MicroRNAs (miRNAs) are a newly identified class of non-coding small RNAs of about 21-24 nucleotides. They play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. To date, a total of 2,043 plant miRNAs are present in the miRNA Registry database (miRBase Release 14.0), and none for tobacco (Nicotiana tabacum). In this research, we used known plant miRNAs against both genomic survey sequence (GSS) and expressed sequence tags (EST) databases to search for potential miRNAs in tobacco. A total of 25 potential miRNAs were identified following a range of strict filtering criteria, and 33 potential targets of miRNAs were predicted by searching the tobacco Unigene database. Most of these miRNA targeting genes were predicted to encode transcription factors which play important roles in tobacco development. Additionally, real-time PCR assays were performed to profile the expression levels of 10 miRNAs after the infection of Cucumber mosaic virus (CMV) and Potato virus X (PVX). The results showed that symptom severity is correlated to the miRNA accumulation, and increased miR168 expression during virus infection is a common, plant- and virus-independent response.

Viral infections generally cause disease symptoms by interfering with the microRNA (miRNA)-mediated regulation of gene expression of host plants. In tomato leaves, the accumulation levels of eleven miRNAs and ten target mRNAs were enhanced by different degrees upon Cucumber mosaic virus (CMV)-Fny and Tomato aspermy virus (TAV)-Bj infections. The ability of CMV-Fny to interfere with miRNA pathway was dramatically suppressed in the addition of the benign satellite (sat) RNA variant (satYn12), but was slightly affected when CMV-Fny was co-inoculated with the aggressive satRNA variant (satT1). In plants harboring the infection of CMV-FnyΔ2b (a CMV-Fny 2b-deletion mutant), the unaltered miRNAs and target mRNAs levels compared with mock inoculated plants indicated that 2b ORF was essential for perturbation of miRNA metabolism. When the amounts of viral open reading frames (ORFs) in these infections were quantified, we found satYn12 caused a higher reduction of CMV-Fny accumulation levels than satT1. These results indicate the complex mechanism by which satRNAs participate in CMV-tomato interaction, and suggest that the severity of disease symptoms positively correlates to some extent with the perturbation of miRNA pathway in tomato.

MicroRNAs (miRNAs) play vital roles in regulating plant growth and development. Recent work has shown that miRNA-mediated regulation of cellular mRNA expression is involved in pathogen-host interactions. However, knowledge about the timing and spatial regulation of plant miRNA expression is still limited. Here, we use stem-loop real-time reverse transcription-polymerase chain reaction to quantify the expression changes of seven miRNAs and their target mRNAs in different tomato tissues during the pathogenic processes. Compared with mock-inoculated plants, the expression levels of investigated miRNAs and mRNAs were enhanced by different degrees upon Cucumber mosaic virus (CMV)-Fny and Tomato aspermy virus-Bj infections, but were almost unchanged in CMVFnyD2b (a CMV-Fny 2b-deletion mutant)-infected tomato seedlings. In addition, the obvious up-regulation of several miRNAs and target mRNAs in some tomato tissues suggested their special roles in these tissues' organogenesis and development. Temporal analyses also revealed that the expressions of these miRNAs and mRNAs were highly regulated by different viral infections. Taken together, the observed spatially and temporally changes in miRNAs and target mRNAs expression levels indicate the abilities of different viruses to interfere with miRNA pathway, and are correlated with their respective functions in phenotype determination in different tomato tissues.

Cucumber mosaic virus (CMV) is widely spread worldwide, causing typical systemic mosaic and other symptoms in tomato (Solanum lycopersicum). Host responses to CMV and molecular mechanisms associated with the development of disease symptoms caused by this virus in tomato are largely unexplored. To investigate plant responses activated during this interaction, we used microarray analysis to monitor changes in host gene expression during disease development. Compared with genes from mock-inoculated control plants, seedlings to adults, 214 of the 3313 tomato genes represented on the array were differentially expressed in CMV-infected plants. Functional classification of CMV-responsive genes revealed that CMV activated typical basal defense responses in the host during the infection process, including induction of defense-related genes, production and scavenging of free oxygen radicals, and hormone synthesis. CMV infection also suppressed a subset of host genes involved in photosynthesis and metabolism. Our results indicate that a wide range of genes play an important role in regulation of the tomato susceptibility response to CMV.