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.

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.

The full-length cDNA encoding Perilla frutescens limonene synthase (PFLS) (603 amino acids, GenBank accession no. D49368) was cloned. To elucidate the role of PFLS in gene regulation, we transiently transformed full-length PFLS into tobacco plants. PFLS mRNA was first detected in the intact leaves of the plants at 6 h, and the LS transcript level increased after 12 h in leaves treated with oxidative stress-related chemicals. The transient overexpression of PFLS resulted in increased transcription of NbPR1 and NbSIP in Nicotiana benthamiana leaves. Thus, our result confirmed that the infiltration of PFLS gene act as a transcriptional regulator of NbPR1 or NbSIP genes in the tobacco.