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Tomato has undergone extensive selections during domestication. Recent progress has shown that genomic structural variants (SVs) have contributed to gene expression dynamics during tomato domestication, resulting in changes of important traits. Here, we performed comprehensive analyses of small RNAs (sRNAs) from nine representative tomato accessions. We demonstrate that SVs substantially contribute to the dynamic expression of the three major classes of plant sRNAs: microRNAs (miRNAs), phased secondary short interfering RNAs (phasiRNAs), and 24-nucleotide heterochromatic siRNAs (hc-siRNAs). Changes in the abundance of phasiRNAs and 24-nucleotide hc-siRNAs likely contribute to the alteration of mRNA gene expression in cis during tomato domestication, particularly for genes associated with biotic and abiotic stress tolerance. We also observe that miRNA expression dynamics are associated with imprecise processing, alternative miRNA-miRNA* selections, and SVs. SVs mainly affect the expression of less-conserved miRNAs that do not have established regulatory functions or low abundant members in highly expressed miRNA families. Our data highlight different selection pressures on miRNAs compared to phasiRNAs and 24-nucleotide hc-siRNAs. Our findings provide insights into plant sRNA evolution as well as SV-based gene regulation during crop domestication. Furthermore, our dataset provides a rich resource for mining the sRNA regulatory network in tomato.

Supplementary Data from Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage–responsive proteins and reverses DNA damage–induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis. Cancer Res; 70(18); 7176–86. ©2010 AACR.

Background: Soybean gene functions cannot be easily interrogated through transgenic disruption (knock-out) of genes-of-interest, or transgenic overexpression of proteins-of-interest, because soybean transformation is time-consuming and technically challenging. An attractive alternative is to administer transient gene silencing or overexpression with a plant virus-based vector. However, existing virus-induced gene silencing (VIGS) and/or overexpression vectors suitable for soybean have various drawbacks that hinder their widespread adoption. Results: We describe the development of a new vector based on cowpea severe mosaic virus (CPSMV), a plus-strand RNA virus with its genome divided into two RNA segments, RNA1 and RNA2. This vector, designated FZ, incorporates a cloning site in the RNA2 cDNA, permitting insertion of nonviral sequences. When paired with an optimized RNA1 construct, FZ readily infects both Nicotiana benthamiana and soybean. As a result, FZ constructs destined for soybean can be first delivered to N. benthamiana in order to propagate the modified viruses to high titers. FZ-based silencing constructs induced robust silencing of phytoene desaturase genes in N. benthamiana, multiple soybean accessions, and cowpea. Meanwhile, FZ supported systemic expression of fluorescent proteins mNeonGreen and mCherry in N. benthamiana and soybean. Finally, FZ-mediated expression of the Arabidopsis transcription factor MYB75 caused N. benthamiana to bear brown leaves and purple, twisted flowers, indicating that MYB75 retained the function of activating anthocyanin synthesis pathways in a different plant. Conclusions: The new CPSMV-derived FZ vector provides a convenient and versatile soybean functional genomics tool that is expected to accelerate the characterization of soybean genes controlling crucial productivity traits.

Marafiviruses, including maize rayado fino virus (MRFV) and oat blue dwarf virus (OBDV), encode two carboxy co-terminal coat proteins, CP1 and CP2, which encapsidate the genome to form icosahedral virions. While CP2 expression is expected to be solely driven from a second start codon of a subgenomic RNA under a marafibox promoter sequence, the larger CP1 with an in-frame N-terminal extension relative to CP2 could potentially be expressed either by proteolytic release from the MRFV polyprotein or from subgenomic RNA translation. We examined MRFV CP expression strategy with a series of mutations in the CP coding region and identified mutants viable and nonviable for systemic plant infection. Polyprotein expression of MRFV CP1 was minimal. Mutants blocking CP2 expression failed to establish systemic infection, while mutants depleted in CP1 exhibited systemic infection and formation of virus-like particles but lost leafhopper transmissibility, indicating that CP1 is required for leafhopper transmission.

Tomato has undergone extensive selections during domestication. Recent progress has shown that genomic structural variants (SVs) have contributed to gene expression dynamics during tomato domestication, resulting in changes of important traits. Here, through comprehensive analyses of small RNAs (sRNAs) from nine representative tomato accessions, we demonstrate that SVs substantially contribute to the dynamic expression of the three major classes of plant sRNAs: microRNAs (miRNAs), phased secondary short interfering RNAs (phasiRNAs), and 24-nt heterochromatic siRNAs (hc-siRNAs). Changes in the abundance of phasiRNAs and 24-nt hc-siRNAs likely contribute to the alteration of mRNA gene expression during tomato’s recent evolution, particularly for genes associated with biotic and abiotic stress tolerance. We also observe that miRNA expression dynamics are associated with imprecise processing, alternative miRNA-miRNA* selections, and SVs. SVs mainly affect the expression of less-conserved miRNAs that do not have established regulatory functions or low abundant members in highly expressed miRNA families, highlighting different selection pressures on miRNAs compared to phasiRNAs and 24-nt hc-siRNAs. Our findings provide insights into plant sRNA evolution as well as SV-based gene regulation during crop domestication. Furthermore, our dataset provides a rich resource for mining the sRNA regulatory network in tomato.

Maize rayado fino virus (MRFV) is the type species of the genus Marafivirus in the family Tymoviridae. It infects maize (Zea mays), its natural host, to which it is transmitted by leafhoppers including Dalbulus maidis and Graminella nigrifrons in a persistent‐propagative manner. The MRFV monopartite RNA genome encodes a precursor polyprotein that is processed into replication‐associated proteins. The genome is encapsidated by two carboxy co‐terminal coat proteins, CP1 and CP2. Cloned MRFV can be readily transmitted to maize by vascular puncture inoculation (VPI), and such virus systems that can be used in maize are valuable to examine plant gene function by gene silencing. However, the efficacy of marafiviruses for virus‐induced gene silencing (VIGS) has not been investigated to date. To this end, MRFV genomic loci were tested for their potential to host foreign insertions without attenuating virus viability. This was done using infectious MRFV clones engineered to carry maize phytoene desaturase (PDS) gene fragments (ZmPDS) at various genomic regions. Several MRFV‐PDS constructs were generated and tested for infectivity and VIGS in maize. This culminated in identification of the helicase/polymerase (HEL/POL) junction as a viable insertion site that preserved virus infectivity, as well as several sites at which sequence insertion caused loss of virus infectivity. Transcripts of viable constructs, carrying PDS inserts in the HEL/POL junction, induced stable local and systemic MRFV symptoms similar to wild‐type infections, and triggered PDS VIGS initiating in veins and spreading into both inoculated and noninoculated leaves. These constructs were remarkably stable, retaining inserted sequences for at least four VPI passages while maintaining transmissibility by D. maidis. Our data thus identify the MRFV HEL/POL junction as an insertion site useful for gene silencing in maize.

Maize rayado fino virus (MRFV) is the type species of the genus Marafivirus in the family Tymoviridae. It infects maize (Zea mays), its natural host, to which it is transmitted by leafhoppers including Dalbulus maidis and Graminella nigrifrons in a persistent‐propagative manner. The MRFV monopartite RNA genome encodes a precursor polyprotein that is processed into replication‐associated proteins. The genome is encapsidated by two carboxy co‐terminal coat proteins, CP1 and CP2. Cloned MRFV can be readily transmitted to maize by vascular puncture inoculation (VPI), and such virus systems that can be used in maize are valuable to examine plant gene function by gene silencing. However, the efficacy of marafiviruses for virus‐induced gene silencing (VIGS) has not been investigated to date. To this end, MRFV genomic loci were tested for their potential to host foreign insertions without attenuating virus viability. This was done using infectious MRFV clones engineered to carry maize phytoene desaturase (PDS) gene fragments (ZmPDS) at various genomic regions. Several MRFV‐PDS constructs were generated and tested for infectivity and VIGS in maize. This culminated in identification of the helicase/polymerase (HEL/POL) junction as a viable insertion site that preserved virus infectivity, as well as several sites at which sequence insertion caused loss of virus infectivity. Transcripts of viable constructs, carrying PDS inserts in the HEL/POL junction, induced stable local and systemic MRFV symptoms similar to wild‐type infections, and triggered PDS VIGS initiating in veins and spreading into both inoculated and noninoculated leaves. These constructs were remarkably stable, retaining inserted sequences for at least four VPI passages while maintaining transmissibility by D. maidis. Our data thus identify the MRFV HEL/POL junction as an insertion site useful for gene silencing in maize.

An East African isolate of the maize-associated polerovirus, maize yellow mosaic virus (MaYMV) was previously shown to cause leaf reddening on singly infected maize plants (Zea mays). Here we describe the construction of a full-length infectious clone of an East African isolate and, for the first time, show infectivity of clone-derived transcripts in the primary host, maize, through vascular puncture inoculation (VPI), as well as in the dicotyledonous research model plant species, Nicotiana benthamiana, through agrobacterium inoculation. Characteristic leaf reddening symptoms were observed in a subset of maize plants inoculated with clone-derived transcripts, and infection was confirmed by RT-PCR and Northern blot analyses. In N. benthamiana plants, infections were entirely asymptomatic even at high virus titers, as was also reported for the cloned Chinese isolate. In this study, however, we demonstrated that N. benthamiana can serve as a clone launching platform for maize infection, as VPI of sap of infected N. benthamiana plants into maize kernels resulted in infection and the typical red leaf symptoms. We further demonstrated that the cloned East African isolate virus was aphid transmissible to maize, with experimental transmission rates up to 97 %, comparable to that shown previously for the native virus. Interestingly, our data additionally showed a definitive correlation of leaf reddening symptoms with increased expression of chalcone synthase, thus suggesting upregulation of the flavonoid biosynthesis pathway as the molecular basis for symptom induction in maize. As the first report of experimental infection of maize with transcripts from a cloned polerovirus, this work constitutes a breakthrough for studies on molecular maize-polerovirus-aphid interactions.

Plant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA pathway. A recent report has attributed viral suppressor-mediated developmental defects to up-regulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence was that the developmental defects in transgenic Arabidopsis (Arabidopsis thaliana) expressing viral suppressors were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. We have reinvestigated the role of ARF8 in viral suppressor-mediated developmental defects, using two independent arf8 mutations and the P1/HC-Pro potyviral suppressor of silencing. Progeny of a cross between P1/HC-Pro transgenic Arabidopsis and the arf8-6 T-DNA insertion mutant showed little effect on the P1/HC-Pro phenotype in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the phenotype in the F2 generation. However, the loss of phenotype in the F2 generation was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA and the appearance of 35S promoter small interfering RNAs. Furthermore, an independent loss-of-function point mutation, Arf8-8, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together, these data argue against the previously reported role of increased ARF8 expression in developmental defects caused by P1/HC-Pro.

Mucormycosis is an opportunistic infection caused by the fungi of the Mucorales order of the class Zygomycetes. Gastrointestinal mucormycosis is an uncommon, fatal condition accounting for only 7% of the cases. We present the case of a gastroduodenal mucormycosis presenting as recurrent massive hematemesis. We report this case to alert clinicians of this rare but fatal condition and to encourage further research into its pathogenesis and management., Highlights • Mucormycosis is rare and often fatal. • Rhino-orbital-cerebral and pulmonary infections dominate literature. • Gastrointestinal mucormycosis is rare and is fatal in 85% of cases. • We encourage further research into its pathogenesis.

*† ‡ SUMMARY The utility of many T-DNA insertion mutant lines of Arabidopsis is compromised by their propensity to trigger transcriptional silencing of transgenes expressed from the CaMV 35S promoter. To try to circumvent this problem, we characterized the genetic requirements for maintenance of 35S promoter homology-dependent transcriptional gene silencing induced by the dcl3-1 (SALK_005512) T-DNA insertion mutant line. Surprisingly, even though DCL3 and RDR2 are known components of the siRNA-dependent transcriptional gene silencing pathway, transcriptional gene silencing of a 35S promoter-driven GUS hairpin transgene did occur in plants homozygous for the dcl3-1 T-DNA insertion and was unaffected by loss of function of RDR2. However, the transcriptional gene silencing was alleviated in dcl2 dcl3 dcl4 triple mutant plants and also by mutations in AGO4, NRPD2, HEN1 and MOM1. Thus, some T-DNA insertion mutant lines induce 35S promoter homologydependent transcriptional silencing that requires neither DCL3 nor RDR2, but involves other genes known to function in siRNA-dependent transcriptional silencing. Consistent with these results, we detected 35S promoter siRNAs in dcl3-1 SALK line plants, suggesting that the 35S promoter homology-dependent silencing induced by some T-DNA insertion mutant lines is siRNA-mediated.

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage–responsive proteins and reverses DNA damage–induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis. Cancer Res; 70(18); 7176–86. ©2010 AACR.

Floral patterning and morphogenesis are controlled by many transcription factors including floral homeotic proteins, by which floral organ identity is determined. Recent studies have uncovered widespread regulation of transcription factors by microRNAs (miRNAs), approximately 21-nucleotide non-coding RNAs that regulate protein-coding RNAs through transcript cleavage and/or translational inhibition. The regulation of the floral homeotic gene APETALA2 (AP2) by miR172 is crucial for normal Arabidopsis flower development and is likely to be conserved across plant species. Here we probe the activity of the AP2/miR172 regulatory circuit in a heterologous Solanaceae species, Nicotiana benthamiana. We generated transgenic N. benthamiana lines expressing Arabidopsis wild type AP2 (35S::AP2), miR172-resistant AP2 mutant (35S::AP2m3) and MIR172a-1 (35S::MIR172) under the control of the cauliflower mosaic virus 35S promoter. 35S::AP2m3 plants accumulated high levels of AP2 mRNA and protein and exhibited floral patterning defects that included proliferation of numerous petals, stamens and carpels indicating loss of floral determinacy. On the other hand, nearly all 35S::AP2 plants accumulated barely detectable levels of AP2 mRNA or protein and were essentially non-phenotypic. Overall, the data indicated that expression of the wild type Arabidopsis AP2 transgene was repressed at the mRNA level by an endogenous N. benthamiana miR172 homologue that could be detected using Arabidopsis miR172 probe. Interestingly, 35S::MIR172 plants had sepal-to-petal transformations and/or more sepals and petals, suggesting interference with N. benthamiana normal floral homeotic gene function in perianth organs. Our studies uncover the potential utility of the Arabidopsis AP2/miR172 system as a tool for manipulation of floral architecture and flowering time in non-model plants.

RNA silencing is a conserved eukaryotic pathway in which double-stranded RNA (dsRNA) triggers destruction of homologous target RNA via production of short-interfering RNA (siRNA). In plants, at least some cases of RNA silencing can spread systemically. The signal responsible for systemic spread is expected to include an RNA component to account for the sequence specificity of the process, and transient silencing assays have shown that the capacity for systemic silencing correlates with the accumulation of a particular class of small RNA. Here, we report the results of grafting experiments to study transmission of silencing from stably transformed tobacco lines in the presence or absence of helper component-proteinase (HC-Pro), a viral suppressor of silencing. The studied lines carry either a tail-to-tail inverted repeat, the T4-IR transgene locus, or one of two different amplicon transgene loci encoding replication-competent viral RNA. We find that the T4-IR locus, like many sense-transgene-silenced loci, can send a systemic silencing signal, and this ability is not detectably altered by HC-Pro. Paradoxically, neither amplicon locus effectively triggers systemic silencing except when suppressed for silencing by HC-Pro. In contrast to results from transient assays, these grafting experiments reveal no consistent correlation between capacity for systemic silencing and accumulation of any particular class of small RNA. In addition, although all transgenic lines used to transmit systemic silencing signals were methylated at specific sites within the transgene locus, silencing in grafted scions occurred without detectable methylation at those sites in the target locus of the scion.

Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed enhanced symptoms after infection with the parental CABMV isolate and also after infection with a heterologous potyvirus, Potato virus Y (PVY) and a comovirus, Cowpea mosaic virus (CPMV). On the other hand, transgenic lines expressing nontranslatable HC-Pro or translatable HC-Pro with a deletion of the central domain showed wild type symptoms after infection with the parental CABMV isolate and heterologous viruses. These results showed that CABMV HC-Pro is a pathogenicity determinant that conditions enhanced sensitivity to virus infection in plants, and that the central domain of the protein is essential for this. The severe symptoms in CABMV-infected HC-Pro expressing lines were remarkably followed by brief recovery and subsequent re-establishment of infection, possibly indicating counteracting effects of HC-Pro expression and a host defense response. One of the HC-Pro expressing lines (h48) was found to contain low levels of transgenic HC-Pro RNA and to be resistant to CABMV and to recombinant CPMV expressing HC-Pro. This indicated that h48 was (partially) posttranscriptionally silenced for the HC-Pro transgene inspite of the established role of HC-Pro as a suppressor of posttranscriptional gene silencing. Line h48 was not resistant to PVY, but instead showed enhanced symptoms compared to nontransgenic plants. This may be due to relief of silencing of the HC-Pro transgene by HC-Pro expressed by PVY.

RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing., Author Summary RNA silencing is an important antiviral defense in plants, and many plant viruses encode proteins that block RNA silencing. However, the mechanism of action of the viral suppressors is complex, and little is known about the role of host plant proteins in the process. Here we report the first example of a host protein that plays a required role in viral suppression of silencing—a transcription factor called RAV2 that is required for suppression of silencing by two different and unrelated viral proteins. Analysis of plant gene expression patterns shows that RAV2 is required for induction of many genes involved in other stress and defense pathways, including genes implicated as plant suppressors of silencing. Overall, the results suggest that RAV2 is an important factor in viral suppression of silencing and that the role of RAV2 is to divert host defenses toward responses that interfere with antiviral silencing.

Small RNAs are the key mediators of RNA silencing and related pathways in plants and other eukaryotic organisms. Silencing pathways couple the destruction of double-stranded RNA with the use of the resulting small RNAs to target other nucleic acid molecules that contain the complementary sequence. This discovery has revolutionized our ideas about host defense and genetic regulatory mechanisms in eukaryotes. Small RNAs can direct the degradation of mRNAs and single-stranded viral RNAs, the modification of DNA and histones, and the inhibition of translation. Viruses might even use small RNAs to do some targeting of their own to manipulate host gene expression. This review highlights the current understanding and new insights concerning the roles of small RNAs in virus infection and host defense in plants.

Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2-but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

Expression of the viral silencing suppressor P1/HC-Pro in plants causes severe developmental anomalies accompanied by defects in both short interfering RNA (siRNA) and microRNA (miRNA) pathways. P1/HC-Pro transgenic lines fail to accumulate the siRNAs that mediate RNA silencing and are impaired in both miRNA processing and function, accumulating abnormally high levels of miRNA/miRNA* processing intermediates as well as miRNA target messages. Both miRNA and RNA silencing pathways require participation of DICER-LIKE (DCL) ribonuclease III-like enzymes. Here, we investigate the effects of overexpressing DCL1, one of four Dicers in Arabidopsis thaliana, on P1/HC-Pro–induced defects in development and small RNA metabolism. Expression of a DCL1 cDNA transgene (35S:DCL1) produced a mild gain-of-function phenotype and largely rescued dcl1 mutant phenotypes. The 35S:DCL1 plants were competent for virus-induced RNA silencing but were impaired in transgene-induced RNA silencing and in the accumulation of some miRNAs. Ectopic DCL1 largely alleviated developmental anomalies in P1/HC-Pro plants but did not correct the P1/HC-Pro–associated defects in small RNA pathways. The ability of P1/HC-Pro plants to suppress RNA silencing and the levels of miRNAs, miRNA*s, and miRNA target messages in these plants were essentially unaffected by ectopic DCL1. These data suggest that P1/HC-Pro defects in development do not result from general impairments in small RNA pathways and raise the possibility that DCL1 participates in processes in addition to miRNA biogenesis.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in animals and plants. Comparative genomic computational methods have been developed to predict new miRNAs in worms, flies, and humans. Here, we present a novel single genome approach for the detection of miRNAs in Arabidopsis thaliana. This was initiated by producing a candidate miRNA-target data set using an algorithm called findMiRNA, which predicts potential miRNAs within candidate precursor sequences that have corresponding target sites within transcripts. From this data set, we used a characteristic divergence pattern of miRNA precursor families to select 13 potential new miRNAs for experimental verification, and found that corresponding small RNAs could be detected for at least eight of the candidate miRNAs. Expression of some of these miRNAs appears to be under developmental control. Our results are consistent with the idea that targets of miRNAs encompass a wide range of transcripts, including those for F-box factors, ubiquitin conjugases, Leucine-rich repeat proteins, and metabolic enzymes, and that regulation by miRNAs might be widespread in the genome. The entire set of annotated transcripts in the Arabidopsis genome has been run through findMiRNA to yield a data set that will enable identification of potential miRNAs directed against any target gene.

The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of HC-Pro expressed with other viral proteins in cowpea protoplasts in a natural CABMV infection, or in protoplasts transfected with a transient expression construct expressing HC-Pro separately from other viral proteins under the control of the 35S promoter. In both cases the protein showed a diffuse cytoplasmic location. Similar localisation patterns were shown in live protoplasts when the transient expression system was used to express HC-Pro as a fusion with the green fluorescent protein as a reporter. In an alternative expression system, the HC-Pro coding region was subcloned in-frame between the movement protein and large coat protein genes of RNA2 of Cowpea mosaic virus (CPMV). Upon transfection of protoplasts with this construct, HC-Pro was expressed as part of the RNA2 encoded polyprotein from which it was fully processed. In this case, the protein localised in broad cytoplasmic patches reminiscent of the typical CPMV induced cytopathic structures in which CPMV replication occurs, suggesting an interaction of HC-Pro with CPMV proteins or host factors in these structures. Finally, recombinant CPMV expressing HC-Pro showed a strongly enhanced virulence on cowpea and Nicotiana benthamiana consistent with the role of HC-Pro as a pathogenicity determinant, a phenomenon now known to be linked to its role as a suppressor of host defense responses based on post-transcriptional gene silencing.

Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed enhanced symptoms after infection with the parental CABMV isolate and also after infection with a heterologous potyvirus, Potato virus Y (PVY) and a comovirus, Cowpea mosaic virus (CPMV). On the other hand, transgenic lines expressing nontranslatable HC-Pro or translatable HC-Pro with a deletion of the central domain showed wild type symptoms after infection with the parental CABMV isolate and heterologous viruses. These results showed that CABMV HC-Pro is a pathogenicity determinant that conditions enhanced sensitivity to virus infection in plants, and that the central domain of the protein is essential for this. The severe symptoms in CABMV-infected HC-Pro expressing lines were remarkably followed by brief recovery and subsequent re-establishment of infection, possibly indicating counteracting effects of HC-Pro expression and a host defense response. One of the HC-Pro expressing lines (h48) was found to contain low levels of transgenic HC-Pro RNA and to be resistant to CABMV and to recombinant CPMV expressing HC-Pro. This indicated that h48 was (partially) posttranscriptionally silenced for the HC-Pro transgene inspite of the established role of HC-Pro as a suppressor of posttranscriptional gene silencing. Line h48 was not resistant to PVY, but instead showed enhanced symptoms compared to nontransgenic plants. This may be due to relief of silencing of the HC-Pro transgene by HC-Pro expressed by PVY.

RNA silencing is a sequence-specific RNA degradation mechanism that occurs in a broad range of eukaryotic organisms including fungi (quelling), animals (RNA interference [RNAi]), and plants (post-transcriptional gene silencing). In all these organisms, the process is triggered by double-stranded RNA

The genomic sequence of a Zimbabwe isolate of Cowpea aphid-borne mosaic virus (CABMV-Z) was determined by sequencing overlapping viral cDNA clones generated by RT-PCR using degenerate and/or specific primers. The sequence is 9465 nucleotides in length excluding the 3' terminal poly (A) tail and contains a single open reading frame (ORF) of 9159 nucleotides encoding a large polyprotein of 3 053 amino acids and predicted Mr of 348. The size of the genome and the encoded polyprotein is in agreement with other potyviruses and contains nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. The P1 and P3 were the most variable proteins while CI, NIb and CP were the most conserved.