Short ORF-Dependent Ribosome Shunting Operates in an RNA Picorna-Like Virus and a DNA Pararetrovirus that Cause Rice Tungro Disease

Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence

Cross-species functionality of pararetroviral elements driving ribosome shunting

Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5'-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.; Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.; Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism. We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany

Viral protein suppresses oxidative burst and salicylic acid-dependent autophagy and facilitates bacterial growth on virus-infected plants

Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection

The classification and nomenclature of endogenous viruses of the family Caulimoviridae

Endogenous members of the family Caulimoviridae have now been found in the genomes of many plant species. Although these sequences are usually fragmented and rearranged and show varying degrees of decay, the genomes of the ancestral viruses can often be reassembled in silico, allowing classification within the existing viral taxonomic framework. In this paper, we describe analyses of endogenous members of the family Caulimoviridae in the genomes of Oryza sativa, Nicotiana tabacum and Solanum spp. and on the basis of phylogeny, genome organization and genetic distance within the pol gene, propose two new virus genera called Orendovirus and Solendovirus. A system of nomenclature for endogenous virus sequences in plants is also proposed

Plant virome reconstruction and antiviral RNAi characterization by deep sequencing of small RNAs from dried leaves

In plants, RNA interference (RNAi) generates small interfering (si)RNAs from entire genomes of viruses, satellites and viroids. Therefore, deep small (s)RNA sequencing is a universal approach for virome reconstruction and RNAi characterization. We tested this approach on dried barley leaves from field surveys. Illumina sequencing of sRNAs from 2 plant samples identified in both plants Hordeum vulgare endornavirus (HvEV) and barley yellow mosaic bymovirus (BaYMV) and, additionally in one plant, a novel strain of Japanese soil-borne wheat mosaic furovirus (JSBWMV). De novo and reference-based sRNA assembly yielded complete or near-complete genomic RNAs of these viruses. While plant sRNAs showed broad size distribution, viral sRNAs were predominantly 21 and 22 nucleotides long with 5'-terminal uridine or adenine, and were derived from both genomic strands. These bona fide siRNAs are presumably processed from double-stranded RNA precursors by Dicer-like (DCL) 4 and DCL2, respectively, and associated with Argonaute 1 and 2 proteins. For BaYMV (but not HvEV, or JSBWMV), 24-nucleotide sRNAs represented the third most abundant class, suggesting DCL3 contribution to antibymovirus defence. Thus, viral siRNAs are well preserved in dried leaf tissues and not contaminated by non-RNAi degradation products, enabling both complete virome reconstruction and inference of RNAi components mediating antiviral defense.Diversification for Tobacco Growing Farms by the alternative crop Stevia rebaudiana Berton

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-α-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function