Differential gene expression and physiological changes during acute or persistent plant virus interactions may contribute to viral symptom differences

Viruses have different strategies for infecting their hosts. Fast and acute infections result in the development of severe symptoms and may cause the death of the plant. By contrast, in a persistent interaction, the virus can survive within its host for a long time, inducing only mild symptoms. In this study, we investigated the gene expression changes induced in CymRSV-, crTMV-, and TCV-infected Nicotiana benthamiana and in PVX- and TMV-U1-infected Solanum lycopersicum plants after the systemic spread of the virus by two different high-throughput methods: microarray hybridization or RNA sequencing. Using these techniques, we were able to clearly differentiate between acute and persistent infections. We validated the gene expression changes of selected genes by Northern blot hybridization or by qRT-PCR. We show that, in contrast to persistent infections, the drastic shut-off of housekeeping genes, downregulation of photosynthesis-related transcripts and induction of stress genes are specific outcomes with acute infections. We also show that these changes are not a consequence of host necrosis or the presence of a viral silencing suppressor. Thermal imaging data and chlorophyll fluorescence measurements correlated very well with the molecular changes. We believe that the molecular and physiological changes detected during acute infections mostly contribute to virus symptom development. The observed characteristic physiological changes associated with economically more dangerous acute infections could serve as a basis for the elaboration of remote monitoring systems suitable for detecting developing virus infections in crops. Moreover, as molecular and physiological changes are characteristics of different types of virus lifestyles, this knowledge can support risk assessments of recently described novel viruses

Additional file 6: Figure S4. of Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis

Nb_miRC8 precursor. A) Secondary structure for Nb_miRC8. The 5′ end of the RNA is marked by a circle. The candidant miRNAs labelled with different colours. B) Northen blot expressions for the two Nb_miRC8 candidants (Nb_miRC8_3p_a, Nb_miRC8_3p_b). An U6-specific probe was used to detect U6 RNA as a loading control for each membrane. (PDF 398 kb

Additional file 1 of Rapid and cost-effective molecular karyotyping in wheat, barley, and their cross-progeny by chromosome-specific multiplex PCR

Supplementary Material 1: Genotypes used for primer design, in silico analysis, and MPCR validatio

Additional file 2: Figure S1. of Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis

Quantification of miRNAs by RT-qPCR. Validation of (A) Nb-miRNA167 and (B) Nb-miR482 expression by RT-qPCR. Northern blot validation of conserved miRNAs (Fig. 4.) compared to RT-qPCR quantification. In both experiments the reference gene was U6. (PDF 134 kb

Additional file 4: Table S2. of Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis

Targets of conserved and other known miRNAs in samples. (XLSX 44 kb

Expression pattern and sequence length composition of 24-nt-long hetsiRNA-producing clusters.

<p>Heat map showing (A) the log2 transformed expression levels (Read Per Million) and (B) sequence length composition of the 50 most abundant clusters, and (C) Northern blot analyses of the expression of Cluster 297979 and Cluster 461037.</p

Small RNA Northern blot analyses of selected miRNAs.

<p>Expression of the conservative (green) and the newly predicted (red) miRNAs. The total RNA samples from the seed, the placenta and the flesh at 28 and 40 days after anthesis (DAA) were run on agarose gels, transferred to nylon membranes, and hybridized with probes detecting miRNAs as indicated. We show the NGS read counts (read/million) in the upper panels, the results of small RNA northern blots in the middle panels, and the rRNA levels as loading controls in the lower panels. We used the same membrane for the hybridization of <i>miR171</i>, <i>miR396</i>, and <i>miR159</i>, <i>can-mir-f13</i> specific probes.</p

Summary of the read numbers along the filtering steps in the small RNA libraries.

<p>Summary of the read numbers along the filtering steps in the small RNA libraries.</p

Heat map showing the expression levels of the differentially expressed miRNAs.

<p>The differential expression analysis between the seed (S), the placenta (P), and the flesh (F) samples at (A) 28 DAA and (B) 40 DAA was conducted using DESeq2. The results were filtered to keep only those miRNAs whose mean normalized expression level was at least 10, and the expression change was at least six-fold. We colored the conservative (black), known (blue) and novel (red) miRNAs. We found 21 differentially expressed miRNAs between different stages (28, 40) in the flesh (C), 28 miRNAs in the seed (D), and none in the placenta. Arrows represent up- (↑) and down-regulation (↓) compared to the two other tissues (for example S↑ means, the expression of the marked miRNA are upregulated in seed compared to both, placenta and flesh. We also listed those changes, which occurred only between two tissues. For example, we marked a miRNA up-regulation between the placenta and the seed like this: P→S↑.</p

Expansion of <i>Capsicum annum</i> fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles

<div><p>Small regulatory RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important transcriptional and post-transcriptional regulators controlling a wide variety of physiological processes including fruit development. Data are, however, limited for their potential roles in developmental processes determining economically important traits of crops. The current study aimed to discover and characterize differentially expressed miRNAs and siRNAs in sweet pepper (<i>Capsicum annuum</i>) during fruit expansion. High-throughput sequencing was employed to determine the small regulatory RNA expression profiles in various fruit tissues, such as placenta, seed, and flesh at 28 and 40 days after anthesis. Comparative differential expression analyses of conserved, already described and our newly predicted pepper-specific miRNAs revealed that fruit expansion is accompanied by an increasing level of miRNA-mediated regulation of gene expression. Accordingly, ARGONAUTE1 protein, the primary executor of miRNA-mediated regulation, continuously accumulated to an extremely high level in the flesh. We also identified numerous pepper-specific, heterochromatin-associated 24-nt siRNAs (hetsiRNAs) which were extremely abundant in the seeds, as well as 21-nt and 24-nt phased siRNAs (phasiRNAs) that were expressed mainly in the placenta and the seeds. This work provides comprehensive tissue-specific miRNA and siRNA expression landscape for a developing pepper fruit. We identified several novel, abundantly expressing tissue- and pepper-specific small regulatory RNA species. Our data show that fruit expansion is associated with extensive changes in sRNA abundance, raising the possibility that manipulation of sRNA pathways may be employed to improve the quality and quantity of the pepper fruit.</p></div