A Viral Satellite RNA Induces Yellow Symptoms on Tobacco by Targeting a Gene Involved in Chlorophyll Biosynthesis using the RNA Silencing Machinery

Abstract

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5′-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.

Inducible expression of magnesium protoporphyrin chelatase subunit I (CHLI)-amiRNA provides insights into cucumber mosaic virus Y satellite RNA-induced chlorosis symptoms

“…These changes include (1) variation in chlorophyll fluorescence and reduced chlorophyll content [3], (2) inhibition of photosynthetic activity [17], (3) disproportionate accumulation of photoassimilates [1,23], (4) alterations in chloroplast ultrastructure and functions [4,25], (5) downregulation of nuclear-encoded chloroplast and photosynthesis-related genes and proteins (CPRGs and CPRPs, respectively) [8,21,34,35], (6) direct binding of viral components with chloroplast factors [4,33], and (7) perturbation of photosynthesis by regulation of host miRNA expression [43].…”

Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis

“…As a result, antiviral silencing is considered to be highly specific and target only the virus that initially triggers the immune response (1,3,4). Silencing of specific host genes occurs when there is nearperfect complementarity between a viral siRNA and the cellular mRNA (7,8).…”

The role of virus-derived small interfering RNAs in RNA silencing in plants

“…All these results indicate that Y-Sat-induced symptoms are caused by the vsiRNAs-mediated silencing of CHLI. Similarly, Shimura et al [62] also revealed the molecular mechanism of the yellowing symptom induced by Y-Sat. They found that transgenic N. benthamiana plants that expressed the inverted-repeat sequence of Y-Sat also developed yellowing symptom similar to the Y-Sat-infected plants.…”

“…Many cases referred to above show clearly that host plants, once invaded by virus, will activate their own RNA silencing system to target the foreign nucleic acid [8,11,12], and the virus will correspondingly express an RNA silencing suppressor to restrict the host defense system [5961]. Also, viral satRNA can utilize the host RNA silencing system to regulate the expression of certain host genes and thus induce symptoms [58,62]. Pantaleo and Burgyan [19] also pointed out that CymRSV could use an RNA silencing mechanism to control the accumulation level of satRNA, while satRNA might assist helper virus to resist the silencing-based defense system of host plants [63,64].…”