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

In recent years, a new class of viral noncoding subgenomic (ncsg)RNA has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant (xr) RNA structure, which blocks the progression of 5’→3’ exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate: (i) absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wildtype RCNMV or an SR1f-deficient mutant RCNMV revealed that wt RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10 to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3’ region of RCNMV RNA1 support the premature mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses. IMPORTANCE Knowledge of how RNA viruses manipulate host and viral gene expression is crucial to our understanding of infection and disease. Unlike viral protein-host interactions, little is known about the control of gene expression by viral RNA. Here we begin to address this question by investigating the noncoding subgenomic (ncsg)RNA of red clover necrotic mosaic virus (RCNMV), called SR1f. Similar exoribonuclease-resistant RNAs of flaviviruses are well-studied, but the roles of plant viral ncsgRNAs, and how they arise, are poorly understood. Surprisingly, we find the likely exonuclease candidate, XRN4, is not required to generate SR1f, and we assess the effects of SR1f on virus accumulation and symptom development. Finally, we compare the effects of infection by wildtype RCNMV vs an SR1f-deficient mutant on host gene expression in Nicotiana benthamiana , which reveals that ncsgRNAs such as SR1f are key players in virus-host interactions to facilitate productive infection.

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Virus-Mediated Targeted DNA Methylation Illuminates the Dynamics of Methylation in an Endogenous Plant Gene

International Journal of Molecular Sciences ◽

10.3390/ijms22084125 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4125

Author(s):

Go Atsumi ◽

Kouki Matsuo ◽

Noriho Fukuzawa ◽

Takeshi Matsumura

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Genomic Dna ◽

Genome Stability ◽

Sequence Identity ◽

Endogenous Sequence ◽

Insight Into ◽

Endogenous Plant

DNA methylation maintains genome stability and regulates gene expression in plants. RNA-directed DNA methylation (RdDM) is critical for appropriate methylation. However, no efficient tools are available for the investigation of the functions of specific DNA methylation. In this study, the cucumber mosaic virus vector was used for targeted DNA methylation. Methylation was rapidly induced but gradually decreased from the 3′ end of the target endogenous sequence in Nicotiana benthamiana, suggesting a mechanism to protect against the ectopic introduction of DNA methylation. Increasing 24-nt siRNAs blocked this reduction in methylation by down-regulating DCL2 and DCL4. RdDM relies on the sequence identity between RNA and genomic DNA; however, this identity does not appear to be the sole determinant for efficient DNA methylation. The current findings provide new insight into the regulation of DNA methylation and promote additional effort to develop efficient targeted DNA methylation in plants.

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Identification of maize mosaic virus by immunoelectron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142827 ◽

1986 ◽

Vol 44 ◽

pp. 240-241

Author(s):

O. E. Bradfute

Keyword(s):

Zea Mays ◽

Mosaic Virus ◽

Immunoelectron Microscopy ◽

Severe Disease ◽

Maize Mosaic Virus ◽

The World ◽

Plant Rhabdovirus

Maize mosaic virus (MMV) causes a severe disease of Zea mays in many tropical and subtropical regions of the world, including the southern U.S. (1-3). Fig. 1 shows internal cross striations of helical nucleoprotein and bounding membrane with surface projections typical of many plant rhabdovirus particles including MMV (3). Immunoelectron microscopy (IEM) was investigated as a method for identifying MMV. Antiserum to MMV was supplied by Ramon Lastra (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela).

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Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PLoS ONE ◽

10.1371/journal.pone.0046451 ◽

2012 ◽

Vol 7 (9) ◽

pp. e46451 ◽

Cited By ~ 176

Author(s):

Deshui Liu ◽

Lindan Shi ◽

Chenggui Han ◽

Jialin Yu ◽

Dawei Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Nicotiana Benthamiana ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Expression Studies ◽

Gene Expression Studies

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Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽

10.3390/plants10030524 ◽

2021 ◽

Vol 10 (3) ◽

pp. 524

Author(s):

Bingqi Wu ◽

Zhiting Chen ◽

Xiaohui Xu ◽

Ronghua Chen ◽

Siwei Wang ◽

...

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Nicotiana Benthamiana ◽

Heterologous Gene Expression ◽

Expression System ◽

Functional Characterization ◽

Transient Gene Expression ◽

High Mobility ◽

Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

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Genome-Wide Identification and Characterization of Long Noncoding RNAs Involved in Chinese Wheat Mosaic Virus Infection of Nicotiana benthamiana

Biology ◽

10.3390/biology10030232 ◽

2021 ◽

Vol 10 (3) ◽

pp. 232

Author(s):

Weiran Zheng ◽

Haichao Hu ◽

Qisen Lu ◽

Peng Jin ◽

Linna Cai ◽

...

Keyword(s):

Virus Infection ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Noncoding Rnas ◽

Long Noncoding Rnas ◽

Differentially Expressed ◽

Genome Wide ◽

Chinese Wheat

Recent studies have shown that a large number of long noncoding RNAs (lncRNAs) can regulate various biological processes in animals and plants. Although lncRNAs have been identified in many plants, they have not been reported in the model plant Nicotiana benthamiana. Particularly, the role of lncRNAs in plant virus infection remains unknown. In this study, we identified lncRNAs in N. benthamiana response to Chinese wheat mosaic virus (CWMV) infection by RNA sequencing. A total of 1175 lncRNAs, including 65 differentially expressed lncRNAs, were identified during CWMV infection. We then analyzed the functions of some of these differentially expressed lncRNAs. Interestingly, one differentially expressed lncRNA, XLOC_006393, was found to participate in CWMV infection as a precursor to microRNAs in N. benthamiana. These results suggest that lncRNAs play an important role in the regulatory network of N. benthamiana in response to CWMV infection.

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Chloroplast Hsp70 Isoform Is Required for Age-Dependent Tissue Preference of Bamboo mosaic virus in Mature Nicotiana benthamiana Leaves

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0012-r ◽

2017 ◽

Vol 30 (8) ◽

pp. 631-645 ◽

Cited By ~ 3

Author(s):

Ying Wen Huang ◽

Chung Chi Hu ◽

Ching Hsiu Tsai ◽

Na Sheng Lin ◽

Yau Heiu Hsu

Keyword(s):

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Transit Peptide ◽

Plant Viruses ◽

Age Dependent ◽

Efficient Replication ◽

Underlying Mechanisms ◽

Lines Of Evidence ◽

Suitable Environment

Plant viruses may exhibit age-dependent tissue preference in their hosts but the underlying mechanisms are not well understood. In this study, we provide several lines of evidence to reveal the determining role of a protein of the Nicotiana benthamiana chloroplast Hsp70 (NbcpHsp70) family, NbcpHsp70-2, involved in the preference of Bamboo mosaic virus (BaMV) to infect older tissues. NbcpHsp70 family proteins were identified in complexes pulled down with BaMV replicase as the bait. Among the isoforms of NbcpHsp70, only the specific silencing of NbcpHsp70-2 resulted in the significant decrease of BaMV RNA in N. benthamiana protopalsts, indicating that NbcpHsp70-2 is involved in the efficient replication of BaMV RNA. We further identified the age-dependent import regulation signal contained in the transit peptide of NbcpHsp70-2. Deletion, overexpression, and substitution experiments revealed that the signal in the transit peptide of NbcpHsp70-2 is crucial for both the import of NbcpHsp70-2 into older chloroplasts and the preference of BaMV for infecting older leaves of N. benthamiana. Together, these data demonstrated that BaMV may exploit a cellular age-dependent transportation mechanism to target a suitable environment for viral replication.

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Zea mays RNA-seq estimated transcript abundances are strongly affected by read mapping bias

BMC Genomics ◽

10.1186/s12864-021-07577-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Shuhua Zhan ◽

Cortland Griswold ◽

Lewis Lukens

Keyword(s):

Gene Expression ◽

Zea Mays ◽

Reference Genome ◽

Transcript Abundance ◽

Gene Transcript ◽

Rna Seq ◽

Individual Genome ◽

Abundance Estimates ◽

Mapping Bias ◽

Quantify Gene Expression

Abstract Background Genetic variation for gene expression is a source of phenotypic variation for natural and agricultural species. The common approach to map and to quantify gene expression from genetically distinct individuals is to assign their RNA-seq reads to a single reference genome. However, RNA-seq reads from alleles dissimilar to this reference genome may fail to map correctly, causing transcript levels to be underestimated. Presently, the extent of this mapping problem is not clear, particularly in highly diverse species. We investigated if mapping bias occurred and if chromosomal features associated with mapping bias. Zea mays presents a model species to assess these questions, given it has genotypically distinct and well-studied genetic lines. Results In Zea mays, the inbred B73 genome is the standard reference genome and template for RNA-seq read assignments. In the absence of mapping bias, B73 and a second inbred line, Mo17, would each have an approximately equal number of regulatory alleles that increase gene expression. Remarkably, Mo17 had 2–4 times fewer such positively acting alleles than did B73 when RNA-seq reads were aligned to the B73 reference genome. Reciprocally, over one-half of the B73 alleles that increased gene expression were not detected when reads were aligned to the Mo17 genome template. Genes at dissimilar chromosomal ends were strongly affected by mapping bias, and genes at more similar pericentromeric regions were less affected. Biased transcript estimates were higher in untranslated regions and lower in splice junctions. Bias occurred across software and alignment parameters. Conclusions Mapping bias very strongly affects gene transcript abundance estimates in maize, and bias varies across chromosomal features. Individual genome or transcriptome templates are likely necessary for accurate transcript estimation across genetically variable individuals in maize and other species.

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