We are presenting a smart RNAi-based solution to knock down the bZIP gene in Phytophthora capsici. We built the siRNAs step by step, ran structure checks, and made sure they target the right gene. To deliver these siRNAs, we propose chitosan as a delivery system, making our solution sustainable and eco-friendly. In the sections below, we break down our solution, highlighting how we designed and validated our siRNAs and the reasons for choosing chitosan as our carrier.
RNA Interference (RNAi) Silencing Pathway
The RNA interference (RNAi) pathway is a conserved post-transcriptional gene regulation mechanism that has been widely utilized for targeted gene silencing. In this pathway, long double-stranded RNA (dsRNA) or synthetic small interfering RNA (siRNA) molecules are processed into 21-23 nucleotide fragments by the Dicer enzyme.
From the resulting duplex, the passenger strand is degraded, while the guide strand is incorporated into the RNA-induced silencing complex (RISC). Guided by this strand, the RISC scans cellular mRNA transcripts for complementary sequences. Upon binding with a perfect match, the target mRNA is cleaved, thereby preventing its translation into a protein. This ultimately results in gene silencing.
siRNA Design Process
Target Gene Selection
After multiple candidate genes were evaluated through a literature review focusing on genes responsible for pathogenicity and survival of Phytophthora spp. and initial off-target screening, the bZIP gene was selected as the final target for siRNA design. This choice was based on evidence demonstrating that silencing of the bZIP gene in Phytophthora spp. resulted in significant impairments to infection-related development. In particular, zoospore motility was disrupted and the ability to form appressoria was completely abolished in the transformants, as shown by comparative assays (Blanco et al., 2005).
Transcriptomic studies on P. capsici were then examined to validate its expression during infection. It was found that bZIP was actively expressed during the various stages of infection (Vijayakumar et al., 2024).
| Id | e_gw1.74.48.1 |
| Base Mean | 157.42027 |
| Fold Change | 116.06497 |
| log2Fold Change | 6.8587882 |
| lfcSE | 0.90449856 |
| stat | 7.58297369 |
| pvalue | 3.3772E-14 |
| padj | 6.5186E-12 |
| NR_ID | KUF95404.1 |
| NR_Description | bZIP transcription factor 1 |
| NR_Organism | Phytophthora nicotianae |
| uniref_id | UniRef100_A0A0W8DGQ2 |
| uniref_description | bZIP transcription factor 1 |
| uniref_organism | Phytophthora nicotianae |
| pfam_id | PF07095.12, PF18709.2 |
| Protein_ID | 128162 |
To obtain the nucleotide sequence, the JGI PhycoCosm database was used, which directed us to the Phyca11 transcript corresponding to bZIP (Grigoriev et al., 2021; Lamour et al., 2012).
siRNA Prediction and Validation
Once the bZIP transcript had been retrieved, possible siRNA candidates were predicted using siRNAPred (Kumar et al., 2005) and siDirect (Naito et al., 2009; Naito et al., 2004). The top-ranking results from both tools were subsequently refined using additional parameters, the selection of which was influenced by insights gained during our Integrated Human Practices (iHP) consultations. These parameters included seed-region stability, GC content, thermodynamic profiles, self-complementarity checks, and off-target minimization.
siRNA Sequence Design
| Candidate No. | Sense Strand | Antisense Strand |
|---|---|---|
| Candidate 1 | 5' --ggauacccucucaacaauaca 3' | 3' uuccuaugggagaguuguuau--5' |
| Candidate 2 | 5' --gcuccuuaccaacgaaugaac 3' | 3' uucgaggaaugguugcuuacu--5' |
| Candidate 3 | 5' --caaucugcuucguaguuuaga 3' | 3' gaguuagacgaagcaucaaau--5' |
| Candidate 4 | 5' --gucaugucguacagcgaaacu 3' | 3'gccaguacagcaugucgcuuu--5' |
The secondary structure predictions (Figure 3) were conducted to validate the efficacy of the selected candidates. The DuplexFold tool (Mathews et al., 2004) was used to assess the stability of siRNA duplexes, while RNAfold (Gruber et al., 2008) was employed to evaluate the accessibility of the target mRNA region by analyzing its structure (Schramm & Ramey, 2005). Such analysis enabled the assessment of silencing efficiency by ensuring that the guide strands could bind to accessible regions of the mRNA and form energetically favourable duplexes.
The inclusion of these computational checks is strongly supported by literature (Heale et al., 2005).
Chitosan - Our Nanoparticle Carrier
To ensure the effective delivery of the designed siRNA molecules, a suitable carrier system was required. Chitosan was selected as the nanoparticle carrier due to its favorable properties for both nucleic acid encapsulation and delivery.
Chitosan is a biopolymer obtained through the deacetylation of chitin, one of the most abundant natural polysaccharides after cellulose. It is widely considered sustainable due to its biodegradable and non-toxic nature. When chitosan degrades, it produces non-toxic residues that can be easily eliminated and biodegraded by nature (Maliki et al., 2022). Its amino and hydroxyl groups provide sites for chemical modification, enabling functional tuning of the nanoparticles. The ability to alter the surface of chitosan nanoparticles as required makes it a very promising delivery system. Under mildly acidic conditions, chitosan becomes positively charged, enabling it to bind strongly to the negatively charged siRNA through electrostatic interactions and form stable complexes or nanoparticles. To enhance stability, cross-linkers are commonly incorporated during the formation of nanoparticles. Sodium tripolyphosphate (TPP) is widely used as a safe, anionic cross-linker. A stable ionic network is formed by the electrostatic interaction between the positively charged amino groups of chitosan and the negatively charged phosphate groups of TPP. Chitosan–TPP nanoparticles are typically smaller, more uniform, and exhibit improved encapsulation efficiency (Yusefi et al., 2021).
By integrating rationally designed siRNA molecules with a sustainable, biodegradable, and customizable carrier such as chitosan, this system presents a highly promising approach for achieving efficient and targeted gene silencing in P. capsici.
References
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