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Василий АлексеевичШекспир и русская культура / ред.: Алексеев, М.П. — М.: Наука, 1965. — 831 с. Якимов (1802—1853) — российский , ,  и ; профессор Харьковского университета по кафедре русской словесности.

Биография

Родился в 1802 году и происходил из духовного звания. По окончании курса в Белгородской духовной семинарии в 1823 году поступил своекоштным студентом в Императорский Харьковский университет — на словесный факультет, который окончил в 1826 году со званием кандидата и получил в то же время золотую медаль за сочинение. В 1827 году был определён учителем словесности в Харьковский институт благородных девиц, а в 1831 году стал преподавать словесность в университете. В 1832 году он получил в Санкт-Петербургском университете степень магистра словесных наук и был утвержден адъюнктом по этому предмету.

Жил и работал в тяжелую для науки эпоху, при весьма печальных условиях личного воспитания и семейной обстановки. Академик М. И. Сухомлинов, слушавший в молодости его лекции, в своем отзыве о нём осторожно замечает о «крайне неблагоприятных условиях его воспитания и жизненной обстановки в годы молодости». Другой его слушатель, М. Ф. Де-Пуле, указал, что Якимов «опустился от расстройства экономических и семейных дел. Он был человек добрый, но какой-то забитый или надломленный, а потому и делом своим занимался спустя рукава; случалось, что по приходе он тотчас уходил с лекции, сознаваясь, что не приготовился к чтению». Другой его слушатель в своих воспоминаниях о харьковских профессорах 1830-х годов отмечал, что семинарское воспитание «наложило на него неизгладимый тип своеобразных воззрений на жизнь и науку». Д. П. Хрущов указывал, что Якимов «читал вяло, бесцветно, голосом медоточивым. Он был, что называется, ума твердого, но простого. Впрочем, он был добрый человек и его мы любили».

Не ограничиваясь преподаванием в учебных заведениях и не довольствуясь содержанием пансионеров, Якимов приобрёл себе дом и открыл свой собственный пансион для детей купеческого сословия, нечто вроде коммерческого училища, с преподаванием предметов гимназического курса, но без древних языков. Потребности в такой школе в то время, по-видимому, совсем не было и пансион, ничего не принёс Якимову, кроме убытков.

По отзывам Де Пуле, профессор Якимов «принадлежал к риторам старой школы, писал рассуждения в стихах и в прозе о красноречии, о красотах и изобилии русского языка и т. п. предметах… Он принадлежал к числу литераторов 20-х и 30-х годов; на тогдашнем миросозерцании он остановился и не пошел далее. Он писал для актов торжественные речи, был убежден в необходимости писать такие же стихотворения и даже песнопения, требовал от студентов того же, хотел, чтобы они изучали Россиаду, но, к счастью, требовал он и хотел всего этого как-то сонно и апатично <…> читал студентам эстетику довольно сносно и историю русской литературы совсем уж плохо».

Он не был совсем замкнут в харьковской провинциальной жизни: бывал в Санкт-Петербурге, входил в общение с некоторыми выдающимися учеными и писателями, например князем Владимиром Фёдоровичем Одоевским, однако заметного влияния на его ученые или литературные труды это не оказало.

Оставил после себя 10 сочинений, из которых, как отмечал автор статьи в «Русском биографическом словаре», лишь одно, «и притом с большой натяжкой», может быть признано учёным исследованием: «О красноречии в России до Ломоносова» — докторская диссертация, которая, по отзыву академика Сухомлинова, «отличается бесцветностью содержания и наивностью критических приёмов». Четыре других его произведения относятся к литературно-мистическим рассуждениям: «О бессмертии человека», «Об отношении просвещения к истинам откровения» и т. п.

Это было время, когда в Харьковском университете попечитель Е. В. Карнеев насаждал благочестие и когда здесь процветало «студенческое библейское сотоварищество», в котором, Якимов изощрялся в благочестивых рассуждениях и в истолкованиях псалмов, описывая в виршах сотворение мира, появление первого человека и прочее. Схожи по содержанию и другие рассуждения автора на темы патриотические и панегирики сильным мира. Якимову принадлежит рассуждение «Дар слова», произнесенное в торжественном собрании университета в 1831 году, являющееся ярким выражением тогдашнего риторического педантизма. Эта речь была напечатана в университетском издании отдельно.

В 1830-х годах Якимовым были изданы переводы Уильяма Шекспира «Король Лир» и «Венецианский купец»; в рукописи же при жизни автора остались: «Отелло», «Цимбалин», «Сон в летнюю ночь» и «Что вам угодно?». Переводы были сделаны с буквальной точностью, но выходившие одновременно переводы Полевого и Кронберга по лёгкости и ясности оставили позади себя тяжёлые переводы Якимова. Однако Якимов дорожил своими переводами из Шекспира, читал их в Санкт-Петербурге у князя В. Ф. Одоевского, который слушать их пригласил даже князя П. А. Вяземского и А. С. Пушкина.

В 1839 году, помимо желания Якимова, к нему был приставлен помощник, а в 1852 году он был забаллотирован и вынужден был выйти в отставку.

Умер в 1853 году.

Примечания

Литература

Русские писатели по алфавиту
Доктора наук
Преподаватели Харьковского института благородных девиц
Переводчики Уильяма Шекспира
Переводчики с английского языка
Переводчики поэзии на русский язык
Члены Общества любителей российской словесности

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EveryWare Global (NASDAQ: EVRY) rose 38.21% to $3.87 after the company reported on the results of ratification votes on revised labor agreements.
BioDelivery Sciences International (NASDAQ: BDSI) jumped 17.60% to $14.10 on positive top-line Phase III results.
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How to Get Pre-Approved for a Mortgage
How to Become a Better Mortgage Rate Shopper
5 Factors that Impact Your Credit Score
Complete Guide to Buying a Home after Foreclosure, Bankruptcy and Short-Sale
FAQs About Getting a Mortgage Pre-Approval
A Video on How Mortgage Interest Rates Move
7 Reasons to Refinance Now
30 Year Fixed vs. 15 Year Fixed – Which One Makes More Sense
Affordability Rears its Head Again
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Let's Do It
Author bio section
Shashank Shekhar
September, 15, 2020
Many affordable housing markets have seen a hike in home prices, making them a tough grab for the buyers, says Redfin.
Daryl Fairweather, Redfin's Chief Economist, stated in a press release that "Home price growth this high is making the housing market especially difficult for first-time homebuyers right now. Rising prices are just one more reason for people to leave expensive urban neighborhoods behind." "Price growth may slow in 2021, but even if it does, high prices are going to continue to make affordability a concern for buyers," added Fairweather.
For the four weeks closing on Sep 6, the median home price sat at $319,171 on average. This is a 13% hike on the year-over-year chart. Such yearly gain hasn't been reported for the last 7 years. Redfin reveals that 28% more homes have been sold compared to the same time, last year. This again is the biggest rise since Aug '15'.
About half the sellers have had to wait less than two weeks after putting their property on the market. This is 15% higher than last year and the highest that the percentage has been since Redfin started tracking the data.
New listings have risen to 86,986- this is a 9% hike on the yearly chart. Such a hike is unprecedented for any four weeks since the last month of 2015.
On a similar note...
Mortgage Rates Come Up, Could Go Down Again
Millennials Continue to be in High Purchasing Spirits
Strengthened by Refinance Volume, Mortgage Applications Shoot Up
Get a Customized Rate Quote in 2 Minutes or Less*
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Shashank Shekhar is a best-selling author and the CEO of InstaMortgage (dba Arcus Lending Inc), offering mortgage loans for home purchase and refinance. For a free consultation and/or rate quote, email him at [email protected] or call his office at 1-855-644-LOAN
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2022 FHA LOAN LIMTS FOR MINNESOTA (MN)
2022 FHA Loan Limits For Minnesota is $420,680 and goes up to $448,500 for high-cost counties for one-unit properties. 2022 FHA Loan Limits For Minnesota for 2-unit properties is $538,650 and goes up to $574,150 for high-cost counties. 2022 FHA Mortgage Loan Limits for Aitkin County 1 Unit – $420,680 2 Unit – $538,650 3 […]
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2022 FHA Loan Limits For Illinois is $420,680 for one-unit properties. 2022 FHA Loan Limits For Illinois is $538,650 for 2-unit properties. 2022 FHA Mortgage Loan Limits for Adams County 1 Unit – $420,680 2 Unit – $538,650 3 Unit – $651,050 4 Unit – $809,150 2022 FHA Mortgage Loan Limits for Alexander County 1 […]
2022 FHA LOAN LIMITS FOR INDIANA (IN)
2022 FHA Loan Limits For Indiana is $420,680 for one-unit properties. 2022 FHA Loan Limits For Indiana is $538,650 for 2-unit properties. 2022 FHA Mortgage Loan Limits for Adams County 1 Unit – $420,680 2 Unit – $538,650 3 Unit – $651,050 4 Unit – $809,150 2022 FHA Mortgage Loan Limits for Allen County 1 Unit […]
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Youth Week is almost here!
All local young people are invited to join us for a youth expo!
We have had a team of youth doing work experience here at the centre, to help us prepare for the event. They have been so helpful in providing feedback and giving their ideas! Thank you Nyssa and James!
Movie vouchers and for the girls, free manicure and make up tips!
This project is a proud project of Belmont Neighbourhood Centre, in partnership with Mary Kay Makeup Artist, APM Employment Services, Community Activities Lake Macquarie, NSW Youth Week and Lifeline Hunter and Central Coast. The event is funded by a grant through Lake Macquarie City Council.


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Viral Virulence Protein Suppresses RNA Silencing–Mediated Defense but Upregulates the Role of MicroRNA in Host Gene Expression
Jun Chen, Wan Xiang Li, Daoxin Xie, Jin Rong Peng and Shou Wei Ding
The Plant Cell May 2004, 16 (5) 1302-1313; DOI: https://doi.org/10.1105/tpc.018986
Jun Chen
Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609Department of Biological Sciences, National University of Singapore, Singapore 117609
Wan Xiang Li
Center for Plant Cell Biology, Institute for Integrative Genome Biology, and Department of Plant Pathology, University of California, Riverside, California 92521
Daoxin Xie
Jin Rong Peng
Shou Wei Ding
American Society of Plant Biologists
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by the ribonuclease Dicer from distinct precursors, double-stranded RNA (dsRNA) and hairpin RNAs, respectively, although either may guide RNA silencing via a similar complex. The siRNA pathway is antiviral, whereas an emerging role for miRNAs is in the control of development. Here, we describe a virulence factor encoded by turnip yellow mosaic virus, p69, which suppresses the siRNA pathway but promotes the miRNA pathway in Arabidopsis thaliana. p69 suppression of the siRNA pathway is upstream of dsRNA and is as effective as genetic mutations in A. thaliana genes involved in dsRNA production. Possibly as a consequence of p69 suppression, p69-expressing plants contained elevated levels of a Dicer mRNA and of miRNAs as well as a correspondingly enhanced miRNA-guided cleavage of two host mRNAs. Because p69-expressing plants exhibited disease-like symptoms in the absence of viral infection, our findings suggest a novel mechanism for viral virulence by promoting the miRNA-guided inhibition of host gene expression.
RNA silencing is a generic term describing related gene silencing processes that include posttranscriptional gene silencing (PTGS) in plants, double-stranded RNA (dsRNA)–mediated RNA interference in animals, and quelling in fungi (Ding, 2000; Matzke et al., 2001; Vance and Vaucheret, 2001). A unifying feature of RNA silencing is the production of small interfering RNAs (siRNAs) that are 21 to 26 nucleotides long and of both sense and antisense polarities (Hamilton and Baulcombe, 1999; Hamilton et al., 2002; Plasterk, 2002). siRNAs are processed from dsRNA precursors by the endoribonuclease Dicer, become incorporated into the RNA-induced silencing complex that contains at least one Argonaute (AGO) protein, and guide specific cleavage of complementary mRNAs to a central position of an siRNA/mRNA duplex (Hammond et al., 2000, 2001; Bernstein et al., 2001; Elbashir et al., 2001). MicroRNAs (miRNAs) are a new class of small RNAs that may control development in animals and plants (Ambros et al., 2003). miRNAs are present in one polarity and are derived from RNA transcripts that have potential to form hairpin structures. miRNAs are also a product of Dicer and found in a complex similar if not identical to the RNA-induced silencing complex, although miRNAs may guide either cleavage or translational arrest of target mRNAs (Ambros et al., 2003).
In plants, PTGS can be induced by transgenes that encode either a highly transcribed sense RNA or an inverted repeat RNA (IR-RNA) that is folded into a long dsRNA. Several genes essential for sense RNA–induced PTGS have been cloned from Arabidopsis thaliana. These include a cellular RNA-dependent RNA polymerase (RdRP) homolog (SGS2/SDE1), AGO1, an RNA helicase gene (SDE3), and genes that encode a coiled-coil protein (SGS3) and a novel protein (HEN1) (Fagard et al., 2000; Mourrain et al., 2000; Boutet et al., 2003). Current models place these genes upstream of dsRNA because none of these genes are required for IR-RNA–induced PTGS (Dalmay et al., 2001; Beclin et al., 2002). In A. thaliana, both Dicer-like 1 (DCL1) and HEN1 are involved in the production of miRNAs (Park et al., 2002; Reinhart et al., 2002), and there is evidence that miRNAs function as siRNAs to specify cleavage of their mRNA targets (Llave et al., 2002; Rhoades et al., 2002; Kasschau et al., 2003).
A biological function for RNA silencing, first established in plants, is as an adaptive defense against RNA and DNA viruses (Covey et al., 1997; Ratcliff et al., 1997; Al-Kaff et al., 1998; Vance and Vaucheret, 2001). Recent work has demonstrated a similar antiviral role for RNA silencing in the animal kingdom (Li et al., 2002, 2004). The antiviral response may be triggered by virus-specific dsRNA intermediates produced during genome replication, by transcription from converging promoters, or through recognition of viral RNAs by host RdRP, as proposed in plants (Voinnet et al., 2000; Szittya et al., 2002). As a counterdefense, viruses encode proteins such as helper component-proteinase (HC-Pro) and Cucumber mosaic virus 2b that suppress RNA silencing at various steps in the pathway (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998; Mlotshwa et al., 2002; Silhavy et al., 2002). The identification of a silencing suppression function typically provides a molecular basis for previously observed defects in viral accumulation and spread of virus mutants that lack a functional suppressor (Li and Ding, 2001; Mlotshwa et al., 2002).
Here, we report the identification of a viral suppressor of RNA silencing that also plays a unique role in the induction of viral disease. The suppressor, p69, is encoded by turnip yellow mosaic virus (TYMV), a plus-strand RNA virus prevalent both in cultivated and wild species of the Brassicaceae family of dicotyledonous plants (Morch et al., 1988; Skotnicki et al., 1992). Previous work has shown that p69 is essential for virus spread and influences viral yields and symptom severity in infected plants but is dispensable for viral replication in single cells (Bozarth et al., 1992; Tsai and Dreher, 1993). Using distinct PTGS systems with defined silencing triggers, we found that p69 represents a unique viral suppressor of RNA silencing by targeting a step upstream of dsRNA production in the cellular RdRP-dependent branch of RNA silencing. However, transgenic A. thaliana plants expressing p69 alone displayed disease-like symptoms, indicating a role of p69 in disease induction that is independent of its suppression of the host PTGS mechanism targeting viral RNAs. Further analyses revealed a significantly upregulated miRNA pathway in p69-expressing plants, including elevated levels of miRNAs and enhanced miRNA-guided cleavages of two host mRNAs that encode putative transcriptional factors. Thus, p69 is distinct to the viral PTGS suppressor HC-Pro that was recently shown to inhibit miRNA silencing (Kasschau et al., 2003). We propose that enhanced miRNA silencing is a consequence of a negative feedback regulation of the RNA silencing pathway triggered by p69 suppression and that miRNAs can play a pathogenic role in the induction of viral diseases.
p69 Inhibits PTGS and DNA Methylation Induced by Sense RNA Transgenes
The first indication that TYMV might encode a suppressor of RNA silencing came from TYMV infection of A. thaliana plants that carry a silencing 35S-β-glucuronidase (GUS) sense RNA transgene at the L1 locus (Elmayan et al., 1998). We found that TYMV infection of L1 plants resulted in a restored GUS expression in the newly emerged leaves as shown by histochemical staining (Figure 1B).
p69 Is a PTGS Suppressor in A. thaliana.
(A) TYMV genome organization.
(B) to (D) Suppression of the GUS RNA silencing in line L1 by TYMV 2 weeks after infection (B), by a 35S-P69 transgene (C), or a TYMV amplicon transgene (D). Photos of the same plants before GUS staining are also shown in the bottom panels in (C) and (D).
TYMV contains three genes, two of which are encoded in out-of-frame overlapping open reading frames (Figure 1A). The viral RdRP and p69 are translated from the genomic RNA with their initiation codons seven nucleotides apart, whereas the coat protein (CP) is produced from a subgenomic RNA that is 3′ coterminal with the genomic RNA (Morch et al., 1988; Skotnicki et al., 1992). We predicted that the suppressor activity of TYMV was encoded by P69 based on its role in viral spread and virulence that is similar to the known viral suppressors (Bozarth et al., 1992; Tsai and Dreher, 1993; Li and Ding, 2001).
To test this possibility, the P69 gene was introduced into A. thaliana ecotype C24 under the control of the 35S promoter of Cauliflower mosaic virus. It is striking that 7 of the 11 35S-P69 transformants obtained showed developmental defects (Figure 2, left; see also Supplemental Figure 1 online). The expression of P69 was hardly detectable in the remaining four lines that were morphologically indistinguishable from either wild-type plants or any of the eight 35S-ΔP69 transformants (data not shown). The pleiotropic defects of the P69 plants cosegregated with the transgene in backcrosses with wild-type plants and included severe dwarf, pale-colored leaves with reduced cell sizes, late flowering, short siliques, and decreased fertility. For example, line 3 homozygous for a single 35S-P69 transgene (P69c) began flowering 48.5 d after sowing with 13.4 rosette leaves on average (n = 65) in contrast with wild-type plants that flowered 42.4 d with 11.8 rosette leaves (n = 61) under our growth conditions. The developmental defects observed in the P69 plants mimicked, but were more severe than, the disease symptoms found in wild-type plants systemically infected with TYMV (Figure 2, right). Thus, p69 alone is able to confer pathogenicity independent of TYMV infection, further extending the finding by Tsai and Dreher (1993).
p69-Conferred Virulence in A. thaliana.
Transgenic line P69c (left) expressing P69 causes pleiotropic developmental defects that resemble the phenotype displayed by a plant infected systemically with TYMV (right).
Further analyses confirmed that p69 is a potent suppressor of sense RNA–induced RNA silencing. High levels of GUS activity (Figure 1C) and GUS mRNA (Figure 3A, top panel) were detected in the F1 progeny plants of crosses between line P69c and line L1. The distinct GUS staining pattern in the L1xP69c plants (Figure 1C) may be caused by the constitutive expression of P69 possibly before or during the GUS transgene was silenced, as compared with that in TYMV-infected L1 plants (Figure 1B) in which GUS silencing was established before virus inoculation. By contrast, 35S-GUS remained silenced in the F1 progeny of a similar cross between the wild type and L1 (Figures 1C and 3A). Furthermore, whereas the GUS-specific siRNAs accumulated to high levels in L1xC24 plants, they were undetectable in L1xP69c plants (Figure 3B). Notably, these RNA analyses showed that suppression of GUS RNA silencing in P69c plants was as effective as in the sgs2 mutant that contains a defective cellular RdRP (Figures 3A and 3B) (Mourrain et al., 2000). Interestingly, silencing suppression as measured by the accumulation of both GUS mRNA and siRNAs, was less efficient in L1xP69d plants that expressed P69 mRNA at a lower level (Figure 3A, cf. lanes 3 and 4). Using 35S constructs directing translation of either the full-length or the first 45 codons of p69 and gene delivery by Agrobacterium tumefaciens coinfiltration, we further showed that p69 actively suppressed RNA silencing targeted against a 35S-controlled green fluorescent protein (GFP) sense transgene in Nicotiana benthamiana (see Supplemental Figure 2 online).
Molecular Characterization of P69 Suppression of PTGS Induced by a Sense RNA Transgene in A. thaliana.
The detection of the GUS mRNA (A), GUS siRNAs (B), and GUS DNA methylation (C) in sgs2(L1), L1xC24, L1xP69c, and L1xP69d seedlings. In contrast with HpaII, MspI cleavage is not blocked to methylation at sites with overlapping CG. Equal loading and completion of DNA digestion was demonstrated by rehybridization of the filter for SPL3 (bottom panel of [C]), whereas equal loading of small RNAs was shown by reprobing for 5S rRNA (B). The positions of 21- and 26-nucleotide RNA markers are indicated (B).
The autonomous RNA silencing in L1 plants is associated with cytosine methylation within the coding sequence of the 35S-GUS transgene (Mourrain et al., 2000). As expected, the GUS DNA in the L1 plants was partially resistant to digestion by the methylation-sensitive restriction enzymes HpaII and MspI (Figure 3C, left panel). By contrast, the GUS DNA from the L1xP69c plants, in which the GUS siRNAs were undetectable, was digested completely by MspI, although the digestion by HpaII, sensitive to methylation at both overlapping and nonoverlapping CG sites, was less efficient (Figure 3C, right panel). This pattern of demethylation was similar to that found previously in the sgs2 mutant in the L1 background (Figure 3C, middle panel) (Mourrain et al., 2000). Thus, p69 expression also prevented the methylation of GUS DNA as completely as in the sgs2 mutant. These results establish p69 as a unique suppressor of RNA silencing because none of the known viral suppressors have been shown to eliminate both siRNA accumulation and transgene methylation associated with a silencing sense RNA transgene (Mallory et al., 2001; Guo and Ding, 2002).
P69 Inhibits PTGS Induced by a Virus-Derived Amplicon Transgene
Viral RNAs are silenced in plants via both the cellular and viral RdRP pathways (Dalmay et al., 2000b; Voinnet et al., 2000). We analyzed p69 suppression of PTGS that targets an amplicon transgene that encodes a replication-competent recombinant potato virus X (PVX) carrying GFP (PVX:GFP) in A. thaliana lines A and GxA (Dalmay et al., 2000a). The autonomous silencing of the amplicon requires active replication of PVX:GFP RNA but is SDE1 independent as shown in line GxA. Line G contains a highly expressed 35S-GFP transgene that is silenced in line GxA by the amplicon. In contrast with amplicon silencing, silencing of the 35S-GFP transgene in line GxA requires SDE1 and is associated with methylation of the GFP and PVX:GFP transgenes (Dalmay et al., 2000a).
RNA gel blot hybridizations detected significant accumulation of PVX:GFP genomic and subgenomic RNAs in both A and GxA plants either after systemic infection with TYMV (Figure 4A, lanes 4 and 5) or after the 35S-P69 transgene was introduced from line P69b, P69c, or P69d through genetic crosses (Figure 4B, lane 3 and lanes 6 to 8). Accumulation of GFP mRNA was also detected in (GxA)xP69 plants (Figure 4B, lanes 6 to 8) and in GxA plants infected with TYMV (Figure 4A, lane 4). Suppression of the amplicon and GFP RNA silencing was weaker in lines (GxA)xP69b and (GxA)xP69d than in (GxA)xP69c and undetectable in (GxA)xP69h and (GxA)xP69i as in lines (GxA)xΔP69a and (GxA)xΔP69b. These results indicate that the silencing suppression activity was determined by the expression levels of the P69 transgene and by whether or not the P69 transgene was translatable in these plants (Figure 4B). As expected from previous work (Dalmay et al., 2000b), the GFP mRNA and the genomic and subgenomic RNAs from PVX:GFP were detected in the sde1(GxA) mutant (Figure 4B, lane 13). Interestingly, the accumulation level was lower for the GFP mRNA and higher for the viral RNAs in (GxA)xP69 plants (Figure 4B, lanes 6 to 8) than in the sde1(GxA) mutant (Figure 4B, lane 13). Thus, p69 suppression of sense RNA silencing is incomplete, suggesting a differential suppression by p69 and the genetic mutation in SDE1of RNA silencing directed against the viral amplicon and sense-transgene transcripts.
Molecular Characterization of p69 Suppression of PTGS Induced by a Virus-Derived Amplicon Transgene.
(A) Silencing suppression in amplicon lines infected with TYMV (lanes 4 to 5). Samples from mock-inoculated lines were included as controls (lanes 1 to 3). PVX:GFP genomic (g) and subgenomic (sg) RNAs as well as GFP mRNA are indicated.
(B) Silencing suppression by the P69 transgene introduced into lines A and GxA by genetic crosses with the maternal parent listed first (this is used throughout the text). Except for G and sde1(GxA), all plants analyzed were hemizygous F1 plants. Total RNA loaded was 5 μg in lanes 1 to 3 and 3 μg in lanes 4 to 13 and equal loading was visualized by methylene blue staining of rRNA.
(C) Detection of siRNAs by a GFP-specific probe in hemizygous F1 amplicon plants crossed with P69c, P69d, or C24. A similar pattern of siRNA accumulation was detected using a PVX-specific probe.
(D) Detection of the GFP DNA methylation. Note that HaeIII cleavage is not blocked to methylation at sites with overlapping CG. The positions of DNA standards (in base pairs) are shown at the right.
(E) Detection of TYMV high and low molecular weight RNAs in either the TYMV-based amplicon plants or wild-type plants infected TYMV. The filters were probed with labeled DNA and RNA sequences corresponding to nucleotides 5591 to 6260, respectively, of the TYMV genome. The amplicon transgene contained an insertion of nonviral sequence (1.6 kb) upstream of the viral CP gene and thus gave rise to a recombinant viral genomic RNA longer than the wild-type TYMV genomic RNA, although the size of the CP subgenomic RNA remained unaltered.
We further examined the methylation status of the 35S-GFP transgene in (GxA)xP69c plants using the methylation-sensitive restriction enzyme HaeIII (Figure 4D). The GFP DNA extracted from (GxA)xP69c plants (lane 3) was completely digested by HaeIII as found for the GFP DNA extracted from either line G (lane 1) or the sde1(GxA) mutant (lane 4). This was in contrast with the GFP DNA extracted from GxA plants that was partially resistant to the digestion by HaeIII (Figure 4D, lane 2), as was found previously (Dalmay et al., 2000a). These results indicate that p69 also suppresses PTGS mediated by the viral amplicon either alone or in combination with a homologous transgene and that p69 suppression is associated with elimination of transgene methylation.
Hybridizations using a probe complementary to the GFP sequence detected the accumulation of siRNAs both in AxP69c and (GxA)xP69c plants (Figure 4C). In fact, the siRNAs became more abundant in these amplicon lines after P69 was introduced (Figure 4C, cf. lanes 3 and 4 with lane 2 or lane 6 with lane 5). Because Ax P69c plants did not carry the 35S-GFP transgene, all of the siRNAs detected in these plants must have come from the PVX:GFP amplicon transgene. Thus, p69 suppression of amplicon silencing led to enhanced accumulation of both viral genomic/subgenomic RNAs and siRNA, as found in the sde1(GxA) mutant (Dalmay et al., 2000b), rather than siRNA elimination observed in L1xP69c plants. This suggests that p69 expression did not prevent Dicer cleavage of virus-derived dsRNAs into siRNAs.
Unlike the PVX-based amplicon that induces consistent silencing in transgenic tobacco and A. thaliana plants (Angell and Baulcombe, 1997; Dalmay et al., 2000a), transgenic A. thaliana plants carrying a TYMV-based amplicon transgene displayed the characteristic TYMV symptoms (Figure 1D, bottom left) and suppression of the GUS transgene silencing as shown by GUS staining after the amplicon was introduced into line L1 by genetic crosses (Figure 1D, top left), suggesting that p69 also suppresses silencing induced by the TYMV amplicon. Compared with the 35S-P69 transgene (Figure 1C), however, suppression of GUS silencing by the TYMV amplicon was less effective (Figure 1D); this was probably caused by silencing of the amplicon transgene as indicated by detection of TYMV-specific siRNAs in the amplicon plants (Figure 4E, bottom panel, lane 3) and much lower levels of the genomic and subgenomic viral RNAs in those plants than in TYMV-infected plants (Figure 4E, top panel, cf. lanes 3 and 4). Consistent with the findings from the PVX amplicon plants, p69 expression from TYMV also did not prevent accumulation of viral siRNAs in TYMV-infected plants (Figure 4E, bottom panel, lane 2).
p69 Does Not Inhibit PTGS Induced by IR-RNA Transgenes
RNA silencing induced by IR-RNA transgenes is distinct to sense and virus RNA silencing because neither the cellular nor viral RdRP is involved in the formation of the dsRNA inducer from a nuclear self-complementary IR-RNA transcript (Beclin et al., 2002). Thus, we next determined if P69 interfered with silencing of the phytoene desaturase (PDS) gene mediated by an IR-RNA transgene (IRPDS), which caused a dominant photobleaching phenotype. An IRPDS/− heterozygote (in Columbia [Col-0] background) that contained a single copy of the transgene was crossed with P69c (in C24 background) and C24, respectively, and the F1 progenies were allowed to grow in the MS medium and MS plus hygromycin (for selection of the IRPDS and/or 35S-P69 positive individuals). The progeny of C24xIRPDS/− segregated 1:1 for hygromycin sensitive (HYGS) to hygromycin resistant (HYGR), but all of the 12 HYGR individuals showed photobleaching (Figure 5B). All of the 19 progenies from P69cxIRPDS/− carried the 35S-P69 transgene and were HYGR; however, only 10 of these F1 plants showed photobleaching (Figure 5C). When grown on MS medium (Figure 5A), both F1 populations from crosses C24xIRPDS/− (n = 29) and P69cxIRPDS/− (n = 52) contained ∼50% individuals displaying the photobleaching phenotype. These results indicate that the presence of 35S-P69 did not suppress the PDS silencing phenotype of IRPDS.
p69 Does Not Suppress the IR-RNA–Induced PTGS Targeting of PDS.
(A) to (C) Genetic segregation of the photobleaching RNA interference phenotype of the IRPDS transgene after crossing an IRPDS heterozygote (IRPDS/−, in Col-0 background) with either C-24 or a P69c homozygote (in the C-24 background). A representative plate is shown for each except for P69cxIRPDS, which did not show difference with or without hygromycin selection. P69c plants appear yellow in contrast with the photobleached plants (C).
(D) Detection of P69 mRNA and PDS mRNA/siRNAs. For comparison to the level of PDS mRNA in the C24xCol-0 genetic background with and without P69, RNA samples from the progeny of C24xCol-0 (lane 3) and P69cxCol-0 (lane 1) were included as controls. Equal loading was monitored for mRNA by rRNA staining and for small RNAs by reprobing for 5S rRNA.
RNA analysis showed that expression of the IRPDS transgene in C24xIRPDS plants resulted in the accumulation of PDS-specific siRNAs (Figure 5D, lane 4) and an ∼75% knockdown of the PDS mRNA (Figure 5D, top panel, cf. lanes 3 and 4). Consistent with lack of P69 suppression indicated by the phenotypic analysis, P69 expression in the IRPDS-expressing plants was not associated with an increased accumulation of PDS mRNA (Figure 5D, cf. lanes 2 and 4). Furthermore, we found that TYMV infection of A. thaliana plants that carry the 35S-GFP transgene targeted by an IR-RNA transgene did not suppress GFP silencing as shown by RNA analyses (see Supplemental Figure 4 online). Thus, potent p69 suppression of RNA silencing triggered by sense RNA transgenes but not by the IR-RNA transgene suggests that p69 disrupts a cellular function leading to dsRNA synthesis.
p69 Enhances miRNA-Mediated Cleavage of Two Target mRNAs
Similar to IR-RNA, the hairpin precursor of miRNAs enters the RNA silencing pathway without new dsRNA synthesis. Thus, we predicted that p69 would not inhibit mRNA cleavage by endogenous miRNAs. We first examined the cleavage of the SCL6-IV mRNA by miR171, which occurs at positions centered in the middle of the miRNA–mRNA duplex so that both the full-length mRNA (SCL6-IVf) and a shorter 3′ fragment of ∼1.3 kb (SCL6-IV3′) are detected by a 3′-proximal probe in wild-type A. thaliana (Llave et al., 2002). We found that this cleavage indeed was not blocked in the presence of p69 because both of the RNA species were detected in P69c plants (Figure 6A). Notably, a decrease in the accumulation of the full-length mRNA and a corresponding increased accumulation of the 3′ fragment was detected in P69c plants as compared with wild-type plants (Figure 6A). This suggests that p69 may enhance RNA silencing mediated by the miRNA pathway.
Reduced Accumulation of Two miRNA-Targeted mRNAs in p69-Expressing Plants.
(A) Detection of full-length SCL6-IV mRNA and its 3′ cleavage product by miR171 in wild-type C-24 and P69c plants.
(B) Accumulation of SPL3 mRNA in P69c and P69d plants as well as in C-24 plants before (Wt) and after systemic TYMV infection (V).
(C) Determination of the miR156 cleavage sites within SPL3 mRNA. The 5′-terminal portion of the predicted 3′ cleavage product for SPL3 mRNA as well as SPL2 and CUC2 mRNAs (Kasschau et al., 2003; used here as controls) was amplified by 5′RACE (top panel), and the dominant products were recovered, cloned, and sequenced. The arrows indicate the positions of two cleavage sites inferred (bottom panel). The number of the 5′RACE clones that correspond to each site is indicated.
We further examined the steady state mRNA levels of SQUAMOSA-promoter binding protein-like gene 3 (SPL3), a predicted target of miR156 (Rhoades et al., 2002). SPL3 transcription is developmentally regulated, and constitutive overexpression of SPL3 resulted in early flowering (Cardon et al., 1997). RNA gel blotting analysis revealed significant decreases (0.7- to 2-fold) in the accumulation of the SPL3 mRNA in two independent p69 lines as compared with wild-type plants (Figure 6B). The reduction was more pronounced in line P69c than in line P69d (cf. lanes 2 and 3), which was correlated with a higher expression level of the P69 transgene in P69c plants as detected by RNA gel blot hybridization (Figure 4B, lanes 7 and 8). A modest decrease in the accumulation of SPL3 mRNA was also observed in wild-type plants infected with TYMV (Figure 6B, lane 4).
miR156 has mismatches to SPL3 mRNA in contrast with both miR171 and siRNAs that are perfectly complementary to their target mRNAs (Llave et al., 2002; Plasterk, 2002). Thus, we determined if there was cleavage of SPL3 mRNA within the predicted miR156/mRNA duplex, which contains one mismatch at the second nucleotide from the 3′ terminus of miR156 in addition to a G:U wobble (Rhoades et al., 2002) (Figure 6C). For this purpose, cDNA corresponding to the predicted 3′ fragment of SPL3 mRNA was amplified using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) essentially as described (Llave et al., 2002). Total mRNAs isolated from P69c plants were ligated to a 45-nucleotide RACE adapter RNA and reverse transcribed using a downstream SPL3-specific primer. Two rounds of nested PCR using the cDNAs obtained as templates and two sets of RACE and gene-specific primers yielded a discrete band that migrated within the predicted size range (Figure 6C, top panel). Sequencing and alignment of the cloned PCR products derived from the SPL3 mRNA identified two cleavage sites in the center of the predicted miR156/mRNA duplex (Figure 6C, bottom panel). Therefore, cleavage at the predicted duplex of miR156 and SPL3 mRNA indeed occurred in the P69c plants.
p69 Expression Increases the Accumulation Level of Seven miRNAs and DCL1 mRNA
The enhanced cleavage of miRNA targets observed in p69-expressing plants might be because of a higher accumulation level of miRNAs. To investigate this possibility, total RNA was extracted from newly bolted inflorescence and analyzed for the accumulation of seven A. thaliana miRNAs, miR156, miR157, miR158, miR162, miR164, miR167, and miR171. We found that each miRNA increased in abundance in the presence of the P69 transgene in P69c plants (Figures 7A and 7B). Similar increases were observed in the P69d plants but not in line G for miR156 and miR171, and we detected no difference in the accumulation of miR156, miR157, miR158, miR167, or miR171 between L1 and the sgs2(L1) mutant (see Supplemental Figure 3 online), suggesting that SDE1 may not be involved in miRNA biogenesis.
Enhanced Accumulation of Seven miRNAs and mRNAs for DCL1 and SDE1/SGS2 in p69-Expressing Plants.
(A) and (B) Detection of miRNAs in P69c and wild-type C-24 plants as described in Methods. We noted that the level of miR162 was much lower in wild-type ecotype C-24 than in either Landsberg erecta (Ler) or Col-0.
(C) Detection of DCL1 and AGO1 mRNAs by RNA gel blot hybridizations. Two micrograms of poly(A)+ mRNAs from P69c and C-24 plants were loaded in each lane, and the filter was probed sequentially for the mRNA of AGO1, DCL1, and β-TUBULIN-1 (as a loading control). The full-length DCL1 and AGO1 mRNAs are 6.2 and 3.5 kb, respectively, as indicated.
(D) Real-time PCR analyses of the mRNA accumulation for AGO1, DCL1, and β-TUBULIN-1 in P69c and C-24 plants.
As the first step to investigate the mechanism of p69-mediated enhancement of miRNA silencing, we examined expression of two genes in the RNA silencing pathway using RNA gel blotting (Figure 7C) and real-time PCR (Figure 7D). No significant differences were detected in the accumulation of either AGO1 or β-TUBULIN-1 mRNA between P69c and wild-type plants. However, both analyses showed that mRNA of DCL1 was at least threefold higher in P69c plants than in wild-type plants.
In this work, we identified TYMV p69 as a unique viral virulence protein that suppresses the host RNA silencing antiviral defense but promotes RNA silencing of host genes mediated by miRNAs. Here, we discuss the mode of action of p69 and the potential role of p69 in viral pathogenesis by miRNAs.
p69 Suppresses RNA Silencing Upstream of dsRNA
Several lines of evidence indicate that among the viral suppressors characterized to date, p69 targets a unique step in the RNA silencing pathway. The fact that p69 suppressed PTGS induced by sense RNA transgenes but not by IR-RNA transgenes indicates that p69 inhibits a cellular function leading to dsRNA production. This conclusion is consistent with the observation that p69 had similar effects on RNA silencing to those of genetic mutations in cellular genes involved in dsRNA production such as SGS2/SDE1. For example, both P69 expression and the sgs2/sde1 mutation eliminated the siRNAs production (Figure 3B) and DNA methylation (Figures 3C) associated with RNA silencing induced by the sense RNA GUS transgene at the L1 locus (Mourrain et al., 2000). In the amplicon-based two-component silencing system (Figure 4), both P69 expression and the sgs2/sde1 mutation suppressed RNA silencing and DNA methylation of the 35S-GFP transgene, but both increased the accumulation of virus-specific siRNAs (Dalmay et al., 2000b). The increase in the production of viral siRNAs is likely from Dicer processing of the more abundant viral RdRP products after p69 suppression of silencing. However, these siRNAs may not be fully functional because of lack of both amplicon silencing and DNA methylation in the amplicon plants either expressing P69 or defective for SDE1 (Dalmay et al., 2000b; Mette et al., 2000).
PVX p25 also suppresses the host RdRP-dependent branch of RNA silencing (Voinnet et al., 2000). Interestingly, both p25 and p69 are essential for virus cell-to-cell movement as compared with a dispensable role for other viral suppressors in the cell-to-cell movement. Unlike p69, however, p25 is not active in the suppression of either established silencing targeted against sense RNA transgenes (Brigneti et al., 1998) or silencing targeted its own amplicon transgene (Angell and Baulcombe, 1997; Dalmay et al., 2000a). Furthermore, it appears that p25 expression also does not prevent DNA methylation of the 35S-GUS transgene in the tobacco host (Mallory et al., 2003). A recent work suggests that p25 acts downstream of dsRNA synthesis by a specific inhibition of the production of the longer siRNA species of 24 to 26 nucleotides (Hamilton et al., 2002). However, the recently described NSs protein of tomato spotted wilt virus (Takeda et al., 2002) may be mechanistically similar to p69.
p69 Suppression May Trigger a Negative Feedback Regulation
Our analysis revealed that P69 expression was associated with enhanced accumulation of all of the seven miRNAs examined and a correspondingly decreased accumulation of two mRNA targets of miRNAs. Increased accumulation was also observed for the mRNA of DCL1 (Figure 7C) and SDE1 (shown by real-time PCR analysis; our unpublished data) but not for the mRNA of AGO1 (Figure 7C). It is not known if p69 expression leads to increased expression of additional RNA silencing pathway genes, such as the homologs of DCL1 and SGS2/SDE1 (Finnegan et al., 2003; Yu et al., 2003) or if any of the p69-induced changes are mechanistically related. Given the known role of DCL1 in the production of miRNAs (Park et al., 2002; Reinhart et al., 2002), however, we propose that the RNA silencing pathway in A. thaliana is under a negative feedback regulation that can be triggered by p69 suppression and that DCL1 and SDE1 represent those genes in the pathway that are responsive to this feedback regulation at the level of transcription. It is possible that an upregulated DCL1 expression will lead to elevated levels of miRNAs by a more efficient processing of miRNA precursors, which may in turn cause enhanced cleavages of those host mRNAs targeted by miRNAs. In this regard, it will be interesting to determine if p69 expression also inhibits host gene expression by miRNA-guided translational repression (Aukerman and Sakai, 2003; Chen, 2003).
Recent work indicates that the DCL1 mRNA is targeted for cleavage in A. thaliana plants by miR162 (Xie et al., 2003). miR162 also accumulated to a higher level in p69-expressing plants than in wild-type plants (Figure 7B), which intriguingly, did not lead to a correspondingly lower level of the DCL1 mRNA as expected, in contrast with miR156 and miR171. It is not clear if this is because of an overall lower accumulation level of miR162 in C24 than in Col-0 and Landsberg erecta (Figure 7B; Xie et al., 2003). Alternatively, miRNA cleavage of mRNAs may be a rate-limiting process so that it becomes ineffective to control mRNA accumulation of those genes that are under transcriptional induction. It is less likely, however, that the increased accumulation of DCL1 mRNA found in p69-expressing plants is a result of p69-mediated inhibition of miRNA cleavage because mRNA cleavages by miR156 and miR171 were not inhibited (Figure 6) and consistent HC-Pro inhibition of miRNA cleavages was observed for 10 target mRNAs (Kasschau et al., 2003; Xie et al., 2003). It should be pointed out that a putative role for DCL1 in elevated levels of miRNAs is based on an assumption that p69 does not inhibit miRNA-mediated translational arrest, which remains to be determined.
Viral Pathogenesis by miRNAs?
Recent work has established a regulatory role for miRNAs in the development of plants and animals by targeting mRNAs for either cleavage or translational repression (Carrington and Ambros, 2003; Bartel, 2004). Although p69 could play a role in viral pathogenesis by miRNA-independent mechanisms, an attractive hypothesis is that the disease symptoms/developmental defects associated with P69 expression in A. thaliana plants represent a consequence of enhanced miRNA-guided inhibition of host gene expression regardless of whether one or both mechanisms are used. For example, the late flowering phenotype of the P69 plants may be in part attributable to the p69-stimulated miR156 knockdown of SPL3 mRNA because it is known that constitutive overexpression of SPL3 results in early flowering (Cardon et al., 1997). Interestingly, the level of P69 expression is highest in the young leaves systemically infected with TYMV but decline as infected leaves expand and mature (Bozarth et al., 1992), possibly because of selective proteolysis by the proteasome (Drugeon and Jupin, 2002). Presumably, young tissues are most sensitive to developmental cues such as miRNAs, a suggestion consistent with the observation that systemic viral symptoms always appear first in young leaves. Thus, miRNAs may play a novel pathogenic role in the induction of viral diseases.
HC-Pro expression in transgenic plants is known to cause developmental defects (Anandalakshmi et al., 2000). Recent studies show that even though miRNAs accumulate to elevated levels in the presence of HC-Pro, miRNA cleavage of target mRNAs is inhibited in HC-Pro–expressing plants, as found previously for PTGS induced by either sense- or IR-RNA transgenes (Anandalakshmi et al., 1998; Kasschau and Carrington, 1998; Mallory et al., 2002, 2003; Kasschau et al., 2003; Xie et al., 2003). Accordingly, it has been proposed that the role of HC-Pro in disease development is to inhibit host developmental pathways that depend on negative regulation by miRNAs (Kasschau et al., 2003). Therefore, our work on p69 of TYMV not only establishes a distinct mechanism in viral suppression of the RNA silencing antiviral defense, but also suggests an alternative model for the induction of viral diseases by upregulating the role of miRNAs in the inhibition of host gene expression.
Plant Materials and Growth Conditions
L1 carries a silenced GUS transgene in the Arabidopsis thaliana ecotype Col-0 background, and sgs2 is a loss-of-function mutant of L1 at the SGS2/SDE1 locus (Mourrain et al., 2000). Lines G, A, and GxA (in C-24 ecotype background) correspond to GFP142, Amp243, and GFP142xAmp243, respectively (Dalmay et al., 2000a). sde1-3 was derived from GxA (Dalmay et al., 2000b).
The P69 coding sequence of TYMV-Blue Lake (BL) (Skotnicki et al., 1992) was cloned into pCambia1301 by replacing the GUS sequence between the 35S promoter and nopaline synthase terminator to give 35S-P69. The start codon (ATG) of the viral RdRP, which is four nucleotides downstream of the start codon for p69, was mutated to ACG in 35S-P69. As a result, the RdRP open reading frame (ORF) is likely not to be translated from 35S-P69 because the next Met codon in the RdRP ORF is 95 codons downstream. A single G-to-T substitution converting the 46th codon of ORF P69 into a stop codon (TAA) was introduced to create 35S-ΔP69. 35S-P69 and 35S-ΔP69 were transferred into A. thaliana ecotype C24 as described (Clough and Bent, 1998). The IR-RNA transgene targeting PDS contained an inverted repeat corresponding to nucleotides 128 to 532 of the PDS mRNA. The IR-RNA targeting GFP corresponded to nucleotides 360 to 716 of the GFP mRNA. Inserted between the repeats of either PDS or GFP was the third intron of A. thaliana actin gene 11 (nucleotides 1957 to 2111; GenBank accession number ATU27981). The PDS and GFP IR-RNA cassettes were cloned into pCambia1300 between 35S promoter and nopaline synthase terminator and transformed into wild-type Col-0 and line G (Dalmay et al., 2000a), respectively. Transformants were selected by hygromycin at 20 mg/L. Lines with a single copy transgene were selected by a 3:1 segregation of HygR and HygS among selfed progeny. Homozygous lines for 35S-P69 or 35S-ΔP69 were used in crosses. For plant inoculation, purified viral particles derived from an infectious plasmid containing the full-length TYMV-BL cDNA (Skotnicki et al., 1992) under the 35S promoter (A. Mackenzie, unpublished data) in the pCass vector as described (Ding et al., 1995) were used. TYMV amplicon plants, which were kindly supplied by N.H. Chua, carried the minimal 35S promoter (−46 to +1)–controlled TYMV-BL cDNA with a 1.6-kb insertion of nonviral sequence between the viral RdRP and CP genes (Aoyama and Chua, 1997). These plants became symptomatic without dexamethasone induction, possibly because of a leaky transcription.
Seeds were either imbibed on wet filter paper at 4°C for 7 d before planting on the Florobella potting compost/sand mix (3:1) or surface sterilized for growth on MS medium with/without antibiotics, but both were maintained in a growth room (16 h light/8 h darkness, 20 to 23°C).
DNA and RNA Analyses
The analysis of DNA methylation of either the GFP coding sequence (Dalmay et al., 2000a) or the GUS coding sequence (Mourrain et al., 2000) was performed as described using 32P-labeled DNA probes corresponding to the full coding sequences. As a control, the GUS filter was rehybridized with a SPL3-specific probe because there is only one HpaII/MspI recognition sequence (CCGG) within the SPL3 genomic DNA.
Total plant RNA extraction and RNA gel blotting analysis were performed as described previously (Li et al., 1999). Hybridization probes were labeled with 32P using the Amersham Megaprime DNA labeling kit (Piscataway, NJ). SPL3, SCL6-IV, and PDS probes corresponded to nucleotides 293 to 532, 774 to 1485, and 128 to 532 of the SPL3, SCL6-IV, and PDS mRNAs, respectively. Total mRNAs were extracted from whole plants including leaves, stems, flowers, and siliques but not roots using the Qiagen Oligotex mRNA Midi kit (Valencia, CA). The probes for DCL1 (Jacobsen et al., 1999), AGO1 (Fagard et al., 2000), and β-TUBULIN-1 corresponded to nucleotides 475 to 2283, 484 to 748, and 500 to 992 of their respective mRNAs.
siRNA detection was as described (Guo and Ding, 2002). These siRNA filters were reprobed for the 5S rRNA to show equal loading. For miRNA detection, total RNA was isolated from newly bolted inflorescence, and 50 μg was loaded in each lane as described (Reinhart et al., 2002) and probed by end-labeled DNA oligonucleotides complementary to miRNA. All experiments on the DNA and RNA analyses were repeated at least twice.
Real-Time RT-PCR Analysis
Total cDNA was first synthesized using the oligo(dT)15 primer from total RNAs and used as templates for the real-time PCR with gene-specific primers. The amplified regions corresponded to nucleotides 721 to 949, 628 to 1057, 484 to 748, and 500 to 992 of the SDE1/SGS2 (RdRP), DCL1, AGO1, and β-TUBULIN-1 mRNAs, respectively. The LightCycler-FastStartDNA Master SYBR Green kit and LightCycler Instrument real-time PCR machine (Roche Applied Science, Penzberg, Germany) were used. The reactions were terminated when PCR was in the log-linear phase but before the later plateau phase, which was 27 cycles for RdRP and 29 for DCL1, AGO1, and β-TUBULIN-1. After confirming the identity by direct sequencing, PCR products were blotted and detected by hybridizations with gene-specific probes.
RLM-RACE
The FirstChoice RLM-RACE kit (Ambion, Austin, TX) was used for RLM-RACE essentially following the manufacturer's instructions. Briefly, poly(A)+ mRNA was obtained from flowering plants using Oligotex mRNA Midi kit (Qiagen) and directly ligated to RLM-RACE 5′RACE RNA oligo adaptor (45 nucleotides) before cDNA synthesis using oligo(dT)15 primer. The first round of nested PCR was performed using 5′RACE outer primer included in the kit and a gene-specific outer primer that was complementary to nucleotides 690 to 710 of SPL3 (At2g33810 complete cDNA), 1166 to 1185 of SPL2 (At5g43270), or 1075 to 1094 of CUC2 (At5g53950). For the second round, 5′RACE inner primer was used with the second set of gene-specific inner primers that were respectively complementary to nucleotides 665 to 685 of SPL3, 1134 to 1152 of SPL2, and 1057 to 1078 of CUC2. In each case, a unique gene-specific DNA fragment was amplified (Figure 6C). The PCR products were gel purified and cloned into pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing.
We thank F. Liu, J. Fei, and H. Guo for helpful discussion and technical support, H. Vaucheret for L1 lines, D. Baulcombe for amplicon lines, A. Mackenzie and A. Gibbs for the TYMV clone, N.H. Chua for the TYMV amplicon line, and Z. Wen, T. Dreher, and the anonymous reviewers for valuable comments on the manuscript. This work was supported first by A-STAR in the Institute of Molecular Agrobiology and then at the Institute of Molecular and Cell Biology. The work conducted at the University of California Riverside was supported by grants from the USDA and the National Institutes of Health.
Online version contains Web-only data.
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Jin Rong Peng (pengjr{at}imcb.a-star.edu.sg) and Shou Wei Ding (shou-wei.ding@ucr.edu).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.018986.
1 These authors contributed equally to this work.
Received November 4, 2003.
Published April 20, 2004.
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The European Union invests billions in research and science. Where exactly is that money going? And to what use? Carlos Moedas is the EU Commissioner for Research, Science and Innovation. He recently launched 'Science4refugees' a project which links up refugee scientists arriving with European scientific institutions. From the migrant crisis to innovations that claim to fight climate change, he speaks to France 24 about some of the scientific solutions funded by the European Union.


The micro-receiver can be stored inside the mouse under the cover.
The device takes 1 AA style battery.
Rechargable battery's appear to operate correctly within the device.
Full details available on HP web site under HP product number WE790AA.


text:
About The Code
Info for Solicitors
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Developer Login Become A Code User Check your Developer
Contact Us About The Code Independent Dispute Resolution Scheme Feedback & Complaints
The Consumer Code for New Homes aims to improve standards of construction in the UK and to ensure that best practice is followed by our registered Developer members in respect of the marketing, selling and purchasing of New Homes and to set expected standards for after sales customer care service, providing consumers with access to low-cost and effective dispute resolution.
This is a Consumer Code which we expect to grow and improve as the industry and consumer habits and expectations change, so in order to ensure we can continually improve, we are always keen to hear from consumers and members about their experiences of the Code in action and buying (or selling) a New Home.
Whether you had an excellent experience when buying a New Home from one of our members, if someone went above and beyond for you or a relative or if you think there are positive improvements we can make to the Code, please don't hesitate to contact us.
In order to provide us with your feedback, please email us at [email protected].
Providing us with Feedback
Although we and our members strive to provide you with a high quality service, we also know there may be times when you feel it is necessary to make a complaint.
If you need to make a complaint to the Consumer Code for New Homes direct, please use the following contact details:
An outline of the overall Complaints & Dispute Resolution Process is available here.
We would recommend that you make a complaint to the Developer in the first instance, since they will be able to deal with your complaint quickly and effectively under their own Complaints Handling Procedure. Find out more about complaining to the Developer below.
Making a Complaint to your Developer
Under the requirements of the Consumer Code for New Homes, registered Developers are required to have a system and procedures for receiving, handling and resolving Buyers' service calls and complaints and must provide the Buyer with clear information about how to access the procedure and systems. We expect that this information should be made available for download via the Developer's website and they should have made you aware of their Complaints Handling Procedure in the pre-purchase information you were provided with before you bought your New Home.
If you want to make a complaint to your Developer, we recommend that you follow their procedures.
You should then expect a response from the Developer within 30 working days of a complaint being made and they should let you know of the estimated time within which a decision will be reached and, where applicable, an estimated time within which required work will be completed.
If you and the Developer cannot reach an amicable resolution to your complaint, then at this point, the complaint becomes a Dispute which will then have the opportunity to refer direct to the independent CCNH Dispute Resolution Scheme or to the Warranty Body for mediation.
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Bill Paxton R.I.P.
That's it, man. Game over.
- Hicks
My favorite Bill Paxton flicks (in alphabetical order) in case you were wondering.
Aliens - As Hicks he earned the audience's contempt and supplied much needed comic relief during the un-ending tension of the film's last three quarters. He did it well, but a lot of that was in the writing. To also make us sorry to see him go? That took a special performer.
Frailty - As star he gave a chilling portrait of religious zealotry and child abuse, as director he announced himself as a talent with a true ear for tone and a taste for material in the bullseye of my sweet spot. Unfortunately, as a director, he never returned to my favorite places.
Near Dark - As the flashy danger in a nomadic-RV-bound tribe of heartless heartland vampires in Kathryn Bigelow's ground breaking flick - still the benchmark for rednecksploitation cool.
Next of Kin - As the youngest brother to Liam Neeson and Patrick Swayze relocated from coal country to the urban wasteland, it's a minor role in what remains, to my knowledge, the only crime flick set amongst Chicago's citybilly underground. ***Dennis McMillan points out Medium Cool's Chicago citybilly setting.
Nightcrawler - As the mentor who would've been turned competitor and then victim of Jake Gyllenhaal's freelance crime scene photographer he put in solid work in a movie yet to really get its due.
One False Move - The first starring role Paxton made an impression on me in. He plays an Arkansas cop with a big heart and a dark secret. Outclassed as a lawman by the FBI and outmatched in ruthlessness by the killers on their way to his town, the character and the performer nevertheless hold their own against the heavyweights (including co-star and screenwriter Billy Bob Thornton) in Carl Franklin's country noir.
A Simple Plan - If a greed-tragedy is to succeed it has to have a convincing innocent to topple - one whose fundamental decency is believable without being off-puttingly pious and whose craven depths are too relatable to ignore. Terrific, haunting thriller from Sam Raimi (based on the novel by Scott Smith) with Paxton front and center.
Tombstone - Probably lost amongst the densely stocked pond that is the cast of George P. Cosmatos' all-flash-bur-really-who-gives-a-shit western, as Morgan Earp his worship of his brother Wyatt gives the audience permission to as well. And his death gives Kurt Russell all the reason this moviegoer needed to justify the bloodbath that followed. Jeez, good thing I'm not in a position of any real-world authority or influence.
Traveller - As a nomadic conman in North Carolina Paxton has to be charming enough to make a living duping marks and world-weary enough to shoulder a little moral weight in this under-exposed drama.
Trespass - The concept of Arkansas firemen looking for buried treasure in abandoned East St. Louis building and getting caught in the crossfire of a gang war with major players including Ice Cube and Ice-T is just fucking rad. Especially for me as a fresh Arkansas transplant to St. Louis when I first saw it. It's pure pulpy exploitation tasteless tastiness served up by Walter Hill (who also cast Paxton in his rock'n' roll fantasy Streets of Fire).
Of interest and yet to be seen...
Mean Dreams - Returning to One False Move territory as a small town policeman with a lot of menace.
Training Day - The TV version finds Paxton stepping into the role Denzel Washington won an academy award for.
Labels: RIP
No Hablo
On The Crime Fix podcast this week Peter Dragovich and I talked about english-language remakes of foreign films inspired by the January release of Sleepless directed by Baran bo Odar (The Silence) and starring Jamie Foxx. It's a remake of the french language original Sleepless Night directed by Frederic Jardin (who's been directing some episodes of the french TV show Braquo - which I've heard Kent Gowran favorably compare to The Shield, so sign me up).
Neither of us have seen Sleepless (though I'm definitely going to check it out soon), but we're both big fans of Sleepless Night and hope that the remake at the very least shines some light on that fantastic original.
Our discussion included english remakes that were better, worse, shot-for-shot identical to their origin material or significant to us for other reasons good or bad. In the order discussed, here's the ground we covered.
Le Dernier Tournant (1939) directed by Pierre Chenal and The Postman Always Rings Twice (1981) directed by Bob Rafelson were both based on the novel by James M. Cain and actually neither are as well known as the 1946 Tay Garnett-directed version starring Lana Turner and John Garfield.
Contraband (2012) directed by Baltasar Kormakur who also produced the original Reykjavik-Rotterdam (2008) which was directed by Oskar Jonasson's original. I apologize for my terrible pronunciations and for mis-crediting (switching) Kormakur and Jonasson on the podcast.
Sorcerer (1977) directed by William Friedkin and Wages of Fear (1953) directed by Henri-Georges Clouzot were both based on the novel by Geroges Arnaud.
The Departed (2006) directed by Martin Scorsese was a remake of Infernal Affairs (2002) directed by Andy Lau and Alan Mak.
The Talented Mr. Ripley (1999) directed by Anthony Minghella and Renet Clement's Purple Noon (1960) were both based on Patricia Highsmith's novel The Talented Mr. Ripley.
Neil Jordan's The Good Thief (2002) is a remake of Jean-Pierre Melville's 1956 original Bob le Flambeur.
Tom Ripley made another appearance in the discussion this time as portrayed by John Malkovich in Liliana Cavani's Ripley's Game (2002) based on the novel of the same name by Patricia Highsmith as was Wim Wenders' The American Friend (1977) featuring a very different performance by Dennis Hopper opposite Bruno Ganz. Anybody out there seen Barry Pepper as Ripley in the 2005 Roger Spottiswoode adaptation of Ripley Under Ground?
Walter Hill's Last Man Standing was a remake of Akira Kurosawa's Yojimbo (1961) as was Sergio Leone's Fistful of Dollars, but I think Dashiell Hammett's novel Red Harvest is probably the real basis for all three.
Michael Haneke's Funny Games (2007) is an english language remake of his own Funny Games (1997).
And Gela Babluani also remade his own feature. 13 (2010) was originally 13 Tzameti (2005).
I'll leave the content of the discussion for the podcast, but here are a few we didn't discuss that I wasn't aware until recently even were remakes...
Before the Russo Brothers were taking over the Marvel Cinematic Universe they made the comic caper film Welcome to Collinwood with a knockout cast that included Sam Rockwell, William H. Macy, Luis Guzman, Isaiah Washington, Michael Jeter, Patricia Clarkson and George Clooney. It's one I just revisited for the first time since catching its theatrical run in 2002 and the first two-thirds really hold up. It kinda falls apart in the final act, but it's well worth killing time with if you want some light-hearted criminal fare.
I had not realized it was a remake of Mario Monicelli's 1958 film Big Deal on Madonna Street. I'll have to seek that one out.
Sean Penn's third directorial effort, 2001's The Pledge starring Jack Nicholson is a pretty terrific tale about a detective disappearing beneath an obsession with an unsolved case.
It's based on the novel by Friedrich Dürrenmatt, which in turn was based on his own screenplay for the 1958 film It Happened in Broad Daylight directed by Ladislao Vajda.
On the Christmas episode Pete and I discussed Daryl Duke's 1978 heist flick The Silent Partner, but it wasn't until this week that I knew it was a remake of the 1969 Danish film Think of a Number directed by Palle Kjærulff-Schmidt.
Both versions are based on the novel Tænk på et tal by Anders Bodelsen.
Plenty of others we didn't get into including Point of No Return/La Femme Nikita, Criminal/9 Queens The Next Three Days/Anything For Her, as well as both versions of Breathless, Nightwatch, Bangkok Dangerous, Oldboy, Pusher, Insomnia, We Are What We Are and The Vanishing. Maybe we'll do something like this again sometime.
Over at Letterboxd I've put together as complete a list as I could think of: English Language Remakes of Foreign Crime Flicks. Lemme know what I missed.
I read Charles Hansen's drug-themed story collection Trips (published under the name Charles Fischer) several years ago and it made an impression. The characters and situations depicted within defied what I thought of as the standard stock and trope of drug literature and turned them into brave explorers and adventure stories. In the book's introduction Kent Anderson says of his own experiences with drugs "I can't recommend drugs to anyone, any more than I can recommend going to war. I'm lucky to be alive, yet those days were rich with life, risk, self-knowledge and smiling death, my companion and teacher. Perhaps it's morally superior to just 'say no' all your life than rot in a nursing home or end up sucking on Dr. Kevorkian's carbon monoxide bottle, but I don't think so," and, as a sheltered kid grown into a naive adult, I found that a compelling, chewy thought.
I asked Charles to recommend me some of his favorite drug stories (books or films) and he hit me up with this little essay about Robert Stone's novel Dog Soldiers and the filmed adaptation, Karel Reisz's Who'll Stop the Rain.
I hope you don't need my endorsement to check out the book and the film, but for heaven's sake, do search out the out of print Trips.
On Dog Soldiers/Who'll Stop the Rain
by Charles Hansen
[Epigraph for Dog Soldiers]:
I've seen the devil of violence and the devil of greed and the devil of hot desire; but, by all the stars!
these were strong, lusty, red-eyed devils that swayed
and drove men—men, I tell you. But as I stood on that
hillside, I foresaw that in the blinding sunshine of that land, I would become acquainted with a flabby, pretending weak-eyed devil
of a rapacious and pitiless folly.
- Joseph Conrad, Heart of Darkness
"I've waited my whole life to fuck up like this."
-John Converse from Dog Soldiers/Who'll Stop the Rain
Spoiler Alert! If you don't already know that a Robert Stone hero is just some fuck-up looking for a noble cause to die for, then his novel Dog Soldiers, from which the film Who'll Stop the Rain was adapted, is good as any place to learn the lesson, I guess.
I recently returned to Who'll Stop the Rain, for an answer to a question that I forgot I'd once asked. It comes up at the finish of one of the very best heroin scenes ever put on the page or screen. Ray Hicks (our hero), Marge Converse (the wife of the book's less tragic hero John Converse), and Eddie Peace- a somewhat degenerate drug dealer (is there any other kind?) and a couple of naïf junkies are thrown together because a two-kilo heroin deal has gone to shit. As the result of some severe miscommunication, Ray tires of the whole sordid scene and decides to cut it short by giving Eddie Peace and the Hollywood naïfs a lethal dose. And here the question arises, did they or didn't they expire? Hint: It's hard to tell.
The central conflict in this war novel Dog Soldiers is not between our 'heroes', such as they are, and the evil scum-sucking and bent narcs whose intent it is to rip them off and kill them. The prime battle is among those who treat life or death situations with the proper seriousness, and the many dilettantes who insist on inserting themselves into situations beyond their ken. The Vietnam War, which forms the setting and the background of this tale is dragged into this debate thusly: America killed a million Vietnamese but were we serious about it? After all, we didn't even slaughter them with the dignity accorded food. Instead we exterminated them like they were noxious weeds. Something to be eliminated for our western democracy nature preserve of the mind. It's why we killed from far above and with, more often than not, napalm, herbicides and high explosives. Vietnam wasn't a war, it was a nation-wide landscaping project.
John Converse (reading from a paper in Saigon): "Elephants declared enemy agents."
We didn't even commit all that many front line troops. Hence, the relatively low America GI death toll of fifty-plus thousand. Still, the more radical elements of the American left promised to "Bring the war back home." Ray Hicks and John Converse, our erstwhile heroes, decide instead to bring back some excellent China White smack and declare victory for themselves.
Early on Hicks and John Converse have a conversation about their inaugural Vietnam-to-America heroin smuggling venture:
Hicks: I didn't know we were that way?
Converse: We're that way. Isn't this where everyone finds out who they are?
Hicks: Yeah. What a bummer for the gooks.
Robert Stone was not a moralist when it comes to drugs. He took them (so to speak) as they came. For instance, when Hicks and Marge Converse go on the run with John Converse's heroin, Hicks introduces Marge to the real shit. Before she was just a pill head, but now she gets a taste:
She hit the other one, and then sat stock-still; tears ran from her closed eyes. Slowly, she bent forward and rested her forehead against the desk. Hicks moved the phone book out of her way.
In a few minutes, she sat up again and turned to him. She was smiling. She put her arms around his waist; her tears and runny nose wet his shirt. He bent down to her; she rested her head on his shoulder. The tension drained from her in small sobs.
"Better than a week in the country, right?"
Holding to him, she stood up and he helped her to the bed. She lay across it, arching her back, stretching her arms and legs toward its four corners.
"It's a lot better than a week in the country," she said. (Dog Soldiers, p. 170.)
Not exactly a stinging indictment against heroin use, is it?
Why is Who'll Stop the Rain, such a great junkie's movie? Maybe it is because heroin is not for dilettantes and Ray Hicks is the embodiment of this truth. He has logical reasons for killing the Hollywood fools that have come to sample his wares, but, really, he does them in because he just hates fools. How better to make them pay than to have their own curiosity do them in.
In the book this central scene takes place in seedy cabins by the beach in Topanga Canyon. In the movie Gerald and Jody's posh home in the hills above LA is where the fun takes place.
Here is Gerald as sets himself up for his own demise:
"I'm a writer," he said.
Eddie Peace joined the tips of his thumb and index finger like a billboard chef and blew him a kiss.
"Now scag is a problem . . . or a phenomenon . . . that's important. It's a subject which has a lot of significance, particularly right now."
"Particularly right now," said Eddie.
"I mean," Gerald told them, "I've done dope like a lot of people have. I've blown acres of pot in my time and I've had some beautiful things with acid. But in all honesty I've never been in a scag environment because it just wasn't my scene."
"But now," Marge suggested, "it's your scene."
Gerald blushed slightly.
"Not exactly. But it's something I feel I should address. As a writer. Because of the significance it has."
"Particularly now," Marge said. (Dog Soldiers, p 189. Kindle edition.)
Then comes what for every needle-freak, dope fiend is climactic:
"Your works or mine, Eddie?"
"Mine," Eddie said. "They're new."
His works were new, a regulation syringe, without improvisations. He had cotton and a jar of surgical alcohol. Hollywood.
"Now that's what I call narcotics paraphernalia, "Hicks said.
"I got better than that," Eddie said. "I got coke to run with it. I don't go for that nowhere noddy feeling."
"I do," Marge said.
"Sure you do. You're a broad."
He assembled the needle and admired its luster. Jody watched him. (Dog Soldiers, pp. 196, Kindle edition.)
And afterwards?
Marge tried to experience Gerald's overdose as a good idea. It was not the way she was used to looking at things.
"So fuck Gerald?"
"That's right," Hicks said. "Fuck Gerald."
"For all the obvious reasons."
"Fuck all the obvious reasons." (Dog Soldiers, pp. 202, Kindle edition.)
And a moment later: "Once, I joined the marines. Never again will I be fucked around by morons."
Missing from the film version is one of Dog Soldiers major characters, Deiter Bechstein. He is a guru of Ray Hicks, a father figure he abandoned for the marines. He is based on author and world-famous acid head Ken Kesey. This was a smart omission by the screenwriters Robert Stone and Judith Rascoe because it makes the film more timeless than the book. Deiter Bechstein and the dialogue between him and Marge Converse and later Hicks roots Dog Soldiers firmly in its time the 60s and early 70s.
Here, Deiter explains Hicks's relationship with reality much in the same way Ken Kesey described Neal Cassady, the real life basis for Hicks:
There was absolutely no difference between thought and action for him." He clapped his hands and held them together in a grip that whitened his fingers. "It was exactly the same. An enormous self-respect. Whatever he believed in he had to embody absolutely."
Marge put a hand to her face and laughed.
"Wow," Dieter said. "Wow is right." (Dog Soldiers, pp. 271, Kindle edition.)
Later Dieter/Kesey explains why it might have all gone wrong when he tried to change the world with mass LSD distribution:
He farted loudly and without embarrassment.
"Then it occurred to me that if I applied the American style—which I didn't really understand—if I pushed a little, speeded things up a little, we might break into something really cosmic. The secular world was falling apart. Nobody knew what they were doing or what they wanted. There was a great ear open. Waiting for something."
Dieter closed his eyes and put clasped hands over the top of his head.
"I was sitting up here hearing it! What they wanted"—with a thrust of his chin he indicated the world below—"I had. I knew! So I thought, a little push, a little shove, a little something extra to shake it loose. And I ended up as Doctor Dope."
He opened his eyes and shrugged.
"It's a fucked-up world. One's a weak vessel."
"Everybody came down."
"Nobody came down," Dieter shouted at her. "We disappeared without a trace. We haven't been seen since."
"Look at Ray," Dieter said. "He's trapped in a samurai fantasy—an American one. He has to be the Lone Ranger, the great desperado—he has to win all the epic battles single handed." He stood up wearily. "It may not be a very original conception, but he's quite good at it."
(Dog Soldiers, pp. 272, Kindle edition.)
Ray Hicks/Neal Cassady was the embodiment of all the hippie words and desires. He was never a junkie and he never sold out, but he did die young and his madness was almost pure.
Here are Ray Hick's last orders for Deiter as their roles reverse:
"When you hear a round, light them up. Get on the mikes—I want a real deluge of weirdness. I want an opera."
"Yes," Dieter said, "I can see that. But in real life, you can't pull it off."
"Well then, fuck real life. Real life don't cut no ice with me." He transferred a couple of clips from the sea bag to his pockets. (Dog Soldiers, pp. 290, Kindle edition.)
And here are our last glimpses of Ray Hicks/Neal Cassady (who famously died walking Mexican railroad tracks in 1968) as filtered through Robert Stone's sensibilities:
He stopped and watched the mountains vibrate.
"You know what's out there? Every goddamn race of shit jerking each other off. Mom and Dad and Buddy and Sis, two hundred million rat-hearted cocksuckers in enormous cars. Rabbits and fish. They're mean and stupid and greedy, they'll fuck you for laughs, they want you dead. If you're no better than them you might as well take gas. (Dog Soldiers, pp. 325, Kindle edition.)
And possibly Ray Hicks expires thinking last what Neal Cassady himself might have:
In the end, there was only the tracks. That's enough, he said, to himself, I can dig tracks.
Out of spite, out of pride, he counted the crossties aloud. He counted hundreds and hundreds of them. When he had to stop, he leaned his head on his rifle and held to the blazing rail with his strong right hand. (Dog Soldiers, pp. 330, Kindle edition.)
A small but not insignificant change in the film version from the book is that at the end in the film Who'll Stop the Rain, the heroin blows away in the desert wind, which seems a typical Hollywood pessimistic cum moralistic take. That is, the decision about what to do with the evil substance is taken from the Converses' hands, so they are free to pursue their future (thanks to Hick's self-sacrifice) bloodied but unbroken. In the book the dope is successfully snatched up by the surviving bent American operative and Mexican narc. It is their reward for treating it with the proper respect. Will they kill each other over it, if time and circumstances offer an opportunity, that is, maybe?
In the book, these words by John Converse serve as well as any summation:
"Nobody knows," Converse told her confidently. "That's the principle we were defending over there. That's why we fought the war." (Dog Soldiers, pp. 306, Kindle edition.)
Having been through the thrill and regret of use and abuse many times myself, the central fear that occurs at some point (after the glorious rush) is, did I push it too far this time. Even though I am not dead or have not killed, have I set in motion doom for myself and my loved ones, which is what I fear most of all. That is the question all dilettantes must ask themselves before it's too late.
Finally, I offer this addenda from the Robert Stone interview in Paris Review No. 90:
Interviewer: There's a lot of dope, and an epic amount of drinking in your books—
Stone: I can't stop them from doing it. I just can't. It's getting ridiculous. I get laughed at for the volume of chemicals that gets consumed, the amount of dope and booze. And now this guy's doing it again in my new book.
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