Fig 1.
Time course of fold-change gene expression of PKS7-1 cluster genes in wild type P. fijiensis isolate 10CR1-24 in infected banana relative to expression in conidia used for inoculation.
PKS7-1 cluster genes assessed include the Zn(II)2 CYS6 transcription factor, PKS7-1, the monooxygenase/hydrolase, the O-methyltransferase, and the ABC transporter genes. To determine gene expression, tissue was harvested from infected banana plants at weekly intervals starting at 2 weeks through 9 weeks post-inoculation. Fold-change gene expression is shown relative to the expression at 0 weeks (in conidia) which is set to 1, with error bars indicating standard error from three biological replicates. W = week.
Fig 2.
Time course of fold-change gene expression of PKS7-1 cluster genes in wild type P. fijiensis isolate 10CR1-24 in infected banana relative to expression at 9 weeks.
PKS7-1 cluster genes assessed include the Zn(II)2 CYS6 transcription factor, PKS7-1, the ABC transporter, the monooxygenase/hydrolase, and the O-methyltransferase genes. Tissue was harvested and assayed weekly starting at 2 weeks post-inoculation through 9 weeks. Samples were normalized with reference genes indicated in Table 1. Fold-change gene expression is shown relative to the expression at 9W post-inoculation that is set to 1. Statistical significance was assessed using one-way ANOVA combined with Dunnett’s multiple comparison analysis using the normalized transcript of the 9W sample as the control group. Differences in gene transcript levels considered statistically significant are denoted by asterisks (*P< 0.05, **P< 0.01, and ***P< 0.001). Error bars indicate standard error from three biological replicates. Each biological replicate was tested with three technical replicates.
Fig 3.
Time course of fold-change gene expression of PKS8-2 cluster genes in wild type P. fijiensis isolate 10CR1-24 in infected banana relative to expression in conidia.
PKS8-2 cluster genes assessed include the transcription factor, PKS8-2, the 5-aminolevulinate synthase, the MFS transporter, the ketosphinganine reductase, and the sphingolipid hydroxylase genes. To determine gene expression, tissue was harvested from infected banana plants at weekly intervals starting at 2 weeks through 9 weeks post-inoculation. Fold-change gene expression is shown relative to the expression at 0 weeks (in conidia) which is set to 1, with error bars indicating standard error from three biological replicates. W = week.
Fig 4.
Time course of fold-change gene expression of PKS8-2 cluster genes in wild type P. fijiensis isolate 10CR1-24 in infected banana relative to expression at 9 weeks.
PKS8-2 cluster genes assessed include the transcription factor, PKS8-2, the 5-aminolevulinate synthase, the MFS transporter, the ketosphinganine reductase, and the sphingolipid hydroxylase genes. Tissue from inoculated banana plants was harvested and assayed weekly starting at 2 weeks post-inoculation through 9 weeks. Samples were normalized with reference genes indicated in Table 1. Fold-change gene expression is shown relative to the expression at 9W post-inoculation that is set to 1. Statistical significance was assessed using one-way ANOVA combined with Dunnett’s multiple comparison analysis using the normalized transcript of the 9W sample as the control group. Differences in gene transcript levels considered statistically significant are denoted by asterisks (*P< 0.05, **P< 0.01, and ***P< 0.001). Error bars indicate standard error from three biological replicates. Each biological replicate was tested with three technical replicates.
Fig 5.
Time course of fold-change gene expression of PKS10-2 cluster genes in wild type P. fijiensis isolate 10CR1-24 in infected banana relative to expression in conidia.
PKS10-2 cluster genes assessed include the NRPS, the cytochrome P450, the oxidoreductase, the monooxygenase, a hypothetical gene encoding a protein of unknown function, and the dehydrogenase genes. To determine gene expression, tissue was harvested from infected banana plants at weekly intervals starting at 2 weeks through 9 weeks post-inoculation. Fold-change gene expression is shown relative to the expression at 0 weeks (in conidia) which is set to 1, with error bars indicating standard error from three biological replicates. W = week.
Fig 6.
Time course of fold-change gene expression of PKS10-2 cluster genes in wild type P. fijiensis isolate 10CR1-24 in infected banana relative to expression at 9 weeks.
PKS10-2 cluster genes assessed include those encoding the NRPS, PKS10-2, the monooxygenase, the oxidoreductase, a protein of unknown function, the dehydrogenase, and the cytochrome P450. Tissue from inoculated banana plants was harvested and assayed weekly starting at 2 weeks post-inoculation through 9 weeks. Samples were normalized with reference genes indicated in Table 1. Fold-change gene expression is shown relative to the expression at 9W post-inoculation that is set to 1. Statistical significance was assessed using one-way ANOVA combined with Dunnett’s multiple comparison analysis using the normalized transcript of the 9W sample as the control group. Differences in gene transcript levels considered statistically significant are denoted by asterisks (*P< 0.05, **P< 0.01, and ***P< 0.001). Error bars indicate standard error from three biological replicates. Each biological replicate was tested with three technical replicates.
Fig 7.
PKS gene expression in silencing transformants, vector control, and wild type.
A) Expression of PKS8-2 from mycelial cultures harvested from four-week-old fungal cultures grown on banana leaf—Murashige and Skoog agar. B) Expression of PKS8-2 in conidia harvested from one-week-old mycelial cultures grown on modified V8 medium. C) Expression of PKS10-2 from mycelial cultures harvested from four-week-old fungal cultures grown on banana leaf—Murashige and Skoog agar. D) Expression of PKS10-2 in conidia harvested from one-week-old mycelial cultures grown on modified V8 medium. Controls included the vector control (VC) (dotted bars) and wild type (WT) (hatched bars). Samples were normalized to the actin gene for the PKS8-2 expression and the ubiquitin gene for the PKS10-2 expression. Fold-change gene expression in the transformed lines is shown relative to expression in the wild type isolate 10CR1-24. Statistical significance was assessed using one-way ANOVA combined with Dunnett’s multiple comparison analysis using the normalized transcript of 10CR1-24 as the control group. Differences in gene transcript levels considered statistically significant are denoted by asterisks (*P< 0.05, **P< 0.01, and ***P< 0.001). Error bars indicate standard error from three biological replicates for mycelial and conidial expression. Each biological replicate was tested with three technical replicates.
Fig 8.
Black Sigatoka disease development in banana inoculated with wild type, vector control, and transformants silenced with the PKS8-2 silencing construct.
A) Black Sigatoka disease response as seen in banana plants 9 weeks after inoculation with the wild type (hatched bar), vector control (dotted bar) and silencing transformants (dark bars). Disease severity caused by each isolate was assessed by the Fiji ImageJ program and is shown relative to wild type isolate 10CR1-24. Data shown are results of two independent experiments with 8 plants per strain in each experiment. The within-group differences between the different PKS8-2 silencing transformants, vector control, and wild type isolate were examined using ANOVA with Bonferroni post hoc test for multiple comparisons. Bars represent Mean ± SE. Different letters on bars indicate statistically significant differences (p ≤ 0.05). B) Symptoms on banana plants inoculated with PKS8-2 silencing transformants 8-2-17, 8-2-2, 8-2-6, and 8-2-28 as compared to symptoms on banana inoculated with the vector control and wild type isolate 10CR1-24. Scale bar represents 1 inch.
Fig 9.
Black Sigatoka disease development in banana inoculated with wild type, vector control, and transformants silenced with the PKS10-2 silencing construct.
A) Black Sigatoka disease response as seen in banana plants 9 weeks after inoculation with the wild type (hatched bar), vector control (dotted bar) and silencing transformants (dark bars). Disease severity caused by each isolate was assessed by the Fiji ImageJ program and is shown relative to wild type isolate 10CR1-24. Data shown are results of two independent experiments with 8 plants per strain in each experiment. The within-group differences between the different PKS10-2 silencing transformants, vector control, and wild type isolate were examined using ANOVA with Bonferroni post hoc test for multiple comparisons. Bars represent Mean ± SE. Different letters on bars indicate statistically significant differences (p ≤ 0.05). B) Symptoms on banana plants inoculated with PKS10-2 silencing transformants 10-2-8, 10-2-4, 10-2-10, and 10-2-9 as compared to symptoms on banana inoculated with the vector control and wild type isolate 10CR1-24. Scale bar represents 1 inch.
Fig 10.
Comparison of PKS10-2 gene clusters from Pseudocercospora species.
Putative biosynthetic clusters from P. fijiensis, P. musae, P. eumusae, P. cruenta, and P. fuligena are shown, with arrows indicating directionality for each gene. Putative orthologous genes are shown in the same color. Additional conserved domains identified in S5 Table are shown in gray arrows for specific hits and checkered arrows for non-specific hits. Scale bar represents 5 kb.
Table 1.
Primer sequences and reference genes used for expression assays.