Turnip crinkle virus-encoded suppressor of RNA silencing suppresses mRNA decay by interacting with Arabidopsis XRN4.

Plant cells employ intricate defense mechanisms, including mRNA decay pathways, to counter viral infections. Among the RNA quality control (RQC) mechanisms, nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop decay (NSD) pathways play critical roles in recognizing and cleaving aberrant mRNA molecules. Turnip crinkle virus (TCV) is a plant virus that triggers mRNA decay pathways, but it has also evolved strategies to evade this antiviral defense. In this study, we investigated the activation of mRNA decay during TCV infection and its impact on TCV RNA accumulation. We found that TCV infection induced the upregulation of essential mRNA decay factors, indicating their involvement in antiviral defense and the capsid protein (CP) of TCV, a well-characterized viral suppressor of RNA silencing (VSR), also compromised the mRNA decay-based antiviral defense by targeting AtXRN4. This interference with mRNA decay was supported by the observation that TCV CP stabilized a reporter transcript with a long 3' untranslated region (UTR). Moreover, TCV CP suppressed the decay of known NMD target transcripts, further emphasizing its ability to modulate host RNA control mechanisms. Importantly, TCV CP physically interacted with AtXRN4, providing insight into the mechanism of viral interference with mRNA decay. Overall, our findings reveal an alternative strategy employed by TCV, wherein the viral coat protein suppresses the mRNA decay pathway to facilitate viral infection.

Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection.

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing-mediated antiviral defence.

Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector.

BACKGROUND: Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. RESULTS: In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants. CONCLUSIONS: The CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

Incomplete DRB4-dependence of the DCL4-mediated antiviral defense.

The double-stranded RNA-binding protein DRB4 of Arabidopsis was shown previously to contribute to the DICER-LIKE 4 (DCL4)-mediated biogenesis of viral small interfering RNAs (vsiRNAs) of 21 nucleotides (nt) in size. However, it is unclear whether all 21-nt vsiRNAs are dependent on this DRB4-DCL4 partnership. To resolve this question, we generated dcl2drb4 and dcl4drb4 double knockout mutants, and subjected them to infections with CPB-CC-PDS, a turnip crinkle virus mutant capable of inducing silencing of the PHYTOENE DESATURASE gene. The dcl2drb4 double knockouts caused a far smaller loss of antiviral silencing than dcl2dcl4. In addition, although both drb4 and dcl4 single mutants permitted a consistent (but small) increase in viral RNA levels, the drb4 mutant correlated with a less pronounced reduction of 21-nt vsiRNAs. Therefore, a substantial subset of DCL4 antiviral activity is DRB4-independent, and may involve other DRB proteins that compensate for loss of DRB4.

Compositional equivalency of RNAi-mediated virus-resistant transgenic soybean and its nontransgenic counterpart.

RNA silencing or RNA interference (RNAi), which is triggered by double-stranded RNA (dsRNA), is an evolutionarily conserved process that is active in a wide variety of eukaryotic organisms. Engineering plants with hairpin construct in which the viral gene is arranged in inverted repeats (IR) renders plants resistant to plant virus infection. However, there is no report on whether biologically important changes occurred by the insertion of IR, which confer transgenic plants virus resistance. In the present study, the compositions of virus-resistant transgenic soybean seeds developed by insertion of three short IRs, each containing the specific, highly conserved sequences derived from one virus, were compared with those of nontransgenic counterparts by applying the principle of substantial equivalence to determine whether significant undesirable biological changes occurred by IR insertion. The results revealed that the nutrient components as well as antinutrient contents of these virus-resistant soybean lines are substantially equivalent to those of the nontransgenic counterparts, and the majority of the measured amounts of nutritional components and antinutrient contents are well within the range of values reported for other commercial soybean lines. The results imply that no biologically important changes occurred by the insertion of IRs in the RNAi-mediated virus-resistant transgenic soybeans. The results can serve as baseline information for developing RNAi-mediated transgenic soybean cultivars or other crops with broader spectrum virus resistance.

Isolation of AtNUDT5 gene promoter and characterization of its activity in transgenic Arabidopsis thaliana.

AtNUDT5 is a cytosol Nudix that catalyzes the hydrolysis of a variety of substrates. In this report, a 1,387-bp 5'-flanking region of the AtNUDT5 gene was isolated from Arabidopsis thaliana. The tissue-specific activity of the 5'-flanking region was investigated by using the GUS gene as a reporter in transgenic A. thaliana plants. Weak GUS activity appeared in vascular tissues of young plants, strong GUS activity appeared in the axial roots, but no GUS activity was observed in the root cap, lateral roots, rosette leaf, mature silique and reproductive tissues such as stamen, pistil, and petal. Furthermore, by using these transgenic A. thaliana plants, results of the histochemical staining and fluorometric assays of GUS activity showed that the AtNUDT5 promoter can be activated by both avirulent Pst avrRpm1 and virulent Pst strains at 5 h post-infiltration and that the activity of AtNUDT5 promoter increased significantly at 24 h post-infiltration. Taken together, our results demonstrated that the AtNUDT5 promoter is pathogen-responsive. The promoter may be used to develop transgenic plants with an increased tolerance to pathogenic stresses.

HbMyb1, a Myb transcription factor from Hevea brasiliensis, suppresses stress induced cell death in transgenic tobacco.

Tapping panel dryness (TPD) is a complex physiological syndrome found widely in rubber tree (Hevea brasiliensis) plantations that causes severe yield loss in natural rubber-producing countries. In an earlier study, we confirmed that there is a negative correlation between HbMyb1 expression and TPD severity. To further investigate the function of HbMyb1 in TPD, HbMyb1 was over-expressed in tobacco controlled by a CaMV 35S promoter. In transgenic plants expressing HbMyb1, cell death induced by UV-B irradiation, paraquat and the hypersensitive reaction to necrotrophic fungal infection (Botrytis cinerea) was suppressed with a close correlation between HbMyb1 protein levels and the extent of suppression. In addition the nuclear condensation and degradation were observed in laticifer cells of TPD trees, while the nucleus of laticifer cells of healthy trees was morphologically normal. On the basis of the results described above, we propose that HbMyb1 maybe suppress stress induced cell death in rubber trees.

Association of decreased expression of a Myb transcription factor with the TPD (tapping panel dryness) syndrome in Hevea brasiliensis.

TPD (tapping panel dryness) is a complex physiological syndrome widely found in rubber tree (Hevea brasiliensis) plantations, which causes severe yield and crop losses in natural rubber-producing countries. The molecular mechanism underlying TPD is not known and there is presently no effective prevention or treatment for this serious disease. To investigate the molecular mechanism of TPD, we isolated and characterized genes for which the change of expression is associated with TPD. We report here the identification and characterization of a Myb transcription factor HbMyb1. HbMyb1 is expressed in leaves, barks, and latex of rubber trees, but its expression is significantly decreased in barks of TPD trees. Our results suggest that the expression of HbMyb1 is likely associated with TPD and that the function of HbMyb1 is associated with the integrity of bark tissue of rubber trees.