Cell surface receptor kinase FERONIA linked to nutrient sensor TORC signaling controls root hair growth at low temperature linked to low nitrate in Arabidopsis thaliana.

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrient availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. We use genetics to address the roles of the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC) in RH growth. We identified that low temperature (10°C) triggers a strong RH elongation response in Arabidopsis thaliana involving FER and TORC. We found that FER is required to perceive limited nutrient availability caused by low temperature. FERONIA interacts with and activates TORC-downstream components to trigger RH growth. In addition, the small GTPase Rho of plants 2 (ROP2) is also involved in this RH growth response linking FER and TOR. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10°C in a NRT1.1-dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER-ROP2-TORC is involved in the control of RH elongation under low temperature and nitrogen deficiency.

TOR represses stress responses through global regulation of H3K27 trimethylation in plants.

Target of rapamycin (TOR) functions as a central sensory hub linking a wide range of external stimuli to gene expression. The mechanisms underlying stimulus-specific transcriptional reprogramming by TOR remain elusive. Here, we describe an in silico analysis in Arabidopsis demonstrating that TOR-repressed genes are associated with either bistable or silent chromatin states. Both states regulated by the TOR signaling pathway are associated with a high level of histone H3K27 trimethylation (H3K27me3) deposited by CURLY LEAF in a specific context with LIKE HETEROCHROMATIN PROTEIN1. The combination of the two epigenetic histone modifications H3K4me3 and H3K27me3 implicates a bistable feature that alternates between an 'on' and an 'off' state, allowing rapid transcriptional changes upon external stimuli. The chromatin remodeler SWI2/SNF2 ATPase BRAHMA activates TOR-repressed genes only at bistable chromatin domains to rapidly induce biotic stress responses. Here, we demonstrate both in silico and in vivo that TOR represses transcriptional stress responses through global maintenance of H3K27me3.

Phosphorylation of a reinitiation supporting protein, RISP, determines its function in translation reinitiation.

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2ß and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2ß, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.

Nuclear export of plant pararetrovirus mRNAs involves the TREX complex, two viral proteins and the highly structured 5' leader region.

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.

GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin.

Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR ROP2 physically interacts with and, when GTP-bound, activates TOR in vitro TOR activation in response to auxin is abolished in ROP-deficient rop2 rop6 ROP4 RNAi plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.

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.

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation.

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator-viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV-TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP--a novel and specific target of TOR/S6K1--in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes.

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.

A new plant protein interacts with eIF3 and 60S to enhance virus-activated translation re-initiation.

The plant viral re-initiation factor transactivator viroplasmin (TAV) activates translation of polycistronic mRNA by a re-initiation mechanism involving translation initiation factor 3 (eIF3) and the 60S ribosomal subunit (60S). QJ;Here, we report a new plant factor-re-initiation supporting protein (RISP)-that enhances TAV function in re-initiation. RISP interacts physically with TAV in vitro and in vivo. Mutants defective in interaction are less active, or inactive, in transactivation and viral amplification. RISP alone can serve as a scaffold protein, which is able to interact with eIF3 subunits a/c and 60S, apparently through the C-terminus of ribosomal protein L24. RISP pre-bound to eIF3 binds 40S, suggesting that RISP enters the translational machinery at the 43S formation step. RISP, TAV and 60S co-localize in epidermal cells of infected plants, and eIF3-TAV-RISP-L24 complex formation can be shown in vitro. These results suggest that RISP and TAV bridge interactions between eIF3-bound 40S and L24 of 60S after translation termination to ensure 60S recruitment during repetitive initiation events on polycistronic mRNA; RISP can thus be considered as a new component of the cell translation machinery.

Functional analogs of mammalian 4E-BPs reveal a role for TOR in global plant translation.

Mammalian/mechanistic target of rapamycin (mTOR) regulates global protein synthesis through inactivation of eIF4E-binding proteins (m4E-BPs) in response to nutrient and energy availability. Until now, 4E-BPs have been considered as metazoan inventions, and how target of rapamycin (TOR) controls cap-dependent translation initiation in plants remains obscure. Here, we present short unstructured 4E-BP-like Arabidopsis proteins (4EBP1/4EBP2) that are non-homologous to m4E-BPs except for the eIF4E-binding motif and TOR phosphorylation sites. Unphosphorylated 4EBPs exhibit strong affinity toward eIF4Es and can inhibit formation of the cap-binding complex. Upon TOR activation, 4EBPs are phosphorylated, probably when bound directly to TOR, and likely relocated to ribosomes. 4EBPs can suppress a distinct set of mRNAs; 4EBP2 predominantly inhibits translation of core cell-cycle regulators CycB1;1 and CycD1;1, whereas 4EBP1 interferes with chlorophyll biosynthesis. Accordingly, 4EBP2 overexpression halts early seedling development, which is overcome by induction of Glc/Suc-TOR signaling. Thus, TOR regulates cap-dependent translation initiation by inactivating atypical 4EBPs in plants.

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 senses and regulates spermidine metabolism during seedling establishment and growth in maize and Arabidopsis.

Spermidine (Spd) is a nitrogen sink and signaling molecule that plays pivotal roles in eukaryotic cell growth and must be finetuned to meet various energy demands. In eukaryotes, target of rapamycin (TOR) is a central nutrient sensor, especially N, and a master-regulator of growth and development. Here, we discovered that Spd stimulates the growth of maize and Arabidopsis seedlings through TOR signaling. Inhibition of Spd biosynthesis led to TOR inactivation and growth defects. Furthermore, disruption of a TOR complex partner RAPTOR1B abolished seedling growth stimulation by Spd. Strikingly, TOR activated by Spd promotes translation of key metabolic enzyme upstream open reading frame (uORF)-containing mRNAs, PAO and CuAO, by facilitating translation reinitiation and providing feedback to polyamine metabolism and TOR activation. The Spd-TOR relay protected young-age seedlings of maize from expeditious stress heat shock. Our results demonstrate Spd is an upstream effector of TOR kinase in planta and provide its potential application for crop protection.

Dissection of a rice OsMac1 mRNA 5' UTR to uncover regulatory elements that are responsible for its efficient translation.

The untranslated regions (UTRs) of mRNAs are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA has three splicing variants of the 5' UTR (UTRa, UTRb, and UTRc), which include a CU-rich region and three upstream open reading frames (uORFs). UTRc contains an additional 38-nt sequence, termed sp38, which acts as a strong translational enhancer of the downstream ORF; reporter analysis revealed translational efficiencies >15-fold higher with UTRc than with the other splice variants. Mutation analysis of UTRc demonstrated that an optimal sequence length of sp38, rather than its nucleotide sequence is essential for UTRc to promote efficient translation. In addition, the 5' 100 nucleotides of CU-rich region contribute to UTRc translational enhancement. Strikingly, three uORFs did not reveal their inhibitory potential within the full-length leader, whereas deletion of the 5' leader fragment preceding the leader region with uORFs nearly abolished translation. Computational prediction of UTRc structural motifs revealed stem-loop structures, termed SL1-SL4, and two regions, A and B, involved in putative intramolecular interactions. Our data suggest that SL4 binding to Region-A and base pairing between Region-B and the UTRc 3'end are critically required for translational enhancement. Since UTRc is not capable of internal initiation, we presume that the three-dimensional leader structures can allow translation of the leader downstream ORF, likely allowing the bypass of uORFs.

Cauliflower mosaic virus transactivator protein (TAV) can suppress nonsense-mediated decay by targeting VARICOSE, a scaffold protein of the decapping complex.

During pathogenesis, viruses hijack the host cellular machinery to access molecules and sub-cellular structures needed for infection. We have evidence that the multifunctional viral translation transactivator/viroplasmin (TAV) protein from Cauliflower mosaic virus (CaMV) can function as a suppressor of nonsense-mediated mRNA decay (NMD). TAV interacts specifically with a scaffold protein of the decapping complex VARICOSE (VCS) in the yeast two-hybrid system, and co-localizes with components of the decapping complex in planta. Notably, plants transgenic for TAV accumulate endogenous NMD-elicited mRNAs, while decay of AU-rich instability element (ARE)-signal containing mRNAs are not affected. Using an agroinfiltration-based transient assay we confirmed that TAV specifically stabilizes mRNA containing a premature termination codon (PTC) in a VCS-dependent manner. We have identified a TAV motif consisting of 12 of the 520 amino acids in the full-length sequence that is critical for both VCS binding and the NMD suppression effect. Our data suggest that TAV can intercept NMD by targeting the decapping machinery through the scaffold protein VARICOSE, indicating that 5'-3' mRNA decapping is a late step in NMD-related mRNA degradation in plants.

Ribosome Shunting, Polycistronic Translation, and Evasion of Antiviral Defenses in Plant Pararetroviruses and Beyond.

Viruses have compact genomes and usually translate more than one protein from polycistronic RNAs using leaky scanning, frameshifting, stop codon suppression or reinitiation mechanisms. Viral (pre-)genomic RNAs often contain long 5'-leader sequences with short upstream open reading frames (uORFs) and secondary structure elements, which control both translation initiation and replication. In plants, viral RNA and DNA are targeted by RNA interference (RNAi) generating small RNAs that silence viral gene expression, while viral proteins are recognized by innate immunity and autophagy that restrict viral infection. In this review we focus on plant pararetroviruses of the family Caulimoviridae and describe the mechanisms of uORF- and secondary structure-driven ribosome shunting, leaky scanning and reinitiation after translation of short and long uORFs. We discuss conservation of these mechanisms in different genera of Caulimoviridae, including host genome-integrated endogenous viral elements, as well as in other viral families, and highlight a multipurpose use of the highly-structured leader sequence of plant pararetroviruses in regulation of translation, splicing, packaging, and reverse transcription of pregenomic RNA (pgRNA), and in evasion of RNAi. Furthermore, we illustrate how targeting of several host factors by a pararetroviral effector protein can lead to transactivation of viral polycistronic translation and concomitant suppression of antiviral defenses. Thus, activation of the plant protein kinase target of rapamycin (TOR) by the Cauliflower mosaic virus transactivator/viroplasmin (TAV) promotes reinitiation of translation after long ORFs on viral pgRNA and blocks antiviral autophagy and innate immunity responses, while interaction of TAV with the plant RNAi machinery interferes with antiviral silencing.

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.