The quantification of messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction requires the availability of reference genes that are stably expressed regardless of the experimental conditions under study. We examined the expression variations of a set of eight candidate reference genes in tomato leaf and root tissues subjected to the infection of five taxonomically and molecularly different plant viruses and a viroid, inducing diverse pathogenic effects on inoculated plants. Parallel analyses by three commonly used dedicated algorithms, geNorm, NormFinder and BestKeeper, showed that different viral infections and tissues of origin influenced, to some extent, the expression levels of these genes. However, all algorithms showed high levels of stability for glyceraldehyde 3-phosphate dehydrogenase and ubiquitin, indicated as the most suitable endogenous transcripts for normalization in both tissue types. Actin and uridylate kinase were also stably expressed throughout the infected tissues, whereas cyclophilin showed tissue-specific expression stability only in root samples. By contrast, two widely employed reference genes, 18S ribosomal RNA and elongation factor 1α, demonstrated highly variable expression levels that should discourage their use for normalization. In addition, expression level analysis of ascorbate peroxidase and superoxide dismutase showed the modulation of the two genes in virus-infected tomato leaves and roots. The relative quantification of the two genes varied according to the reference genes selected, thus highlighting the importance of the choice of the correct normalization method in such experiments.

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Viral infections interfere with the microRNA (miRNA)-mediated regulation of gene expression, determining developmental defects. In tomato leaves, the accumulation levels of six miRNA species and their target transcripts corresponding to transcription factors with roles in plant development and leaf morphogenesis and two genes involved in the short RNA processing, DCL1 and AGO1, were significantly enhanced upon infection with the severe strain Cucumber mosaic virus (CMV)-Fny, while that of AGO4 was reduced. In plants harboring the infection of the mild strain CMV-LS, the effects on miRNA pathway were reduced, although AGO1, DCL1, and NAC1 also were shown to overaccumulate during infections exhibiting a mild phenotype. The use of the recombinant strain CMV-Fny(LS2b), in which the 3'-terminal region of CMV-Fny RNA 2, including the 2b coding sequence, was replaced with the corresponding region of CMV-LS RNA 2, provided evidence that the exchanged region was implicated in the perturbation of miRNA metabolism. In tomato plants infected with CMV-Fny supporting the ameliorative satellite (sat)RNA variant Tfn-satRNA, the symptomless phenotype correlated, with the exception of NAC1 upregulation, with the absence of effects on mitochondrial RNA and miRNA expression. Some of the aspects of miRNA pathway perturbation described were peculiar to CMV-tomato interactions and involved in the etiology of the disease phenotype elicited in this host.