MIMV) is an economically important virus in several regions of Iran. In addition to maize, it infects wheat, barley, rice, and several other gramineous plant species [6]. The virus is transmitted by the planthopper Laodelphax striatellus in a persistent-propagative manner [7, 8]. There is no close serological relationship between MIMV and other rhabdoviruses infecting gramineous plants such as maize mosaic virus (MMV), barley yellow striate mosaic virus (BYSMV), and cynodon chlorotic streak virus (CCSV), but a close evolutionary relationship of MIMV with MMV and taro vein chlorosis virus (TaVCV) was reported based on genome sequence identity [9]. MIMV genome has been completely sequenced and encodes six genes in the order 3′-N-P-P3-M-G-L-5′ with P3 representing a putative cell-to-cell movement protein [8, 10].

RNA-Seq technology provides a powerful high-throughput tool to comprehensively assess genome-wide transcript expression. Also, RNA-Seq can be used to determine single-nucleotide polymorphisms (SNPs) [11] of viral genomes within a replicating population. Mutations that occur during viral replication can accumulate, forming microvariants that deviate from a consensus master sequence by one or more SNPs within a population. These SNPs may be the basis for the formation of viral quasi-species that can lead to resistance-breaking strains in some hosts [12, 13].

Considering the lack of reverse genetics systems for most negative-sense RNA plant viruses, localization and interaction studies of several nucleo- and cytorhabdovirus proteins have provided valuable information on potential functions of viral proteins in the host cell during replication and movement [14, 15, 16, 17, 18, 19]. However, this information is neither available for MIMV nor the related nucleorhabdoviruses MMV and TaVCV. The present paper addresses these questions in regard to MIMV.

Materials and methods

MIMV infection of maize plants

Seeds of maize (Zea mays) cultivar 704 (Agriculture and Natural Resources Research Center of Fars, Shiraz, Iran) were germinated and grown in a greenhouse on a cycle of 16 h light at 30 °C and 8 h dark at 25 °C. Viruliferous Laodelphax striatellus were used to infect maize seedlings with MIMV. Three days after germination (at the two-leaf stage) ten plants were exposed for 3 days to 20 viruliferous planthopper nymphs that were infected with MIMV isolate ‘Fars’ (Genbank accession MF102281) and had been maintained on MIMV-infected barley plants in an insect-proof cage. Control plants of the same age were exposed to 20 planthoppers from a virus-free colony. Plants were maintained in insect-proof cages until appearance of MIMV symptoms.

RNA extraction, and transcript sequencing and analysis

Leaf tissues for RNA extraction were individually taken from three uninfected and three infected plants after the appearance of the first symptoms, 14 days post inoculation (dpi). Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Libraries were prepared and sequenced using an Illumina-Hiseq 2500 machine (Macrogen, Seoul, South Korea). Cleaned raw transcript reads from MIMV-infected maize were mapped to the MIMV complete genome (accession number MF102281) using CLC Genomics Workbench version 9 software. SNPs were determined using Geneious R10 software (Biomatters, New Zealand).

MIMV protein localization and interactions

Complementary DNA was synthesized using SuperScript III First-Strand Synthesis SuperMix (Life Technologies) and random primers following the manufacturer’s instructions. Complete N, P, and M ORFs were amplified using gene-specific primers that incorporated attB recombination sites using Phusion High Fidelity polymerase (New England Biolabs). PCR amplicons were gel-purified (Wizard gel purification kit, Promega) and cloned into plasmid pDONR221 (Life Technologies) using BP Clonase II Enzyme Mix. Chemically competent Omnimax Escherichia coli (Life Technologies) were transformed using the heat shock method and recombinant colonies were selected on Luria–Bertani agar containing 50 mg/L kanamycin. Plasmid DNA was extracted using the GeneJet plasmid purification kit (Thermo Fisher Scientific). Sequence-validated Gateway entry clones were recombined into pSITE destination vectors using LR Clonase II Enzyme Mix (Life Technologies) as fusions to the C-terminus of green fluorescent protein (GFP) for localization, and yellow fluorescent protein (YFP) fragments for bimolecular fluorescence complementation (BiFC) assays. Recombinant pSITE vectors carrying N, P, and M ORFs of MIMV were identified by colony PCR (AmpliTaq Gold- Fast PCR, Applied Biosystems, Thermo Fisher Scientific) and purified plasmids were individually transformed into Agrobacterium tumefactions LBA 4404. For transient expression of fluorescently labeled fusion proteins, agrobacterium cells harboring the desired pSITE vector were infiltrated at 0.7–0.8 optical density (OD600) units into the abaxial side of N. benthamiana leaves using 1-mL syringe and gentle pressure. Expression of fusion proteins and determination of intracellular localization and interactions were done two days after agroinfiltration as previously described (Tsai et al. [18]). For localization studies, transgenic N. benthamiana expressing red florescent protein (RFP) fused to histone 2B (RFP-H2B) in the nucleus [20] or plants co-infiltrated with mCherry-ER plasmid [21] and for BiFC protein–protein interaction assays, cyan fluorescent protein fused to histone 2B (CFP-H2B) transgenic marker plants were used. For BiFC assays viral proteins were expressed as C-terminal fusions to the amino- or carboxy-terminal portions of YFP using pSITE-BiFC-nEYFP and pSITE-BiFC-cEYFP vectors [20]. At least four independent repetitions were done for each experiment and three images captured for each. Confocal laser scanning microscopy was done on a Zeiss LSM-700 microscope; images were acquired using Zen 2012 Lite software and exported as tiff or jpeg files [22].

Results and discussion

Expression of MIMV genes in maize during virus infection

Transcription of the rhabdoviral genome is controlled by a transcriptase complex composed of viral N, P, and L proteins [3, 23]. Rhabdovirus transcript expression levels are controlled by disconnection of the transcriptase complex from the genome template once it reaches the transcription termination polyadenylation signal at the end of each viral gene [3]. We used RNA-Seq data to determine the number of viral transcripts that accumulated in maize during a systemic infection with MIMV. A total of 8,981,914 out of 130,219,916 reads (6.9%) from three biological replicates mapped to the MIMV genome. MIMV N, P, M, and P3 genes that are located towards the 3′ terminus of the genome were consistently more highly expressed than the 5′ terminal G and L genes, based on the number of specific transcripts identified in RNA-Seq (Fig. 1). One-way ANOVA p < 0.01 showed no significant difference in viral transcript levels between N, P, M, and P3, whereas their levels were significantly higher than those of G and L transcripts. The L gene showed the lowest transcript level of all MIMV genes (Fig. 1). These results partly agree with previous studies on vesicular stomatitis Indiana virus (VSIV, genus Vesiculovirus) [23] and a recent study on MIMV using quantitative PCR [24] which showed that the relative abundance of each viral mRNA was inversely related to the gene distance from the genomic 3′ end [23, 24]. However, in our study at 14 dpi, we did not find statistically significant differences in the expression of the first four mRNAs nearest the 3′ end. This may be due to the stage of systemic infection or the detection method used, or it may reflect mRNA stability or turnover. Interestingly, in the other MIMV study [24], descending transcript levels were clearly seen at 21 and 28 dpi, but the data at 14 dpi were similar to our results.
Fig. 1

Expression levels (Reads per kilobase of transcript per million mapped reads, RPKM) of maize Iranian mosaic virus (MIMV) ORFs in RNA-seq data of maize infected with MIMV, 14 days post planthopper-mediated inoculation. The mean and standard deviation of 3 biological replicates is shown. One-way ANOVA (p < 0.01) was used for statistical analysis. Values with common letters are not significantly different at 1% probability level

Genome variation in an MIMV population in maize

In the RNA-Seq analysis, some of the reads could not be mapped completely (nucleotide mismatch) to the reference genome indicating a polymorphic population of genome sequences. Detailed sequence analysis of the population of almost 9 million MIMV reads identified 12 SNPs. Positions of SNPs in the genome were in ORF L (4 SNPs), ORF P (2 SNPs), ORF P3 (1 SNP), 5′ trailer (3 SNPs), 3′ leader (1 SNP), and 3′ UTR of P3 gene (1 SNP). A SNP A → G (49.50% variant frequency across all MIMV-specific reads) occurred in P3 ORF and represented the only polymorphism that led to a change in the encoded amino acid from arginine to glycine (Table 1). P3 is a putative movement protein [4], but it is not known if the identified amino acid change in P3 ORF may affect cell-to-cell movement of this variant in maize. A two-nucleotide substitution TG to GA (12.7% variant frequency) was identified in the 5′ trailer. The variant frequency seen in this study ranged from 12.7 to 49.50%, when we applied a threshold for SNP detection of 12%, which is in the range of 5–40% used in previous studies [25, 26, 27]. This variation may be required for infection and/or adaptation to a particular cell type. Genetic diversity within virus populations is the basis of quasi-species [28]. SNPs in non-coding regions of MIMV may affect binding to putative host factors during replication and transcription, but this has not yet been studied in plant rhabdoviruses.
Table 1

Single-nucleotide polymorphisms (SNPs) among a population of maize Iranian mosaic virus genome RNA-Seq reads in maize

Nucleotide position in (+) sense (3′ to 5′)

Amino acid change

Genome position

Nucleotide change

Codon change

Variant frequency (%)


3′ leader

A –› C




T –› C





T –› C




R –› G


A –› G




P3 utr

T –› A




G –› T





C –› T





C –› T





T –› C




5′ trailer

T –› G



5′ trailer

G –› A



5′ trailer

T –› G


Intracellular localization of MIMV structural proteins N, P, and M in N. benthamiana

A number of nuclear and cytoplasmic plant rhabdoviruses have been studied for the intracellular localization and interactions of their proteins [15, 16, 17, 18, 19, 29]. Here, we studied the localization and interactions of the structural proteins N, P, and M of MIMV because of their importance in rhabdovirus replication and transcription [3]. Current data support the assumption that nucleorhabdoviruses establish virus replication factories in the nuclei of infected plant cells [16, 17, 18, 19]. MIMV N, P, and M ORFs were transiently expressed as fusions to the C-terminus of GFP to determine intracellular localization by live cell imaging using confocal laser scanning microscopy. Free GFP localized to the cell periphery and diffused into the nucleus due to its small size. GFP-N fusion localized exclusively in the nucleus (Fig. 2), similar to the localization reported for N protein of several other nucleorhabdoviruses [17, 18, 19]. Also, N protein of dichorhaviruses orchid fleck virus (OFV) and coffee ringspot virus (CoRSV) that are related to nucleorhabdoviruses [2] accumulated in the nucleus [30, 31]. In contrast, N proteins of cytorhabdoviruses such as lettuce necrotic yellows virus (LNYV) and alfalfa dwarf virus (ADV) localized in the cytoplasm of plant cells [15, 29]. GFP-P localized mostly not only in the nucleus as many punctate aggregates (95% of nuclei observed) or uniformly distributed (5%), but also in the cell periphery (Fig. 2). This localization pattern was similar to that of P protein of datura yellow vein virus (DYVV), sonchus yellow net virus (SYNV), and maize fine streak virus (MFSV) that accumulated both in the nucleus and cell periphery when overexpressed. The P protein of potato yellow dwarf virus (PYDV), like that of MIMV, accumulated in punctate loci within nuclei [16, 17, 18, 19].
Fig. 2

Confocal micrographs showing intracellular localization in Nicotiana benthamiana cells of transiently expressed maize Iranian mosaic virus (MIMV) proteins. Green fluorescent protein (GFP) fusions to MIMV N, P, and M proteins, and N+P proteins (co-expression of N and P proteins that were both GFP-tagged) were individually expressed from pSITE-2CA vector in agroinfiltrated N. benthamiana epidermal leaf cells and confocal laser scanning microscope images taken after 2–3 days. Cultures of recombinant agrobacteria harboring MIMV constructs were infiltrated into transgenic red fluorescent protein-Histone 2B marker plants. The left panel shows GFP-fusions, middle panel red nuclear marker, and right panel the merged images of both channels. Experiments were repeated 3 times and at least 50 cells were viewed per construct. Scale bars represent 20 μM

Previous studies have shown that co-expression of N and P proteins of nucleorhabdoviruses alters their individual localization patterns and may lead to import of P into the nucleus [15, 16, 18, 19, 29, 32]. On the other hand, in cytorhabdoviruses, N and P proteins form aggregates in the cytoplasm. In the present study, co-expression of GFP-tagged MIMV N and P proteins led to the accumulation of both N and P proteins in the nucleus (Fig. 2), suggesting a potential interaction between these proteins.

The localization of MIMV M protein appears to be unique among nucleorhabdoviruses and similar to M protein of CoRSV and cytorhabdoviruses ADV and LNYV [16, 18, 19, 29, 30]. GFP-M localized mostly to endoplasmic reticulum (ER) membranes, including the nuclear membrane and cell periphery (Fig. 2), strongly co-localizing with the mCherry-ER marker (Fig. S1). On the other hand, M proteins of nucleorhabdoviruses DYVV, SYNV, PYDV, and MFSV localized exclusively in the nucleus [16, 18, 19, 33]. Localization of MIMV M protein on the plasma membrane and nuclear membrane supports its role in condensation of the viral ribonucleoprotein complex during virion assembly [2].

Intracellular interactions of MIMV structural proteins N, P, and M in N. benthamiana

BiFC is an established technology that shows both the strongest protein–protein interactions and their localizations in plant cells, and has been widely applied to the study of plant rhabdovirus proteins [14, 15, 16, 17, 18, 19, 29, 34]. None of the tested MIMV proteins showed any interaction with glutathione S-transferase (GST) non-binding control in either orientation (Fig. 3, panels N/GST, P/GST, and data not shown). We detected both homotypic (self) and heterotypic interactions between MIMV core proteins. Self-interaction of MIMV N protein was detected exclusively in the nucleus (Fig. 3, panel N/N), similar to that of PYDV N protein [17]. N:N self-interaction is thought to be essential for RNA binding and it has been demonstrated for all rhabdoviruses studied to date. N proteins of rhabdoviruses are known to be involved in regulating replication and transcription of the viral genome and to be tightly associated with the genomic RNA at all times [2, 3].
Fig. 3

Confocal micrographs showing maize Iranian mosaic virus N, P, and M protein–protein interactions determined by bimolecular fluorescence complementation (BiFC). Interaction assays were conducted in leaf epidermal cells of transgenic Nicotiana benthamiana expressing cyan fluorescent protein fused to the nuclear marker histone 2B (CFP-H2B). Shown are the localization of CFP-H2B (nucleus, column 1) interaction assay (BiFC, column 2), and an overlay of the two preceding panels (merge, column 3). Proteins listed first in the pair of interactors were expressed as fusions to the amino-terminal half of yellow fluorescent protein (YFP). Those listed second were expressed as fusions to the carboxy-terminal half of YFP. Protein fusions to each half of YFP were tested in all pairwise combinations, of which a subset is shown here. Representative results using glutathione S-transferase (GST) fusions as non-binding negative controls with the MIMV proteins are shown as N/GST and P/GST. Experiments were repeated at least 3 times and a minimum of 50 cells viewed per construct. Scale bars represent 20 μM

Self-interaction of MIMV P protein in aggregates in the nucleus, where it excluded nuclear DNA (Fig. 3, panel P/P), was similar to that reported for P proteins of SYNV, ADV, and LNYV [15, 19, 29]. Transiently expressed GFP-P was detected in both nucleus and cytoplasm (Fig. 2), but MIMV P:P interaction was observed only in the nucleus (Fig. 3). MIMV P:P interaction appeared as punctate aggregates in the nucleus similar to P protein self-interaction of ADV, an unusual cytorhabdovirus with nuclear P protein accumulation [29].

We detected MIMV N:P interaction (Fig. 3, panels N/P, P/N) that is conserved in all rhabdoviruses where protein–protein interactions have been studied [15, 16, 17, 18, 19, 29, 30, 31, 35]. This interaction was localized in the nucleus as in ADV, SYNV, PYDV, CoRSV, and OFV [14, 17, 29, 30, 31] but different from LNYV N:P interaction which was localized in the cytoplasm [15]. N:P:L complex has an essential role in genome replication and binding of nascent RNA to the N protein via sugar-phosphate interactions [35]. MIMV P and M proteins interacted in the nucleus in small aggregates (Fig. 3, panels P/M, M/P) similar to the P:M interaction seen for ADV; P:M interaction has however not been reported for the nucleorhabdoviruses PYDV and SYNV [14, 15, 17, 29].

MIMV M:M interaction was not detected although this interaction is otherwise conserved in all plant rhabdoviruses [14, 15, 17]. This may be due to the low concentration of M protein in the nucleus as MIMV M protein mostly accumulated in the cell periphery. Heterotypic interaction of N:M which has been reported for PYDV [17] was not observed in MIMV. The demonstrated P:P, P:N, P:M, and N:N protein interactions support the essential role of these viral nucleocapsid core proteins in regulation of MIMV genome structure, replication, and transcription.

In conclusion, our study of viral gene expression and MIMV genome diversity indicated the genome plasticity of this nucleorhabdovirus, and sequence variability and genetic potential of MIMV in maize. MIMV core protein intracellular localization and interactions were slightly different from those of other nucleorhabdoviruses and this may be related to partial accumulation of virus particles in the cytoplasm as reported by Massah and colleagues [7]. This study increased our knowledge about MIMV N, P, and M proteins and provided evidence of a polymerase complex composed of at least N, P, and M proteins (L protein was not included in this study) thought to be involved in viral transcription and replication.



This research was jointly supported by the Queensland Department of Agriculture and Fisheries and the University of Queensland through the Queensland Alliance for Agriculture and Food Innovation. AG was supported by a scholarship from Shiraz University. Funding for RNA-Seq analysis was provided by Plant Virology Research Center of Shiraz University. We thank Kerry Vinall (University of Queensland) for training AG and expert advice in confocal microscopy.

Author contributions

AG, KI, and RGD designed the study; AG performed the experiments; AG and RGD analyzed the data; AG drafted the manuscript, and all authors edited and approved the final version.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11262_2018_1540_MOESM1_ESM.docx (1.4 mb)
Supplementary material 1 (DOCX 1387 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Abozar Ghorbani
    • 1
    • 2
  • Keramatollah Izadpanah
    • 1
  • Ralf G. Dietzgen
    • 2
  1. 1.College of AgriculturePlant Virology Research Center, Shiraz UniversityShirazIran
  2. 2.Queensland Alliance for Agriculture and Food InnovationThe University of QueenslandSt. LuciaAustralia

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