In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5′-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3′ half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.antiviral immunity | cucumber mosaic virus | RNA silencing | RNAdependent RNA polymerase | secondary small interfering RNA R NA silencing or RNA interference in fungi, nematodes and plants requires amplification of small interfering RNAs by eukaryotic RNA-directed RNA polymerases (RDRs) (1-3). Plant and fungal RDRs convert transcripts of target genes into dsRNA that is subsequently processed into secondary siRNAs by a Dicer or Dicer-like (DCL) nuclease. In contrast, Caenorhabditis elegans RDRs, such as RRF-1, may directly manufacture secondary siRNAs without dicing a dsRNA precursor (1-3). The genome of Arabidopsis thaliana encodes six RDRs that are grouped into four clusters (1, 2, 4), among which little is known about cluster III, consisting of RDRs 3a, 3b, and 3c. RDR2 and RDR6 are both required for the short-distance spread of transgene silencing and for the perception, but not the production, of the long-distance mobile silencing signal (1, 5-9). RDR2 is also essential for the biogenesis of the DCL3-dependent 24-nucleotide (nt) repeat-associated siRNAs (rasiRNAs) derived from transposons, retroelements, and other elements, which are the most abundant endogenous small RNAs in A. thaliana (3). Similarly, RDR6 coupled with DCL4 or DCL1 is responsible for the biogenesis of transacting siRNAs (tasiRNAs) and natural antisense siRNAs (nat-siRNAs), which silence expression of their target genes like microRNAs (miRNAs) (1-3).RNA silencing controls antiviral immunity in fungi, plants, and invertebrates by producing virus-derived siRNAs to be loaded in an Argonaute protein for antiviral silencing (10-12). In A. thaliana, DCL4 and DCL2 produce viral siRNAs against distinct positive (+)-strand RNA viruses in a hierarc...

We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real-time PCR assay. The concentration of the bacterium was monitored at 5-day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca. L. asiaticus-infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus-infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25-, 360-and 130-fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.

The c-met protooncogene is a growth factor receptor with tyrosine kinase activity. Recently the hepatocyte growth factor was identified as the ligand for this receptor. Because the hepatocyte growth factor is a most potent mitogen in hepatocytes, possible involvement of c-met expression in hepatocarcinogenesis is suspected. In this study, we examined c-met expression in 23 hepatocellular carcinoma cases by means of Northern-blot analysis and an immunohistochemical study. Northern-blot analysis revealed c-met mRNA expression in the tumors of 6 of 19 patients (31.6%); in the immunohistochemical study, c-met protein was detected in 16 of 23 patients (69.6%). With both methods, c-met was found to be overexpressed in hepatocellular carcinoma compared with the surrounding normal liver. Comprehensive analysis showed that c-met protein expression was correlated with poor-to-moderate differentiation of cancer cells (p < 0.05). Tumor proliferative activity of hepatocellular carcinoma was evaluated by means of Ki-67 labeling index. All cases with increased tumor proliferative activity showed c-met protein expression, although the elevation of proliferative activity in the c-met-positive group was not statistically significant. These data suggest that the overexpression of c-met plays an important role in the development of hepatocellular carcinoma.

No abstract

Sequential polyacrylamide gel electrophoresis analyses showed many viroid-like RNAs in samples collected from citrus trees in Japan. Reverse transcription polymerase chain reaction and sequencing analyses of the amplified fragments verified that they were derived from variants of six citrus viroids, Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd) including CVd-I-LSS (a distinct variant of CBLVd), Hop stunt viroid, Citrus viroid III, Citrus viroid IV, and Citrus viroid OS. The samples induced symptoms with variable severity in Arizona 861-S1 'Etrog' citrons (Citrus medica L.) likely due to the varying accumulation patterns produced by the different viroids. Some of the symptoms caused by the samples harboring the citrus viroids other than CEVd were as severe as those caused by CEVd. Some source citrus trees showing the severe bark scaling characteristic of exocortis disease in trifoliate orange (Poncirus trifoliata (L.) Raf.) rootstocks contained only citrus viroids other than CEVd in complex. This indicates that certain exocortis-like diseases in Japan were caused by some combination of citrus viroids not including CEVd.

No abstract

The 3'-terminal regions of the genomes of apple stem grooving virus (ASGV), isolated from apple, Japanese pear, and European pear plants, and citrus tatter leaf virus (CTLV), isolated from citrus plants, were amplified by reverse transcription-polymerase chain reaction. The DNA products were cloned and sequenced. The results indicated that the ASGV isolates from apple, Japanese pear, and European pear comprise at least two to four "sequence variants" that differ considerably from each other in nucleotide sequence. Comparison of the amino acid (aa) sequences of the open reading frame (ORF) 1- and 2-encoded proteins among isolates or sequence variants, including isolates of ASGV and CTLV reported previously, showed that the aa sequences of the ORF2-encoded protein (320 aa) and the putative coat protein (CP) (237 aa), which was equivalent to aa positions 1869 to 2105 of the ORF1-encoded protein of ASGV (P-209), were highly conserved among isolates and sequence variants. Identities ranged from 92.8 to 100% for the ORF2-encoded protein among 15 isolates and sequence variants and from 92.4 to 100% for the CP among 21 isolates and sequence variants. On the other hand, the aa sequence identities of a variable 284-aa region (designated the V-region), equivalent to aa positions 1585 to 1868 of the ORF1-encoded protein (which also encoded the ORF2-encoded protein in another frame), were highly variable (53.2 to 99.3%) among 15 isolates and sequence variants. The percentage of conserved V-region aa in all 15 isolates was only 20.4%. Phylogenetic trees constructed from the aa sequences of the V-region, coat, and ORF2-encoded proteins showed that isolates and sequence variants were separated into several clusters, regardless of the source host plant species (apple, Japanese pear, European pear, citrus, and lily). In a tree constructed for the V-region proteins, two CTLV isolates were grouped into two clusters, each of which contained isolates and sequence variants from apple and Japanese pear. These results suggest that both CTLV from citrus and ASGV from Rosaceous plants should be considered isolates of ASGV.

A new viroid was obtained from a viroid sample, named 'OS', collected from a citrus plant. The viroid consists of 330-331 nucleotides, contains the central conserved region (CCR) characteristic of the genus Apscaviroid, and has the highest sequence similarity (only 68%) with Citrus III viroid (CVd-III) among known viroids. This viroid, by itself, caused only mild petiole necrosis and characteristically very mild leaf bending in Arizona 861-S1 Etrog citrons (Citrus medica L.), the degree of which differed from that induced by other citrus viroids. This viroid could be a new citrus viroid species belonging to the genus Apscaviroid; for convenience, it was tentatively named Citrus viroid OS (CVd-OS) after the original sample. CVd-OS has chimeric features related to other viroids. In particular, CVd-OS has high sequence similarity with CVd-III and Apple dimple fruit viroid in the putative central and terminal left domains, including a duplicative sequence from the lower CCR of the genus Pospiviroid in the left terminus. Further, CVd-OS shares high sequence similarity with Citrus exocortis viroid (CEVd) in the lower strand of the putative variable and terminal right domains, including the sequence identical to that of the right termini of CEVd and of Citrus IV viroid.