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/lp/springer-journals/suppression-of-phytophthora-capsici-using-double-stranded-rnas-haqfDQNXhj
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- Springer Journals
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- Copyright © The Author(s) 2023
- ISSN
- 2468-0834
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- 2468-0842
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- 10.1186/s13765-023-00768-4
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Abstract
RNA interference (RNAi) is a gene regulatory mechanism that involves the interaction of small interfering RNAs (siR- NAs) and RNA-induced silencing complex (RISC). Dicer cleaves exogenous double-stranded RNA (dsRNA) into siRNAs, which get incorporated into RISC and bind to complementary sequences on the target mRNA to induce its degrada- tion. In this study, we adopted RNAi technology using dsRNAs to suppress Phytophthora capsici, which causes diseases in solanaceous crops, including pepper. We designed and synthesized dsRNAs targeting the P. capsici effector genes PcNLP2 and PcNLP6, respectively. These genes encode necrosis and ethylene-inducing peptide 1-like proteins in P. capsici, which are known to promote oomycete infection. Nicotiana benthamiana leaves were first infiltrated with dsR- NAs and inoculated with P. capsici 2 days later. We confirmed significant suppression of P. capsici and PcNLP2, PcNLP6 expression in dsRNA-treated leaves. In addition, we found that downregulation of PcNLP2 and PcNLP6 distinctly affected the expression of some defense-related genes. These results suggest that dsRNA mediated RNAi technology can be used to suppress various pathogens, and may contribute toward crop protection. Keywords Phytophthora capsici, Double-stranded RNA, Exogenous RNA application, RNA interference is not used commercially [2, 3]. Moreover, the continuous Introduction use of chemical fungicides containing mefenoxam cause Pepper is an important crop used worldwide as a veg- P. capsici to develop resistance to them [4, 5]. Therefore, etable, spice, and a source of pharmacological agents. new strategies are necessary to manage P. capsici. Phytophthora capsici is an oomycete plant pathogen that RNA interference (RNAi), induced by exogenous dou- infects solanaceous crops like pepper, causing root rot ble-stranded RNA (dsRNA), can be used for crop protec- leading to yield losses [1, 2]. Criollo de Morelos 334 is a tion and other processes as a gene silencing mechanism pepper landrace known to be resistant to P. capsici but it in plants [6]. Dicer-like endonucleases cleave dsRNA into 20–25-nucleotide small interfering RNAs (siRNAs) Minsu Park and Yujin Kweon are authors contributed equally to this work in plant cellular system. The guide strand of siRNAs is incorporated into the Argonaute protein to form RNA- *Correspondence: Chanseok Shin induced silencing complex (RISC), which post-transcrip- cshin@snu.ac.kr tionally silences target genes through sequence-specific Department of Agricultural Biotechnology, Seoul National University, base pairing [6–8]. Target gene silencing via exogenous Seoul 08826, Republic of Korea Research Institute of Agriculture and Life Sciences, Seoul National dsRNA is influenced by the length of dsRNA, regions in University, Seoul 08826, Republic of Korea the target mRNA, and the method of application [9–14]. Plant Genomics and Breeding Institute, Seoul National University, In previous studies, we suppressed green fluorescent pro - Seoul 08826, Republic of Korea Research Center for Plant Plasticity, Seoul National University, tein and pepper mottle virus in plants using dsRNAs var- Seoul 08826, Republic of Korea ying in their length and position of their targets [12–14]. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Park et al. Applied Biological Chemistry (2023) 66:5 Page 2 of 7 These results suggest that P. capsici can be effectively 13]. Therefore, we designed and synthesized two distinct suppressed via exogenous dsRNA. dsRNAs targeting PcNLP2 and PcNLP6, respectively, and Plant pathogens carry various effector proteins that applied them at the optimum time for effective P. capsici contribute to microbial fitness and pathogen virulence suppression. by manipulating plant immune responses at infection interface [15–18]. Necrosis and ethylene-inducing pep- Materials and methods tide 1-like proteins (NLPs) secreted by oomycetes induce Plant materials and growth conditions leaf necrosis distinct from immune-related programmed The wild-type N. benthamiana was used in this study. cell death in plants. They have been suggested to play Its seeds were sown in 200-plug tray filled with horti - roles of both an immune response trigger and a toxin- cultural soil (Seoulbio, Republic of Korea). Two-week- like virulence factor [19]. Most of the NLPs in P. sojae old N. benthamiana plants were transplanted into are highly expressed during cyst germination and infec- pots and grown at 25 ℃ under 16 h of light per day and tion stages [20]. In P. capsici, PcNLP2 and PcNLP6 were 50% humidity in a growth chamber. Three-week-old highly expressed at infection stage. Moreover, the larg- N. benthamiana plants were selected for the following est necrotic area in Capsicum annuum and Nicotiana experiments. benthamiana leaves were produced in agroinfection assays that delivered PcNLP2 and PcNLP6, respectively, Design and synthesis of dsRNAs indicating the importance of these genes for virulence For each of PcNLP2 and PcNLP6, two dsRNAs were during infection stages [21]. Thus, inhibiting PcNLP2 and designed to target the 5′ and 3′ regions of the mRNA. PcNLP6 may suppress P. capsici infection in plants. They had a length of 400 bp and 540 bp with overlaps The aim of this study was to suppress P. capsici via of 59 bp and 63 bp between each dsRNA, respectively RNAi using dsRNAs targeting PcNLP2 and PcNLP6. (Fig. 1). A 500 bp-long dsRNA targeting the Renilla lucif- For efficient application of dsRNAs, location on the tar - erase gene was used as a mock. For dsRNA synthesis, get gene and time of application are crucial factors [12, their corresponding DNA templates, including the T7 PcNLP2 Phytophthoracapsici Necrosis and ethylene-inducing 741 bp peptide 1-like protein 2 dsRNA_PcNLP2_5′ (PcNLP2) 400 bp dsRNA_PcNLP2_3′ 400 bp 59 bp PcNLP6 Phytophthoracapsici Necrosis and ethylene-inducing 1017 bp peptide 1-like protein 6 dsRNA_PcNLP6_5′ (PcNLP6) 540 bp dsRNA_PcNLP6_3′ 540 bp 63 bp Fig. 1 Design of double-stranded RNAs (dsRNAs) targeting PcNLP2 and PcNLP6. a dsRNA_PcNLP2_5′ and dsRNA_PcNLP2_3′ were designed to target the 5′ and 3′ regions of the PcNLP2 mRNA sequence, respectively. Each dsRNA was 400 bp in length, with a 59 bp overlap between them. b dsRNA_PcNLP6_5′ and dsRNA_PcNLP6_3′ were designed to target the 5′ and 3′ regions of the PcNLP6 mRNA sequence, respectively. Each dsRNA was 540 bp in length, with a 63 bp overlap between them P ark et al. Applied Biological Chemistry (2023) 66:5 Page 3 of 7 Quantitative real‑time PCR (qRT‑PCR) promoter sequences (5′-TAA TAC GAC TCA CAT ATA Complementary DNA was synthesized from 1 μg of AGA GAG -3′), were synthesized by polymerase chain each RNA sample using PrimeScript Reverse Tran- reaction (PCR) using Phusion High-Fidelity DNA poly- scriptase (Takara, Japan) and oligo (dT) primers merase (Thermo Scientific, United States), according (Thermo Scientific, United States), according to the to the manufacturer’s protocol. The PCR products were manufacturer’s instructions. qRT-PCR was performed then used for dsRNA synthesis using the MEGAscript using Light Cycler 480 SYBR Green I Master (Roche, RNAi Kit (Invitrogen, United States), following the man- United States) with SYBR Green detection and gene- ufacturer’s protocol (Additional file 1: Fig. S1). The prim - specific primers. The Ct values for genes were obtained ers for PCR are listed in Additional file 1: Table S1. using NbL23 as a control [23], and relative expression values were calculated using the ΔΔCt method. The Maintenance of P. capsici primers for defense-related genes were kindly provided The KACC 40476 strain of P. capsici was kindly provided by Dr. Doil Choi’s Laboratory (Seoul National Univer- by Dr. Doil Choi’s Laboratory (Seoul National University, sity, Seoul, Republic of Korea) and sequences of all the Seoul, Republic of Korea). It was grown on V8 juice agar primers are listed in Additional file 1 : Table S2. media for 8 days in the dark at 23 °C. Results Infiltration of dsRNAs and infection of N. benthamiana Design of dsRNAs targeting PcNLP2 and PcNLP6 in P. capsici with P. capsici In our previous study [12, 13], treating plants with dsR- According to our previous study [12], 2 mL of dsRNAs NAs targeting different regions in the target mRNA (25 ng/μL) were infiltrated 2 days before P. capsici infec- sequence yielded different effects on the target gene. tion into the abaxial side of N. benthamiana leaves using Likewise, we first examined which region of the PcNLP2 a 1 mL needle-free syringe. One day later, the mycelium and PcNLP6 mRNAs should be targeted to most effec - of P. capsici on V8 juice agar media was scraped and tively suppress P. capsici infection. We designed incubated overnight at 23 °C under continuous light to two dsRNAs for each gene: dsRNA_PcNLP2_5′ and form sporangia. The next day, the plate was filled with dsRNA_PcNLP2_3′ targeting the 5′ and 3′ regions of 10 mL of distilled water and incubated at 4 °C for 1 h PcNLP2, dsRNA_PcNLP6_5′ and dsRNA_PcNLP6_3′ to harvest the zoospores. The zoospore suspension was targeting the 5′ and 3′ regions of PcNLP6, respectively counted with a hemocytometer and adjusted to a con- 4 −1 (Fig. 1). A total of four dsRNAs were synthesized to centration of 5 × 10 zoospores mL . The abaxial side evaluate their suppressive effects on P . capsici infection. of detached N. benthamiana leaves was infected with a 12 μL droplet of zoospore suspension. Infected leaves were incubated in the dark at 23 °C for about 24 h and P. Determination of the treatment time of dsRNAs and P. capsici capsici infection was confirmed via phenotypic observa - In our previous study, pepper mottle virus was sup- tion before sampling the leaves. pressed the most when dsRNAs were infiltrated 2 days before viral inoculation [12], suggesting that pre-treat- Observation of chlorophyll fluorescence expression ment of dsRNAs provides sufficient time for siRNA Chlorophyll fluorescence was confirmed by the fluores - production and RISC formation to counteract virus cence in vivo imaging system FOBI (Neoscience, Repub- infection. Hence, we injected dsRNAs into N. bentha- lic of Korea) under blue light combined with a yellow miana leaves 2 days before P. capsici inoculation as filter. This method was used to confirm P. capsici infec - well (Additional file 1 : Fig. S2a). tion because infected lesions do not exhibit chlorophyll We performed qRT-PCR at 3, 6, 12, 24, 27, 30, and fluorescence. The size of infected lesions was quantified 36 h post-inoculation (hpi) of P. capsici (without dsR- using ImageJ [22]. NAs) to examine the kinetics of PcNLP2 and PcNLP6 expression during the infection stage in N. benthami- ana leaves. The highest PcNLP2 and PcNLP6 expres - Total RNA extraction sions were observed at 24 hpi (Additional file 1: Fig. After observing chlorophyll fluorescence, N. benthami- S3). Therefore, we measured the size of infected lesions ana leaves were sampled and ground in liquid nitrogen. and PcNLP2/PcNLP6 expression in P. capsica-inocu- Total RNA was isolated using RiboEx (GeneAll, Republic lated leaves at 24 hpi using FOBI and qRT-PCR, respec- of Korea) and treated with recombinant DNase I (Takara, tively (Additional file 1 : Fig. S2). Japan) to eliminate single-stranded and double-stranded DNA, according to the manufacturer’s instructions. Park et al. Applied Biological Chemistry (2023) 66:5 Page 4 of 7 P. capsici via suppression dsRNAs targeting PcNLP2 and PcNLP6 dsRNAs targeting PcNLP2, PcNLP6, or mock (50 µg dsRNA_ each) were infiltrated into the abaxial side of 3 week- Mock PcNLP6_3′ old N. benthamiana leaves, which were inoculated + P. capsici with P. capsici zoospore drops 2 days later. At 24 P. capsici 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 Mock dsRNA_ lesion size was observed in dsRNA_PcNLP2_3′-treated PcNLP6_3′ leaves (Additional file 1: Fig. S4a). On the contrary, dsRNA_PcNLP6_3′-treated leaves showed significantly b c suppressed lesions (Fig. 3a, b), while dsRNA_PcNLP6_5′- PcNLP6 1.5 1.5 treated leaves did not (Additional file 1: Fig. S4b). These results indicate that dsRNA-mediated gene suppression 1 1 varies with the region targeted, highlighting the impor- tance of dsRNA design. 0.5 0.5 ** * Additionally, we analyzed PcNLP2 and PcNLP6 tran- script levels in leaves-treated with dsRNA_PcNLP2_5´ 0 0 Mock dsRNA_ Mock dsRNA_ and dsRNA_PcNLP6_3´, respectively. Compared with PcNLP6_3′ PcNLP6_3′ Fig. 3 Suppression of P. capsici infection and PcNLP6 expression via PcNLP6-targeting dsRNA. a The left figure shows the scheme of dsRNA treatment and P. capsici infection, and the right figure shows the phenotype result of P. capsici infection after treatment with mock and dsRNA_PcNLP6_3′, respectively, in 3 week-old N. dsRNA_ Mock benthamiana leaves using FOBI. Scale bar = 1 cm. b Quantification of PcNLP2_5′ P. capsici infection lesion size using ImageJ. c Quantification of PcNLP6 P. capsici transcript level using qRT-PCR. Mock: Treated with dsRNA targeting P. capsici Renilla luciferase. dsRNA_PcNLP6_3′: Treated with dsRNA_PcNLP6_3′. Data represent mean ± SEM (N = 3). Significance is determined by Student’s t-test, *P < 0.05 and **P < 0.01 dsRNA_ Mock PcNLP2_5′ mock-treated leaves, PcNLP2 and PcNLP6 expression b c were more than two-fold lower in dsRNA_PcNLP2_5′- PcNLP2 1.5 1.5 treated leaves and dsRNA_PcNLP6_3′-treated leaves, respectively (Figs. 2c and 3c). Therefore, the exogenous 1 1 application of these designed dsRNAs could significantly suppress P. capsici infection and inhibit PcNLP2 and ** 0.5 0.5 PcNLP6 expression in N. benthamiana. 0 0 Mock dsRNA_ Mock dsRNA_ Expression of defense‑related genes in N. benthamiana PcNLP2_5′ PcNLP2_5′ treated with dsRNAs Fig. 2 Suppression of Phytophthora capsici infection and PcNLP2 We investigated the expression of some well-known plant expression via PcNLP2-targeting dsRNA. a The left figure shows defense-related genes in response to targeting PcNLP2 the scheme of dsRNA treatment and P. capsici infection, and the right figure shows the phenotype result of P. capsici infection and PcNLP6 with dsRNAs. We performed qRT-PCR on after treatment with mock and dsRNA_PcNLP2_5′, respectively, three defense-related genes in N. benthamiana: Patho- in 3 week-old Nicotiana benthamiana leaves using a fluorescence genesis-related 1 (PR1), a defense factor that responds to in vivo imaging system FOBI. Scale bar = 1 cm. b Quantification of P. pathogen infection; WRKY8, a disease-associated gene capsici infection lesion size using ImageJ. c Quantification of PcNLP2 that interacts with mitogen-activated protein kinase transcript level using quantitative real-time PCR (qRT-PCR). Mock: Treated with dsRNA targeting Renilla luciferase; dsRNA_PcNLP2_5′: involved in plant innate immunity; and Harpin-induced Treated with dsRNA_PcNLP2_5′. Data represent mean ± standard 1 (Hin1), a marker gene for hypersensitive response error of mean (SEM; N = 3). Significance is determined by Student’s [24–26]. t-test, *P < 0.05 and **P < 0.01 Lesion size Relative expression level Lesion size Relative expression level P ark et al. Applied Biological Chemistry (2023) 66:5 Page 5 of 7 Compared with mock-treated leaves, the expression of different effects on P. capsici suppression (Figs. 2, 3, and NbPR1 and NbHin1 decreased by six-fold each and that Additional file 1: File. S4). In our previous study [12], of NbWRKY8 by two-fold in dsRNA_PcNLP2_5′-treated differential cleavage tags of target genes were observed leaves. Interestingly, dsRNA_PcNLP6_3′ treatment only in dsRNA-treated samples. Therefore, the different reduced the expression of NbHin1 by three-fold, while effects of dsRNAs on P. capsici may be because different the expressions of NbPR1 and NbWRKY8 were not amounts of siRNAs are produced from distinct dsRNA affected significantly (Fig. 4). These results suggest that sequences, which can be confirmed by analyzing the pool suppression of PcNLP2 and PcNLP6 by dsRNAs may play of small RNAs in dsRNA-treated samples. distinct roles in the expression of NbPR1 and NbWRKY8. Since NLPs are known to affect plant defense and immune responses [19], we explored the expression of Discussion three well-known defense-related genes in response to Chemical fungicides have been traditionally used to con- PcNLPs suppression using qRT-PCR [27]. Interestingly, trol P. capsici [2, 4], but their long-term use and accumu- inhibition of PcNLP2 significantly downregulated NbPR1, lation may induce unknown mutations in the pathogen. NbWRKY8, and, NbHin1, whereas inhibition of PcNLP6 dsRNA-mediated RNAi technology is now being widely downregulated NbHin1 only (Fig. 4). The expression of used to target plant genes, insects, viruses, and fungi [6]. Hin1, a hypersensitive response marker gene [24], was This approach enables us to respond to plant diseases expected to change in the same pattern as the size of P. faster than any other technology, especially in an ecosys- capsica-infected lesions (Figs. 2b, 3b, and 4c). PR genes tem where disease-related mutations are frequent. When have been shown to induce systemic acquired resist- a specific gene mutation leads to the emergence of a new ance, increasing the defensive capacity of plants against plant disease that resists existing chemical pesticides, it necrotizing infections [25]. Silencing of WRKY8, which can be quickly managed by using dsRNAs targeting the interacts with mitogen-activated protein kinase, has been specific mutated gene. shown to increase the burden of pathogen-induced dis- To date, no study has demonstrated the suppression ease [26]. Therefore, we suggest that dsRNA_PcNLP6_3′ of P. capsici via the suppression of PcNLPs. Our study is more effective than dsRNA_PcNLP2_5′ in maintain - showed that P. capsici was effectively suppressed using ing the expression of NbWRKY8 and NbPR1 to retain dsRNAs targeting the P. capsici effector genes PcNLP2 the defense capacity of plants against other pathogen- and PcNLP6. In particular, dsRNAs targeting different induced diseases. We also suggest that PcNLP2 and mRNA regions in PcNLP2 and PcNLP6 brought about PcNLP6 have distinct functions in the plant immune a b c NbPR1 NbWRKY8 NbHin1 12,000,000 70 12,000 *** ** 10,000,000 10,000 8,000,000 8,000 6,000,000 6,000 4,000,000 4,000 2,000,000 2,000 0 0 0 Fig. 4 Changes in expression of defense-related genes after treatment with dsRNAs targeting PcNLP2 and PcNLP6, respectively. Quantification of the expression of defense-related genes a NbPR1, b NbWRKY8, and c NbHin1 using qRT-PCR. WT: Uninfected wild-type N. benthamiana; Mock: P. capsica-infected N. benthamiana treated with dsRNA targeting Renilla luciferase; dsRNA_PcNLP2_5′: P. capsica-infected N. benthamiana treated with dsRNA_PcNLP2_5′; dsRNA_PcNLP6_3′: P. capsica-infected N. benthamiana treated with dsRNA_PcNLP6_3′. Data represent mean ± SEM (N = 3). Significance is determined by Student’s t-test, **P < 0.01 and ***P < 0.001 Relative expression level WT WT Mo Mock ck dsRNA_ dsRNA_PcNLP PcNLP2_5' 2_5′ Mock Mock dsRNA_ dsRNA_PcNLP PcNLP6_3' 6_3′ WT WT Mo Mock ck dsRNA_PcNLP2_5′ dsRNA_PcNLP2_5' Mock Mock dsRNA_ dsRNA_PcNLP PcNLP6_3' 6_3′ WT WT Mo Mock ck dsRNA_ dsRNA_PcNLP PcNLP2_5' 2_5′ Mo Mock ck dsRNA_ dsRNA_PcNLP PcNLP6_3' 6_3′ Park et al. Applied Biological Chemistry (2023) 66:5 Page 6 of 7 Author contributions system after P. capsici infection, since their suppression CS conceived the project. MP, YK, DL performed experiments. MP, YK, and CS distinctly influenced the expression of NbWRKY8 and wrote the manuscript. All authors read and approved the final manuscript. NbPR1. In the future, a transcriptome-wide analysis of Funding dsRNA-treated plants would allow us to profile disease- This study was supported by the National Research Foundation of Korea (NRF) relevant genes, including other defense-related genes, fol- grant funded by the Korea government (MSIT ) (No. 2021R1A5A1032428 and lowing suppression of PcNLPs by dsRNAs. This approach No. 2022R1A2C1011032). This was also supported by a grant from the New breeding technologies development Program (Project No. PJ01652102), Rural would help uncover the relationship between PcNLP Development Administration, Republic of Korea. effector genes of P. capsici and plant defense genes. In conclusion, this study demonstrated the successful Availability of data and materials Not applicable. suppression of P. capsici using dsRNAs. In addition, we suggest that PcNLP2 and PcNLP6 have distinct relation- Declarations ships with plant defense-related genes. Based on these results, RNAi applications using dsRNAs can be used to Competing interests replace chemical pesticides as well as to screen the func- The authors declare that they have no competing interests. tion of a gene of interest. Received: 14 December 2022 Accepted: 8 January 2023 Abbreviations RNAi RNA interference dsRNA Double-stranded RNA siRNA Small interfering RNA References RISC RNA-induced silencing complex 1. Rehrig WZ, Ashrafi H, Hill T, Prince J, Van Deynze A (2014) CaDMR1 NLP Necr osis and ethylene-inducing peptide 1-like protein cosegregates with QTL Pc5.1 for resistance to Phytophthora capsici in qRT-PCR Quantitativ e real-time polymerase chain reaction pepper (Capsicum annuum). Plant Genome. https:// doi. org/ 10. 3835/ hpi Hours post-inoculation plant genom e2014. 03. 0011 2. Barchenger DW, Lamour KH, Bosland PW (2018) Challenges and strate- gies for breeding resistance in Capsicum annuum to the multifarious Supplementary Information pathogen Phytophthora capsici. 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New Phytol 229:532–547 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
Journal
Applied Biological Chemistry – Springer Journals
Published: Jan 21, 2023
Keywords: Phytophthora capsici; Double-stranded RNA; Exogenous RNA application; RNA interference