Design of dsRNAs targeting PcNLP2 and PcNLP6 in P. capsici
In our previous study [12, 13], treating plants with dsRNAs targeting different regions in the target mRNA sequence yielded different effects on the target gene. Likewise, we first examined which region of the PcNLP2 and PcNLP6 mRNAs should be targeted to most effectively suppress P. capsici infection. We designed two dsRNAs for each gene: dsRNA_PcNLP2_5′ and dsRNA_PcNLP2_3′ targeting the 5′ and 3′ regions of PcNLP2, dsRNA_PcNLP6_5′ and dsRNA_PcNLP6_3′ targeting the 5′ and 3′ regions of PcNLP6, respectively (Fig. 1). A total of four dsRNAs were synthesized to evaluate their suppressive effects on P. capsici infection.
Determination of the treatment time of dsRNAs and P. capsici
In our previous study, pepper mottle virus was suppressed the most when dsRNAs were infiltrated 2 days before viral inoculation [12], suggesting that pre-treatment of dsRNAs provides sufficient time for siRNA production and RISC formation to counteract virus infection. Hence, we injected dsRNAs into N. benthamiana leaves 2 days before P. capsici inoculation as well (Additional file 1: Fig. S2a).
We performed qRT-PCR at 3, 6, 12, 24, 27, 30, and 36 h post-inoculation (hpi) of P. capsici (without dsRNAs) to examine the kinetics of PcNLP2 and PcNLP6 expression during the infection stage in N. benthamiana leaves. The highest PcNLP2 and PcNLP6 expressions were observed at 24 hpi (Additional file 1: Fig. S3). Therefore, we measured the size of infected lesions and PcNLP2/PcNLP6 expression in P. capsica-inoculated leaves at 24 hpi using FOBI and qRT-PCR, respectively (Additional file 1: Fig. S2).
P. capsici via suppression dsRNAs targeting PcNLP2 and PcNLP6
dsRNAs targeting PcNLP2, PcNLP6, or mock (50 µg each) were infiltrated into the abaxial side of 3 week-old N. benthamiana leaves, which were inoculated with P. capsici zoospore drops 2 days later. At 24 hpi, infected lesions were significantly suppressed in dsRNA_PcNLP2_5′-treated leaves compared with mock-treated leaves (Fig. 2a, b), but no significant change in lesion size was observed in dsRNA_PcNLP2_3′-treated leaves (Additional file 1: Fig. S4a). On the contrary, dsRNA_PcNLP6_3′-treated leaves showed significantly suppressed lesions (Fig. 3a, b), while dsRNA_PcNLP6_5′-treated leaves did not (Additional file 1: Fig. S4b). These results indicate that dsRNA-mediated gene suppression varies with the region targeted, highlighting the importance of dsRNA design.
Additionally, we analyzed PcNLP2 and PcNLP6 transcript levels in leaves-treated with dsRNA_PcNLP2_5´ and dsRNA_PcNLP6_3´, respectively. Compared with mock-treated leaves, PcNLP2 and PcNLP6 expression were more than two-fold lower in dsRNA_PcNLP2_5′-treated leaves and dsRNA_PcNLP6_3′-treated leaves, respectively (Figs. 2c and 3c). Therefore, the exogenous application of these designed dsRNAs could significantly suppress P. capsici infection and inhibit PcNLP2 and PcNLP6 expression in N. benthamiana.
Expression of defense-related genes in N. benthamiana treated with dsRNAs
We investigated the expression of some well-known plant defense-related genes in response to targeting PcNLP2 and PcNLP6 with dsRNAs. We performed qRT-PCR on three defense-related genes in N. benthamiana: Pathogenesis-related 1 (PR1), a defense factor that responds to pathogen infection; WRKY8, a disease-associated gene that interacts with mitogen-activated protein kinase involved in plant innate immunity; and Harpin-induced 1 (Hin1), a marker gene for hypersensitive response [24,25,26].
Compared with mock-treated leaves, the expression of NbPR1 and NbHin1 decreased by six-fold each and that of NbWRKY8 by two-fold in dsRNA_PcNLP2_5′-treated leaves. Interestingly, dsRNA_PcNLP6_3′ treatment only reduced the expression of NbHin1 by three-fold, while the expressions of NbPR1 and NbWRKY8 were not affected significantly (Fig. 4). These results suggest that suppression of PcNLP2 and PcNLP6 by dsRNAs may play distinct roles in the expression of NbPR1 and NbWRKY8.