Alteration of tomato microRNAs expression during fruit development upon Cucumber mosaic virus and Tomato aspermy virus infection

The economic importance of Solanaceae plant species is well documented, and tomato has become a model for fleshy fruit development and ripening studies. Plant microRNAs (miRNAs) are small endogenous RNAs that are involved in a variety of activities including plant development, signal transduction and protein degradation, as well as response to environment stress and pathogen invasion. Here in this study, we aimed at quantifying the expression alterations of nine miRNAs and target mRNAs in tomato flower and fruit development upon Cucumber mosaic virus (CMV) and Tomato aspermy virus infections. Three different CMV strains CMV-Fny, CMV-FnyΔ2b and CMV-Fny-satT1 were used in our investigation, and the miRNA/mRNA expression alterations were analyzed by real-time quantitative RT-PCR. The results shown the levels of several miRNA/mRNA pairs were increased upon virus infections. However, the increased level of individual miRNA differed for different virus strains, reflecting differences in severity of symptom phenotypes. The altered expression patterns of these miRNA/mRNA pairs and their predicted functions indicate the possible roles in flower and fruit development, and provide experimental data for understanding the miRNA-mediated phenotype alterations in tomato fruit.     

Characterization of the interactions between Cucumber mosaic virus and Potato virus Y in mixed infections in tomato

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.     

THO2, a core member of the THO/TREX complex,is required for microRNA production in Arabidopsis

The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA‐dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA‐targeted mRNAs. Yeast two‐hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA‐processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine‐rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.     

Molecular Detection and Complete Genome Sequences of Tomato chlorosis virus Isolates from Infectious Outbreaks in China

Tomato chlorosis virus (ToCV) is a whitefly‐transmitted, phloem‐limited, bipartite Crinivirus. In 2012, severe interveinal symptoms characteristic of ToCV infections were observed in greenhouse tomato plants in the Shandong province of China. High levels of infestation by whiteflies (Bemisia tabaci), which transmit ToCV, were also observed on tomato plants in all the greenhouses investigated. The presence of ToCV was confirmed by specific RT‐PCR either in the sampled plants or in the whiteflies collected from the ventral surface of the leaves of diseased plants. The complete genomic nucleotide sequences (RNA1 and RNA2) of the Shandong isolate of ToCV (ToCV‐SDSG) were determined and analysed. ToCV‐SDSG RNA1 consisted of 8594 nucleotides encompassing four open reading frames (ORFs). ToCV‐SDSG RNA2 consisted of 8242 nucleotides encompassing nine ORFs. Phylogenetic analysis suggests that the Chinese ToCV‐SDSG isolate is most similar to the ToCV‐Florida isolate.     

Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner

Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3′ untranslated region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3′ UTR of viral genome did. Our work provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly in other crops.     

The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development

The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR₁₈₀NK box of HC-Pro to FI₁₈₀NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMV^FRNK caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA “passenger” strands (miRNA*s), in systemically infected leaves than the attenuated ZYMV^FINK did. HC-Pro^FRNK specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-Pro^FINK. Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HC^FINK mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.     

Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microrna in host gene expression

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by the ribonuclease Dicer from distinct precursors, double-stranded RNA (dsRNA) and hairpin RNAs, respectively, although either may guide RNA silencing via a similar complex. The siRNA pathway is antiviral, whereas an emerging role for miRNAs is in the control of development. Here, we describe a virulence factor encoded by turnip yellow mosaic virus, p69, which suppresses the siRNA pathway but promotes the miRNA pathway in Arabidopsis thaliana. p69 suppression of the siRNA pathway is upstream of dsRNA and is as effective as genetic mutations in A. thaliana genes involved in dsRNA production. Possibly as a consequence of p69 suppression, p69-expressing plants contained elevated levels of a Dicer mRNA and of miRNAs as well as a correspondingly enhanced miRNA-guided cleavage of two host mRNAs. Because p69-expressing plants exhibited disease-like symptoms in the absence of viral infection, our findings suggest a novel mechanism for viral virulence by promoting the miRNA-guided inhibition of host gene expression.     

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants

The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.