It has been proved that agroinfiltration-based temporary expression of coatomer subunit alpha (COPA) gene from Tetranychus urticae hairpin RNA induces RNA interference (RNAi) and lethality to T. urticae. To establish detailed protocols for agroinfiltration, the efficiency of agroinfiltration to the soybean and kidney bean was determined with respect to different Agrobacterium delivery methods (sea sand, carborundum and syringe) and the spacial expression patterns of hairpin RNA was investigated following Agrobacterium delivery. Sea sand and syringe showed the highest expression level in soybean and kidney bean, respectively. Considering the resulting tissue damage, syringe appeared the best choice for agroinfiltration in both soybean and kidney bean. The apical region of a leaf showed more relative expression levels in both soybean and kidney bean compared to the basal region. Following agroinfiltration, adjacent untreated leaves were determined to express hairpin RNA though the expression level was low, suggesting that hairpin RNA can be translocated to other leaves. In conclusion, Agrobacterium delivery by syringe and use of whole leaf were recommended for T. urticae bioassay following agroinfiltration.

Recently, RNA interference (RNAi) technology has been emerged as a potent tool for pest control strategy. Based on the previous studies on RNAi via leaf disc-mediated systemic delivery of dsRNA and in planta expression of hairpin RNA by agroinfiltration, the coatomer subunit alpha (COPA) gene has been found to be a crucial target for RNAi against Tetranychus urticae. In current study, transgenic plants of Arabidopsis thaliana expressing COPA hairpin RNA were generated by the floral dip method. Putative transgenic plants were screened by PCR and positive transformants were subjected to bioassay using age-synchronized and host-adapted T. urticae. T. urticae feeding on plants expressing dsRNA/siRNA showed more than 80% mortality as compared to the mites feeding on control plants at 6 days post-infestation. Our data shows that in planta expression of hairpin gene such as COPA may serve as an effective way for the control of this important pest in ornamental and economically important plants.

In our previous study, COPA (coatomer subunit alpha) gene from the two-spotted spider mite, Tetranychus urticae, exhibited RNA interference (RNAi)-based lethality when its double-stranded RNA (dsRNA) was systemically delivered via multi-unit chambers or its hairpin RNA was in planta-expressed by agroinfiltration. The cumulative mortality of T. urtcae was 55.0 ±14.2% in soybean plants agroinfiltrated with COPA gene. To investigate the temporal expression profiles of hairpin RNA following agroinfiltration, the amount of hairpin RNA expressed in plants was quantified over time by quantitative real-time PCR. Relative transient expression levels of T. urticae COPA hairpin RNA was highest at 46 h post-agroinfiltration and the extent of COPA gene knockdown was lowest at 12 h post-infestation on soybean plants. To investigate small interference RNA (siRNA) profiling, northern blot assay is currently under progress.

RNA interference (RNAi) has been proven as an operative technique for efficient gene silencing in many organisms. In our study, Tetranychus urticae, an extremely polyphagous and rapidly resistance developing mite against acaricides, was screened by double-stranded RNA (dsRNA) delivery method using multi-unit chambers. Among several lethal genes of T.urticae, COPA (the coatomer subunit alpha), a gene involved in membrane transport between the endoplasmic reticulum (ER) and the Golgi complex, showed the highest mortality rate [median lethal time (LT50)=54h]. To investigate the effect of dsCOPA treatment to lysosome formation, we used the Lysotracker green DND26 dye, selective to acidic cellular compartments such as lysosome. The result revealed that the dsEGFP-treated T. urticae has 1.3-fold more of lysosome than that of dsCOPA treated, indicating that downregulation of COPA affected lysosomes function and autophagy, thereby resulting in lethality. To investigate the further detailed toxic mechanism of COPA knockdown, investigation on histological changes in T. urticae fed COPA dsRNA is currently on going.

Expression of hairpin RNA corresponding to the part of COPA transcript was done by agroinfiltration in soybean plants and was confirmed by qRT-PCR. In a pot experiment, T. urticae was infested on agroinfiltrated soybean plants and T. urticae mortality was observed and compared with control plants overtime. Significantly higher mortalities of T. urticae were observed in the COPA-agroinfiltrated soybean plants from post-infestation day 2 (15 ±5%), day 4 (50 ±10 %). At post-infestation day 6, mortality reached to (70 ± 15%). To validate the observed COPA silencing effect in T. urticae fed on the agroinfiltrated soybean plant expressing COPA hairpin RNAs, qRT-PCR analysis was performed. The transcript level of COPA gene was decreased in T. urticae fed on agroinfiltrated soybean plants expressing COPA hairpin RNA from post-infestation day 2. At post-infestation day 2, 4 and 6, COPA transcript levels were reduced by 23.8, 20.7 and 18.8 fold, respectively compared to post-infestation day 1 (control). The results obtained in this study also ruled that the plant mediated production and uptake of silencing (dsRNAs/siRNAs) is an effective way to trigger RNAi in the T. urticae.