Figure 1.
Symptoms of PMeV infection in Carica papaya.
As opposed to most viruses, PMeV appears to reside primarily in lactifers, where it modifies potassium levels and osmotic balance, leading to rupture of cells and exudation of fluid and translucent latex from the fruits (A) and young leaves. The latex oxidises after atmospheric exposure, resulting in small necrotic lesions on the edges of young leaves (B) and a sticky latex on the fruits (C) that makes them unacceptable for consumption.
Figure 2.
The pipeline of miRNAs search procedure and putative targets in the C. papaya genome.
Flowchart of the principal steps for prediction of secondary structures of known miRNA and their putative targets in the C. papaya genome. Part of the flowchart represents the MirCheck pipeline.
Figure 3.
Analysis of C. papaya miRNA precursors.
Number of precursors identified in each miRNA family (only miRNAs with more than 5 precursors are presented).
Figure 4.
Secondary structures of selected MIR156a ortholog genes in C. papaya, Arabidopsis, soybean and Populus.
The mature miRNA is highlighted (yellow) in stem-loop structures obtained using Mfold.
Figure 5.
Phylogeny of C. papaya MIR156 orthologous precursors with dicot species.
R-coffee alignment of C. papaya identified precursors (cpa-MIR156a-I and cpa-MIR156a-II) with MIR156a orthologs from A. thaliana, B. napus, G. max, N. tabacum, P. trichocarpa, S. lycopersicum and V. vinifera. Nucleotides marked in red and orange have better alignment (A); resulting relationship tree of R-coffee alignment (B).
Figure 6.
Gene Ontology enriched terms of putative miRNA targets.
Bars indicate the number of GOs annotated as unique GO terms in C. papaya tentative consensus sequences.
Figure 7.
Relationship of miRNAs to proteasomal degradation.
A schematic showing the relationship of miRNAs 156, 398, 408 and 162 to proteasomal degradation. The red arrows indicate the target genes of miRNAs. Proteasome-ubiquitin degradation involves the binding of ubiquitin (Ubi) to substrate (SUB). Polyubiquitinated proteins are recognised and degraded by the 26S proteasome, which consists of the 19S regulatory particle that recognises, selects and binds the polyubiquitinated proteins, cleaves the polyubiquitin chains and forwards the target polypeptide into the lumen of the 20S core particle, where proteolytic degradation takes place.
Figure 8.
Relative quantification of PMeV load in C. papaya leaf samples.
Relative quantification of PMeV following real time RT-PCR. The amplification curve was assessed, and cyclophilin was used for normalisation. The normalised level of No PMeV load (control) was set as 1.0, and the other PMeV loads were calculated in relation to the control. The following four samples were selected: 1 - No detectable virus, no symptoms; 2 - Low viral load (Log10RQ ∼1.4), no symptoms; 3 - High viral load (Log10RQ ∼4), with symptoms of sticky disease; and 4 - Very high viral load (Log10RQ ∼5.5), with symptoms of sticky disease.
Figure 9.
Relative quantification of miRNAs related to the UPS system in C. papaya leaf samples.
MiRNAs were isolated from leaves, and the accumulation of a group of miRNAs associated with proteasome-ubiquitin degradation was assessed by RT-PCR using a TaqMan Small RNA Assay kit (Life Technologies). Comparison of the four samples: i) No PMeV load, ii) Very low PMeV load, iii) High PMeV load and iv) Very high PMeV load. The miRNA levels were normalized to cyclophilin (CYP) mRNA. The normalised miRNA level of No PMeV load (control) was set as 1.0, and the other PMeV loads were calculated in relation to the control.
Figure 10.
Relative quantification of miRNAs related to stress response pathways in C. papaya leaf samples.
MiRNAs were isolated from leaves, and the accumulation of a group of miRNAs associated with stress response pathways was assessed by RT-PCR using a TaqMan Small RNA Assay kit (Life Technologies). Comparison of the four samples: i) No PMeV load, ii) Very low PMeV load, iii) High PMeV load and iv) Very high PMeV load. The miRNA levels were normalized to cyclophilin (CYP) mRNA. The normalised miRNA level of No PMeV load (control) was set as 1.0, and the other PMeV loads were calculated in relation to the control.
Figure 11.
Relative quantification of RNAs in C. papaya leaf samples.
mRNAs were isolated from leaves, and the accumulation of a group of RNAs associated with stress response pathways was assessed by RT-PCR using a SYBR Green (Life Technologies) with a set of two PCR primers that flank the target region to analyse target genes (Life Technologies). Comparison of the four samples: i) No PMeV load, ii) Very low PMeV load, iii) High PMeV load and iv) Very high PMeV load. The mRNA levels were normalized to cyclophilin (CYP) mRNA. The normalised mRNA level of No PMeV load (control) was set as 1.0, and the other PMeV loads were calculated in relation to the control.