Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600 nt leader. We found that this leader region is transcribed into long sense- and antisense-RNAs and spawns a massive quantity of 21, 22 and 24 nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding region

Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600 nt leader. We found that this leader region is transcribed into long sense- and antisense-RNAs and spawns a massive quantity of 21, 22 and 24 nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding regions

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

Molecular dissection of the prototype foamy virus (PFV) RNA 5′-UTR identifies essential elements of a ribosomal shunt

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs

TOR acts as a metabolic gatekeeper for auxin-dependent lateral root initiation in Arabidopsis thaliana

Plant organogenesis requires matching the available metabolic resources to developmental programs. In Arabidopsis, the root system is determined by primary root-derived lateral roots (LRs), and adventitious roots (ARs) formed from non-root organs. Lateral root formation entails the auxin-dependent activation of transcription factors ARF7, ARF19, and LBD16. Adventitious root formation relies on LBD16 activation by auxin and WOX11. The allocation of shoot-derived sugar to the roots influences branching, but how its availability is sensed for LRs formation remains unknown. We combine metabolic profiling with cell-specific interference to show that LRs switch to glycolysis and consume carbohydrates. The target-of-rapamycin (TOR) kinase is activated in the lateral root domain. Interfering with TOR kinase blocks LR initiation while promoting AR formation. The target-of-rapamycin inhibition marginally affects the auxin-induced transcriptional response of the pericycle but attenuates the translation of ARF19, ARF7, and LBD16. TOR inhibition induces WOX11 transcription in these cells, yet no root branching occurs as TOR controls LBD16 translation. TOR is a central gatekeeper for root branching that integrates local auxin-dependent pathways with systemic metabolic signals, modulating the translation of auxin-induced genes.The authors gratefully acknowledge [...] the Cluster of Excellence Cellular Networks of the University of Heidelberg (CellNetworks) through grant EcTOP6 “Metabolism and Development” and the German Research Foundation (DFG) through grants INST 35/1314-1 FUGG, INST 35/1503-1 FUGG and FOR2581. Open Access funding enabled and organized by ProjektDEAL. Open Access funding enabled and organized by Projekt DEAL.Peer reviewe

TOR acts as a metabolic gatekeeper for auxin-dependent lateral root initiation in Arabidopsis thaliana [Dataset]

Appendix Figure S1. Lateral root deficiency leads to starch hyperaccumulation in leaves. -- Appendix Figure S2. Comparable starch content in foliage at the end of the light period in plants producing LR or not. -- Appendix Figure S3. Glucose and Sucrose levels in shoots of IAA-treated Col-0 and slr seedlings. -- Appendix Figure S4. Auxin/slr-dependent signaling reconfigures the carbon metabolism-related transcriptome during LR formation is influenced. -- Appendix Figure S5. TOR over-activation leads to longer primary roots, whereas impairment of the TOR machinery results in reduced primary root length. -- Appendix Figure S6. Silencing efficiency in UB10pro>>amiR-TOR line. -- Appendix Figure S7. IAA or external carbohydrate sources in TOR-deficient seedlings can not rescue lateral root formation. -- Appendix Figure S8. Foliar accumulation of starch upon TOR silencing. -- Appendix Figure S9. Transcriptome analysis upon auxin-induced induction of lateral root formation in UB10pro>>amiR-TOR. -- Appendix Figure S10. Expression of TOR, GATA23, and ARF7 transcripts upon inhibition of TOR via AZD8055. -- Appendix Figure S11. IAA-responsive genes detected during ribosome profiling and TOR inhibition IAA induced in the RNA-seq experiment under TOR deficiency vastly overlap. -- Appendix Figure S12. WOX11 expression upon TOR-knockdown or TOR inhibition. -- Expanded view figures EV1-EV3. -- Dataset EV1. -- Dataset EV2.embj2022111273-sup-0001-appendix.pdfembj2022111273-sup-0002-evfigs.pdfembj2022111273-sup-0003-datasetev1.xlsxembj2022111273-sup-0004-datasetev2.xlsxembj2022111273-sup-0006-sdataev.zipPeer reviewe

Auxin Signaling in Regulation of Plant Translation Reinitiation

The mRNA translation machinery directs protein production, and thus cell growth, according to prevailing cellular and environmental conditions. The target of rapamycin (TOR) signaling pathway—a major growth-related pathway—plays a pivotal role in optimizing protein synthesis in mammals, while its deregulation triggers uncontrolled cell proliferation and the development of severe diseases. In plants, several signaling pathways sensitive to environmental changes, hormones, and pathogens have been implicated in post-transcriptional control, and thus far phytohormones have attracted most attention as TOR upstream regulators in plants. Recent data have suggested that the coordinated actions of the phytohormone auxin, Rho-like small GTPases (ROPs) from plants, and TOR signaling contribute to translation regulation of mRNAs that harbor upstream open reading frames (uORFs) within their 5′-untranslated regions (5′-UTRs). This review will summarize recent advances in translational regulation of a specific set of uORF-containing mRNAs that encode regulatory proteins—transcription factors, protein kinases and other cellular controllers—and how their control can impact plant growth and development

Integrity of the stem base secondary structure is essential for RTSV shunting.

<p>Twelve point mutations in either ascending (Disrupt L) or descending (Disrupt R) arm of the RTSV stem base secondary structure are shown on the left side. A combination of these mutations (Restore L+R) restores stable secondary structure. On the right side, relative values of CAT expression downstream of the wild type (“Wild type”) and the stem base-mutated versions (“Stem Disrupt L”, “Stem Disrupt R” and “Stem Restore L+R”) of the RTSV leader [or its variant with the Kozak stem (KS) sequence in the middle part] in <i>O. sativa</i> (rice) protoplasts are given. CAT expression from the wild-type leader construct is set to 100%.</p