Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of EXORIBONUCLEASE4/THYLENE-INSENSITIVE5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed.
Cucumber Mosaic Virus (CMV) is a highly infectious cucumovirus, which infects more than 800 plant species and causes major diseases in greenhouse and field crops worldwide. Parasitic weeds such as Phelipanche aegyptiaca are a major constraint to the production of many crops in the world and the parasite's lifestyle makes control extremely difficult. The parasite seeds can germinate after conditioning and perceiving strigolactones secreted by the host roots. Strigolactones are rhizosphere signaling molecules in plants that are biosynthesized through carotenoid cleavage. In the present study we investigated the possibility of reducing ?-carotene and then strigolactone production in the host roots by blocking carotenoid biosynthesis using CMV-infected tobacco. It was found that CMV downregulated the enzyme phytoene desaturase(PDS) and reduced significantly both carotenoid production and Phelipanche infection in tobacco host roots infected with both CMV and P. aegyptiaca. Based on our results (decrease of ?-carotene and repression of PDS transcripts in tobacco roots), we hypothesized that the reduction of Phelipanche tubercles and shoots occurred due to an effect of CMV on secondary metabolite stimulators such as strigolacetones. Our study indicated that mass production of the host roots was not affected by CMV; however, most inflorescences of Phelipanche grown on CMV-infected tobacco developed abnormally (deformed shoots and short nodes). Carotenoid biosynthesis inhibitors such as CMV can be used to reduce the production of strigolactones, which will lead to decreased Phelipanche attachment. Interestingly, attenuated CMV strains may provide a safe means for enhancing crop resistance against parasitic weeds in a future plan.
In the early 2000s, two cucurbit-infecting begomoviruses were introduced into the eastern Mediterranean basin: the Old World Squash leaf curl virus (SLCV) and the New World Watermelon chlorotic stunt virus (WmCSV). These viruses have been emerging in parallel over the last decade in Egypt, Israel, Jordan, Lebanon and Palestine.
IMPORTANCE Physicians can demonstrate mastery of the knowledge that supports continued clinical competence by passing a maintenance of certification examination (MOCEX). Performance depends on professional learning and development, which may be enhanced by informal routine interactions with colleagues. Some physicians, such as those in solo practice, may have less opportunity for peer interaction, thus negatively influencing their examination performance. OBJECTIVE To determine the relationship among level of peer interaction, group and solo practice, and MOCEX performance. DESIGN, SETTING, AND PARTICIPANTS Longitudinal cohort study of 568 surgeons taking the 2008 MOCEX. Survey responses reporting the level of physicians' peer interactions and their practice type were related to MOCEX scores, controlling for initial qualifying examination scores, practice type, and personal characteristics. EXPOSURES Solo practice and amount of peer interaction. MAIN OUTCOMES AND MEASURES Scores on the MOCEX and pass-fail status. RESULTS Of the 568 surgeons in the study sample, 557 (98.1%) passed the examination. Higher levels of peer interaction were associated with a higher score (??=?0.91 [95% CI, 0.31-1.52]) and higher likelihood of passing the examination (odds ratio, 2.58 [1.08-6.16]). Physicians in solo (vs group) practice had fewer peer interactions (??=?-0.49 [95% CI, -0.64 to -0.33), received lower scores (??=?-1.82 [-2.94 to -0.82]), and were less likely to pass the examination (odds ratio, 0.22 [0.06-0.77]). Level of peer interaction moderated the relationship between solo practice and MOCEX score; solo practitioners with high levels of peer interaction achieved an MOCEX performance on a par with that of group practitioners. CONCLUSIONS AND RELEVANCE Physicians in solo practice had poorer MOCEX performance. However, solo practitioners who reported high levels of peer interaction performed as well as those in group practice. Peer interaction is important for professional learning and quality of care.
Particle bombardment is an efficient method for virus inoculation of intact plants. This technique enables inoculation with full-length infectious clone cDNA, PCR products, virus from sap or virus preparation, and in vitro viral transcripts. The inoculation of some phloem-limited RNA and circular DNA viruses is also possible. The technique of bombardment without the use of vacuum permits the inoculation of soft-leaved plants that do not usually survive bombardment inoculation, the investigation of viral recombination in planta, promoter analysis, monitoring virus movement using an infectious clone bearing a reporter gene and the inoculation of large numbers of plants. The inoculation of whitefly-borne circular DNA begomoviruses is now possible due to direct genome amplification by Rolling Circle Amplification (RCA), followed by bombardment using a device that does not require a vacuum for operation. Here we describe the inoculation of intact plants with (a) RNA virus infective clones and (b) begomoviruses after direct genome amplification by RCA, using a handheld bombardment device.
Gene-silencing has been used to develop resistance against many plant viruses but little is known about the transgenic small-interfering RNA (t-siRNA) that confers this resistance. Transgenic cucumber and melon lines harboring a hairpin construct of the Zucchini yellow mosaic potyvirus (ZYMV) HC-Pro gene accumulated different levels of t-siRNA (6 to 44% of total siRNA) and exhibited resistance to systemic ZYMV infection. Resistance to Watermelon mosaic potyvirus and Papaya ring spot potyvirus-W was also observed in a cucumber line that accumulated high levels of t-siRNA (44% of total siRNA) and displayed significantly increased levels of RNA-dependent RNA (RDR)1 and Argonaute 1, as compared with the other transgenic and nontransformed plants. The majority of the t-siRNA sequences were 21 to 22 nucleotides in length and sense strand biased. The t-siRNA were not uniformly distributed throughout the transgene but concentrated in "hot spots" in a pattern resembling that of the viral siRNA peaks observed in ZYMV-infected cucumber and melon. Mutations in ZYMV at the loci associated with the siRNA peaks did not break this resistance, indicating that hot spot t-siRNA may not be essential for resistance. This study shows that resistance based on gene-silencing can be effective against related viruses and is probably correlated with t-siRNA accumulation and increased expression of RDR1.
Little is known about the translocation of proteins and other macromolecules from a host plant to the parasitic weed Phelipanche spp. Long-distance movement of proteins between host and parasite was explored using transgenic tomato plants expressing green fluorescent protein (GFP) in their companion cells. We further used fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite. Accumulation of GFP was observed in the central vascular bundle of leaves and in the root phloem of transgenic tomato plants expressing GFP under the regulation of AtSUC2 promoter. When transgenic tomato plants expressing GFP were parasitized with P. aegyptiaca, extensive GFP was translocated from the host phloem to the parasite phloem and accumulated in both Phelipanche tubercles and shoots. No movement of GFP to the parasite was observed when tobacco plants expressing GFP targeted to the ER were parasitized with P. aegyptiaca. Experiments using fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite demonstrated that Phelipanche absorbs dextrans up to 70 kDa in size from the host and that this movement can be bi-directional. In the present study, we prove for the first time delivery of proteins from host to the parasitic weed P. aegyptiaca via phloem connections, providing information for developing parasite resistance strategies.
Tomato plants transformed with a cDNA clone encoding the inhibitor-of-virus-replication (IVR) gene were partially resistant to Botrytis cinerea. This resistance was observed as a significant reduction in the size of lesions induced by the fungus in transgenic plants compared with the lesions on the nontransgenic control plants. This resistance was weakened when plants were kept at an elevated temperature, 32 degrees C, before inoculation with B. cinerea compared with plants kept at 17 to 22 degrees C prior to inoculation. Resistance correlated with the presence of IVR transcripts, as detected by reverse transcription-polymerase chain reaction. This is one of the few cases in which a gene associated with resistance to a virus also seems to be involved in resistance to a fungal disease.
Broomrapes (Phelipanche, formerly Orobanche) are parasitic plants that physically connect with the vascular systems of their hosts through haustorial structures. In this study, we found that Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV), Potato virus Y (PVY), and Tomato yellow leaf curl virus (TYLCV) translocate from infected host plants to Phelipanche aegyptiaca. In order to examine whether these viruses, and specifically CMV, replicate in the parasite, we tested several replication parameters. We detected accumulation of both plus and minus strands of CMV genomic RNA and CMV-derived siRNAs in the shoots of Phelipanche grown on CMV-infected tobacco and tomato plants. We purified CMV particles from Phelipanche grown on CMV-infected plants. These particles were present in amounts comparable to those found in the hosts leaves. These data indicate that CMV replicates in Phelipanche tissues. In addition, viable ToMV and PVY were observed, and the plus and minus strand RNAs of ToMV were detected in Phelipanche shoots grown on infected hosts. However, we found only low levels of ToMV coat protein and did not detect any PVY coat protein. We also detected genomic TYLCV DNA in shoots of Phelipanche grown on TYLCV-infected tomato. Thus, for the first time, we demonstrate that broomrape is a host for at least one plant virus CMV, and possibly various other viruses.
Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%-80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.
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