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.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells 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 the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.transfection | plant pathogen adaptation | host species jump

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.

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.

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Grafting of commercial tomato varieties and hybrids on the tomato ecotype Manduria resulted in high levels of tolerance to the infection of Sw5 resistance-breaking strains of tomato spotted wilt virus and of severe cucumber mosaic virus strains supporting hypervirulent satellite RNAs that co-determine stunting and necrotic phenotypes in tomato. To decipher the basis of such tolerance, here we used a RNAseq analysis to study the transcriptome profiles of the Manduria ecotype and of the susceptible variety UC82, and of their graft combinations, exposed or not to infection of the potato virus Y recombinant strain PVY c-to. The analysis identified graft-and virus-responsive mRNAs differentially expressed in UC82 and Manduria, which led to an overall suitable level of tolerance to viral infection confirmed by the appearance of a recovery phenotype in Manduria and in all graft combinations. The transcriptome analysis suggested that graft wounding and viral infection had diverging effects on tomato transcriptome and that the Manduria ecotype was less responsive than the UC82 to both graft wounding and potyviral infection. We propose that the differential response to the two types of stress could account for the tolerance to viral infection observed in the Manduria ecotype as well as in the susceptible tomato variety UC82 self-grafted or grafted on the Manduria ecotype.