SUMMARY: 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.

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.

Significance: RNAi is used to elucidate gene functions also in phytopathogenic fungi, where it is expressed by hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. These approaches have drawbacks ranging from instability of hairpin constructs to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in phytopathogenic fungi by direct transfection with a plant virus-based vector and that the approach also can be used to obtain foreign protein expression in fungi. This technique could find wider application for functional genomics in filamentous fungi of biomedical and phytopathological interest.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVY-SON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

Grafting is routinely implemented in modern agriculture to manage soilborne pathogens such as fungi, oomycetes, bacteria, and viruses of solanaceous crops in a sustainable and environmentally friendly approach. Some rootstock/scion combinations use specific genetic resistance mechanisms to impact also some foliar and airborne pathogens, including arthropod or contact-transmitted viruses. These approaches resulted in poor efficiency in the management of plant viruses with superior virulence such as the strains of tomato spotted wilt virus breaking the Sw5 resistance, strains of cucumber mosaic virus carrying necrogenic satellite RNAs, and necrogenic strains of potato virus Y. Three different studies from our lab documented that suitable levels of resistance/tolerance can be obtained by grafting commercial tomato varieties onto the tomato ecotype Manduria (Ma) rescued in the framework of an Apulian (southern Italy) regional program on biodiversity. Here we review the main approaches, methods, and results of the three case studies and propose some mechanisms leading to the tolerance/resistance observed in susceptible tomato varieties grafted onto Ma as well as in self-grafted plants. The proposed mechanisms include virus movement in plants, RNA interference, genes involved in graft wound response, resilience, and tolerance to virus infection.