Citrus exocortis viroid causes ribosomal stress in tomato plants.

Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.

Honeysuckle-derived microRNA2911 directly inhibits varicella-zoster virus replication by targeting IE62 gene.

Varicella-zoster virus (VZV) leads to chicken pox on primary infection and herpes zoster on reactivation. Recent studies suggest that microRNA2911 (MIR2911), honeysuckle (HS)-encoded atypical microRNA, has potential as a therapeutic agent against influenza and EV71 virus infections. Here, we report that MIR2911 directly inhibits VZV replication by targeting the IE62 gene. The luciferase reporter assay and bioinformatics prediction revealed that MIR2911 could target the IE62 gene of VZV. The VZV-encoded IE62 protein expression was inhibited significantly by synthetic MIR2911, while the expression of the mutants, whose MIR2911-binding sites were modified, was not inhibited. The RNA extracted from HS decoction and synthetic MIR2911 considerably suppressed VZV infection. However, it did not influence viral replication of a mutant virus with alterations in the nucleotide sequences of IE62. At the same time, the RNA extracted from HS decoction treated with the anti-MIR2911 antagomir could not inhibit the VZV replication, demonstrating that VZV replication was specifically and sufficiently inhibited by MIR2911. These results indicated that, by targeting the IE62 gene, MIR2911 may effectively inhibit VZV replication. Our results also suggest a potential novel strategy for the treatment and prevention of diseases caused by VZV infection.

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses.

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells.

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

Transcript suppression of TaGW2 increased grain width and weight in bread wheat.

Bread wheat (Triticum aestivum L.) is a major staple crop in the world. Grain weight is a major factor of grain yield in wheat, and the identification of candidate genes associated with grain weight is very important for high-yield breeding of wheat. TaGW2 is an orthologous gene of rice OsGW2 that negatively regulates the grain width and weight in rice. There are three TaGW2 homoeologs in bread wheat, TaGW2A, TaGW2B, and TaGW2D. In this study, a specific TaGW2-RNA interference (RNAi) cassette was constructed and transformed into a Chinese bread wheat variety 'Shi 4185' with small grain. The transcript levels of TaGW2A, TaGW2B, and TaGW2D were simultaneously downregulated in TaGW2-RNAi transgenic wheat lines. Compared with the controls, TaGW2-underexpressing transgenic lines displayed significantly increases in the grain width and weight, suggesting that TaGW2 negatively regulated the grain width and weight in bread wheat. Further transcript analysis showed that in different bread wheat accessions, the transcript abundance of TaGW2A was negatively associated with the grain width.

Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.).

MicroRNAs (miRNAs) play important roles in growth, development, and response to environmental changes in plants. Based on the whole-genome shotgun sequencing strategy, more and more wheat miRNAs have been annotated. Now, there is a need for an effective technology to analyse endogenous miRNAs function in wheat. We report here that the modified barley stripe mosaic virus (BSMV)-induced miRNAs silencing system can be utilized to silence miRNAs in wheat. BSMV-based miRNA silencing system is performed through BSMV-based expression of miRNA target mimics to suppress miR159a and miR3134a. The relative expression levels of mature miR159a and miR3134a decrease with increasing transcript levels of their target genes in wheat plants. In summary, the developed approach is effective in silencing endogenous miRNAs, thereby providing a powerful tool for biological function analyses of miRNA molecules in common wheat.

Isolation of lilin, a novel arginine- and glutamate-rich protein with potent antifungal and mitogenic activities from lily bulbs.

From the dried bulbs of the lily (Lilium brownii), a protein with strong antifungal and mitogenic activities was isolated. It also exhibited an inhibitory action on the activity of HIV-1 reverse transcriptase. The protein was single-chained and possessed a molecular weight of 14.4 kDa and an N-terminal sequence distinct from chitinases and antimicrobial proteins of garlic, leek and onion which belong to a family closely related to lily. However, there was a small degree of resemblance to cyclophilins and a considerable extent of identity to the 6.5 kDa arginine/glutamate-rich polypeptide from Luffa cylindrica seeds. A nearly homogeneous preparation was obtained after the extract was fractionated on DEAE-cellulose and Affi-gel Blue gel since subsequent chromatography on Mono S and Superdex 75 both yielded a single peak.

Nitrosomethylurea induced streptomycin resistance in Lycopersicon esculentum Mill.

High frequency of streptomycin resistant variants of Lycopersicon esculentum were isolated on selective shoot regeneration medium supplemented with IAA (0.5 mg/L), zeatin (1.5 mg/L) and streptomycin sulphate (500 mg/L). Nonmutagenized (controls) and NMU treated cotyledons were placed on shoot regeneration medium supplemented with antibiotic streptomycin. Resistant shoots appeared at a high frequency in mutagenized cotyledons, whereas in controls morphogenesis was suppressed, accompanied by bleaching. Shoot regeneration occurred from the nodular tissues developed at the cut ends of cotyledons. Resistant shoots developed into complete plantlets on rooting medium containing selective concentration of antibiotic. Stability of streptomycin resistance was confirmed by leaf assay and reciprocal crosses between streptomycin-resistant and sensitive plants.

Defective RNA processing enhances RNA silencing and influences flowering of Arabidopsis.

Many eukaryotic cells use RNA-directed silencing mechanisms to protect against viruses and transposons and to suppress endogenous gene expression at the posttranscriptional level. RNA silencing also is implicated in epigenetic mechanisms affecting chromosome structure and transcriptional gene silencing. Here, we describe enhanced silencing phenotype (esp) mutants in Arabidopsis thaliana that reveal how proteins associated with RNA processing and 3' end formation can influence RNA silencing. These proteins were a putative DEAH RNA helicase homologue of the yeast PRP2 RNA splicing cofactor and homologues of mRNA 3' end formation proteins CstF64, symplekin/PTA1, and CPSF100. The last two proteins physically associated with the flowering time regulator FY in the 3' end formation complex AtCPSF. The phenotypes of the 3' end formation esp mutants include impaired termination of the transgene transcripts, early flowering, and enhanced silencing of the FCA-beta mRNA. Based on these findings, we propose that the ESP-containing 3' end formation complexes prevent transgene and endogenous mRNAs from entering RNA-silencing pathways. According to this proposal, in the absence of these ESP proteins, these RNAs have aberrant 3' termini. The aberrant RNAs would enter the RNA silencing pathways because they are converted into dsRNA by RNA-dependent RNA polymerases.

Ricin A-chain: kinetics, mechanism, and RNA stem-loop inhibitors.

Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a kcat/Km of 4.5 x 10(5) M-1 s-1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower kcat values, but all have Km values of approximately 5 microM. Both the 10- and 18-base substrates have kcat/Km near 10(4) M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem-loop DNA (10 bases) of the same sequence is also a substrate with a kcat/Km of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O-riboside at the depurination site is not a substrate, but binds tightly to the enzyme (Ki = 0.34 microM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pKa of the adenine analogue at N7. Binding of this analogue (Ki = 9.4 microM) is weaker than substrate which indicates that the altered pKa at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (Ki = 0.18 microM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.