BACKGROUND

One of the key limiting factors for using RNA interference technology to develop new pest control practices is the toxicological effectiveness. In order to explore potential methods to improve the efficiency of RNAi in insects, we cloned two dsRNA binding proteins from Laodelphax striatellus and investigated their functions.



RESULTS

In this study, the genes homologous to the dsRNA binding proteins TRBP and STAU in L. striatellus were first cloned and designated as LsTRBP and LsSTAU. Using the chitinase gene Chi, which has been confirmed to have lethal effects when knocked down by RNAi, as a reporter, the functions of these two dsRNA binding proteins were then investigated by using dsRNA feeding. The results showed that feeding with dsChi alone caused significant lethal effects, with a mortality four-fold higher than that of the control, whereas feeding with dsTRBP or dsSTAU alone did not yield any significant lethal effects. When dsChi was fed in the mixture 1:1 with dsSTAU, it also resulted in high mortality, which was a little lower than feeding with dsChi alone, but not statistically significant. However, when fed in the 1:1 mixture with dsTRBP, the lethal effect of dsChi was completely eliminated. Further analysis of gene expression found that single dsRNA depressed only its own target gene. When treated in 1:1 mixture, dsSTAU or dsChi did not change significantly the depression of the other dsRNA, but dsTRAB counteracted almost all the effect of dsChi.



CONCLUSION

These results indicate that the loss of LsTRBP can significantly inhibit dsChi-mediated genetic interference. These findings not only suggest that the LsTRBP gene in L. striatellus plays an important role in regulating RNAi but also imply that the metabolic regulation of dsRNAs with respect to genetic interference in L. striatellus is similar to that in higher animal species. © 2013 Society of Chemical Industry

A functional study of two dsRNA binding protein genes in Laodelphax striatellus

OF DISSERTATION Role of plasmodesmata localizingand double-stranded RNA binding-proteins in systemic immunity and plant defense The various modes of defense induced upon the recognition of pathogen-derived molecules provide species level resistance to non-host pathogens, local and systemic resistance to race-specific pathogens, and basal resistance to virulent pathogens. Resistance (R) genemediated or species-specific immunity is induced when a strain-specific avirulence (avr) effector from the pathogen associates directly/indirectly with a cognate plant R protein. Plant viruses often encode suppressors of host RNA silencing machinery. In turn, plants recognize such suppressors via R proteins. Recognition of the Turnip Crinkle Virus (TCV) coat-protein (CP, RNA silencing suppressor) induces HRT-mediated resistance in Arabidopsis thaliana. This requires the RNA silencing component, double-stranded RNAbinding protein (DRB)4, even though HRT-mediated resistance neither requires the RNA silencing suppressor function of CP, nor is it associated with the accumulation of TCVspecific small-RNA. The drb4 mutant plants contain reduced HRT protein and induce spreading hypersensitive-response to TCV. HRT interacts with DRB4, but only its cytosolic fraction. TCV infection increases the cytosolic DRB4 pool, but also inhibits the HRT-DRB4 interaction. The virulence inducing R8A CP mutation also disrupts this interaction, but alters DRB4 localization partially. This suggests that subcellular compartmentalization of DRB4 may contribute to HRT-derived resistance signaling. DRB4 is likely a universal defense regulator because it also regulates R-mediated defense to bacteria. Consequently, drb4 plants are compromised in systemic acquired resistance (SAR). SAR involves symplastic intracellular transport between adjacent cells occurs via channels known as plasmodesmata (PD). I show that proteins that regulate PD gating, such as the PD localizing proteins (PDLP) 1 and 5 are essential for SAR. Furthermore, while phloem loading of the SAR-inducing azelaic acid (AzA) and glycerol-3-phosphate (G3P) occurs via the symplast, that of salicylic acid (SA) occurs via the apoplast. Plants overexpressing PDLP5 have constitutively closed PD and are impaired for SAR because their PD closure impaired the transport of AzA and G3P, but not SA. Together, these results identify the transport routes of SAR signals and highlight an important regulatory role for DRB and PDLP proteins in plant defense and systemic immunity. Notably, the pdlp1/5 mutants with constitutively open PD, are also compromised for SAR. PDLP1 interacts with a protein (AZI1) that is required for AzA/G3P-induced SAR and contributes to the partitioning of AZI1 between the plastids and cytoplasm.

Role of Plasmodesmata Localizing- and Double-Stranded RNA Binding-Proteins in Systemic Immunity and Plant Defense

Global papaya production is severely affected by papaya ringspot disease caused by Papaya ringspot virus (PRSV). Management of this potyvirus is challenging, due to 1) its non-persistent transmission by numerous aphid species and 2) the diversity of PRSV strains that exists within a country or between different geographical regions. Papaya cultivars with transgenic resistance have reduced the impact of the disease. There are no effective alternatives to transgenic resistance available in areas where disease pressure is high. In Hawaii, transgenic papayas such as “SunUp” and “Rainbow” have remained resistant to PRSV more than two decades saving the commercial papaya industry. Following the success in Hawaii, researchers from other countries have focused on developing PRSV-resistant transgenic papaya. These transgenic cultivars often demonstrated an initial transitory resistance that was ultimately overcome by the virus. For other cases, resistance was inconsistent. That is, some transgenic lines were resistant while others were not. Transgenic cultivars are now losing PRSV-resistance for various reasons in China and Taiwan. In this review, we present an update on work with transgenic papaya with resistance to PRSV. The focus is on factors affecting transgenic resistance in papaya and our attempt to explain why the Hawaiian scenario of complete and durable resistance has not been replicated in other regions. The utilization of more recent technologies to the development of virus resistance in papaya is also discussed.

Transgene-mediated resistance to Papaya ringspot virus: challenges and solutions

Double stranded RNA(dsRNA) is a trigger of cellular responses to viral infections.An essential prerequisite is that dsRNA acts on enzymes and functional proteins which can specifically recognize dsRNA and elicit cellular response.Some data indicated that dsRNA-binding domain(DRBD) not only binds with dsRNA,but also interacts with DNA or RNA,or only acts as a regulator without binding any nucleic acid.In addition,DRBD can interact with each other in the same protein.Among different proteins,DRBD can also interact with each other.These interactions result in complicated protein-protein complexes,which participate in multiple cell metabolic pathways.Therefore,DRBD has diverse functions.

Double stranded RNA binding protein and dsRNA binding domain

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Double stranded RNA binding protein and dsRNA binding domain