Fig 1.
Schematic diagram describing different strategies used in the assembly the H. vastatrix genome (race XXXIII).
Blue rectangles refer to the first assembly strategy, which used 17 SMRTcells with P5-C3 chemistry obtained on the PacBio RS2 platform in the HGAP assembler. Green rectangles refer to the second assembly strategy, which included a de novo assembly using reads obtained on the Illumina platform with SOAP de novo 2 assembler and subsequently improved with 32 SMRT cells of PacBio reads using PBJelly software. Red rectangles refer to the third strategy, which performed a de novo assembly using reads obtained from both next generation sequencing platforms with additional improvement with PBJelly software.
Table 1.
Genomic features for the Hemileia vastatrix assembled genome (race XXXIII).
Table 2.
Structural annotation analysis of Hemileia vastatrix genome (race XXXIII).
Fig 2.
Functional clustering analysis of secreted proteins identified in H. vastatrix genome, race XXXIII.
Secreted proteins were found to be significantly similar (e-value < 10−5) to protein sequences obtained from NCBI and UNIPROT databases by using the Blast2GO bioinformatics platform. The y-axis consists of GO-terms described in the molecular function category for the hierarchical level #3 and the x-axis consists of protein sequences found for each GO-term in this category.
Table 3.
Prediction of subcellular localization of Hemileia vastatrix (race XXXIII) secretome using Wolf Psort.
Fig 3.
Functional clustering analysis of secreted proteins found in the genome of H. vastatrix race XXXIII, which specific signatures in KOG categories.
The y-axis consists of KOG categories, and the x-axis consists of protein sequences found for each KOG category.
Fig 4.
Quantitative RT-PCR-based analysis of gene expression performed for six EHv33 genes that encode putative candidate secreted effector proteins of H. vastatrix, race XXXIII.
The relative expression pattern of target genes was estimated in plant samples of the hybrid of Timor and Caturra. Data were recorded at 12, 24, 48 and 72 hours after inoculation. The period of 12 hours after inoculation was used as reference sample. The expression level of target genes was normalized by using two endogenous genes of H. vastatrix, namely, β-tubulin and CytIII. (A) EHv33_15: the highest level of gene expression was recorded at 24 hours after inoculation (hai). (B) EHv33_1: the highest level of gene expression was recorded at 24 hai and, then, decreased over time. (C) EHv33_17: the highest level of gene expression was recorded at 24 hai and, then, decreased over time. (D) EHv33_13: the highest level of gene expression was recorded at 24 hai and, then, decreased at 48 hai and, at the end, the expression estimate kept constant at 72 hai. (E) EHv33_8: the highest level of gene expression was recorded at 24 hai and, then, decreased at 48 hai and increased again at 72 hai. (F) EHv33_12: the increase of gene expression was recorded at 48 hai but, the highest level of gene expression was only recorded at 72 hai.
Fig 5.
Quantitative RT-PCR-based analysis of gene expression performed for four EHv33 genes that encode putative candidate secreted effector proteins of H. vastatrix, race XXXIII.
The relative expression pattern of target genes was estimated in plant samples of the hybrid of Timor and Caturra. Data were recorded at 12, 24, 48 and 72 hours after inoculation. The period of 12 hours after inoculation was used as reference sample. The expression level of target genes was normalized by using two endogenous genes of H. vastatrix, namely, β-tubulin and CytIII. (A) EHv33_6: the highest level of gene expression was recorded at 24 hours after inoculation (hai). (B) EHv33_5: the highest level of gene expression was recorded at 24 hai and, then, decreased over time. (C) EHv33_9: the highest level of gene expression was recorded at 24 hai and, then, decreased at 48 hai, and increased again at 72 hai. (D) EHv33_11: the highest level of gene expression was recorded at 24 hai and, then, decreased at 48 hai, and increased again at 72 hai.
Fig 6.
Quantitative RT-PCR-based analysis of gene expression performed for five EHv33 genes that encode putative candidate secreted effector proteins of H. vastatrix, race XXXIII.
The relative expression pattern of target genes was estimated in plant samples of the hybrid of Timor and Caturra. Data were recorded at 12, 24, 48 and 72 hours after inoculation. The period of 12 hours after inoculation was used as reference sample. The expression level of target genes was normalized by using two endogenous genes of H. vastatrix, namely, β-tubulin and CytIII. (A) EHv33_7: the highest level of gene expression was recorded at 48 hours after inoculation (hai). (B) EHv33_2: the highest level of gene expression was recorded at 48 hai and, then, decreased over time. (C) EHv33_10: the highest level of gene expression was recorded at 48 hai and, then, decreased over time. (D) EHv33_4: the highest level of gene expression was recorded at 48 hai and, then, decreased over time. (E) EHv33_16: the highest level of gene expression was recorded at 72 hai.
Fig 7.
Heat map of 17 genes expression profile obtained from quantitative real time PCR.
(A): genes expression profile (17) in resistant plant (Híbrido de Timor) during 24, 48 and 72 hai. (B): genes expression profile (17) in susceptible plant (Caturra) during 24, 48 and 72 hai. Red indicates a relatively high level of up-regulated, whereas green indicates a relatively low level of up-regulation.