DefinePK

Research Objective

To identify and validate microRNAs (miRNAs) responsive to infection by the fungal pathogen Fusarium oxysporum f. sp. lycopersici in tomato roots and to study their regulatory roles in resistance.

Methodology

1. Plant/Pathogen System: Tomato cultivar Pusa Early Dwarf (PED, moderately susceptible) infected with F. oxysporum f. sp. lycopersici strain 4471 via root incubation. Control plants were mock-inoculated with water.
2. RNA Isolation and Sequencing: Total RNA was isolated from infected and control roots using TRIzol reagent. sRNA sequences were compared against known plant miRNAs using miRBase software to identify conserved miRNAs (103 conserved miRNAs from 24 families found).
3. miRNA/Target Validation: Expression patterns of two novel miRNAs (miR30 and miR33) and two known miRNAs (miR46 and miR49), along with their target genes (P4 and -1,3-glucanase), were validated using quantitative RT-PCR (qRT-PCR) 30 days post-inoculation (dpi).
4. Novel miRNA Cloning and Sequencing: miR30 and miR33 fragments were cloned into pGEM®-T Easy vector, transformed into E. coli, and sequenced.
5. Secondary Structure Prediction: Secondary structures of miR30 and miR33 were predicted using the Vienna RNA package program V.1.7, calculating Minimum Free Energy (MFE).
6. Statistical Analysis: qRT-PCR results were analyzed using one-tailed paired Student's t-test (P $\le$ 0.05 considered significant).

Methodology Flowchart

                        graph TD;
A[Obtain Tomato cv. PED & F. oxysporum f. sp. lycopersici] --> B[Inoculation & Sample CollectionRoots];
B --> C[Total RNA Isolation];
C --> D[sRNA Sequencing & In Silico Analysis miRBase];
D --> E[Identify Differentially Expressed miRNAs miR30, miR33, miR46, miR49];
E --> F[Clone & Sequence Novel miRNAs miR30, miR33];
F --> G[Predict Secondary Structure Vienna RNA Package];
E --> H[qRT-PCR Validation of miRNAs & Targets P4, -1,3-glucanase];
H --> I[Analyze Expression Patterns & Correlations];
I --> J[Conclusion on miRNA Role in Resistance];                    

Discussion

MiRNAs are crucial fine-tuning regulators in plant immunity, involved in both PTI and ETI responses. The differential expression of miR46, miR33, and miR49 suggests their involvement in the tomato defense response against F. oxysporum f. sp. lycopersici. The inverse expression pattern observed between miR46 and its target P4, and between miR33/miR49 and the defense enzyme -1,3-glucanase, indicates a regulatory mechanism where miRNA activity influences the expression of pathogenesis-related genes. The moderate susceptibility of PED suggests its miRNA-mediated resistance mechanisms are not fully effective. The high conservation of miR30/miR33 (as miR166) across species highlights their fundamental role.

Key Findings

- Four miRNAs (two novel: miR30, miR33; two known: miR46, miR49) showed differential expression during pathogen infection.
- miR46 was down-regulated (fold decrease of 33.33).
- miR33 and miR49 were up-regulated (fold increase of 0.4 and 0.15, respectively).
- miR30 expression was not significantly altered.
- Target genes P4 and -1,3-glucanase showed inverse correlation with their corresponding miRNAs (P4 with miR46; -1,3-glucanase with miR33 and miR49).
- Tomato cv. Pusa Early Dwarf (PED) was confirmed to be moderately susceptible to the fungus.
- Novel miRNAs miR30 (20 nt) and miR33 (21 nt) showed 100% homology to conserved miR166 in other plant species.
- MFE for miR30 was -1.2 kcal/mol and for miR33 was -0.4 kcal/mol.

Conclusion

Two novel miRNAs (miR30 and miR33) and two known miRNAs (miR46 and miR49) were identified as responsive to F. oxysporum f. sp. lycopersici infection in tomato cv. PED roots. The differential expression of these miRNAs and their target genes (-1,3-glucanase and P4) provides insight into the miRNA-mediated regulatory network governing tomato resistance to Fusarium wilt.

Fact Check

1. Four miRNAs (two novel: miR30, miR33; two known: miR46, miR49) were validated as differentially expressed. (Confirmed in Abstract/Results).
2. miR46 was down-regulated with a fold decrease of 33.33 upon fungal infection. (Confirmed in Results).
3. The total free energy (MFE) of miRNA30 was -1.2 kcal/mol and miR33 was -0.4 kcal/mol. (Confirmed in Abstract/Table 2).

Mind Map