Fig 1.
Comparison of PKS8-1 gene clusters across different species.
For P. fijiensis, P. musae, P. eumusae, and P. pini-densiflorae, PKS8-1 and adjacent genes in the genome or their putative orthologs are shown by arrows indicating their direction. Putative orthologous genes are shown with the same color of arrow. A description of the putative function of each corresponding protein is shown under the P. fijiensis arrows. Scale bar indicates 5 kb.
Fig 2.
Phylogenetic analysis of PKS8-1.
RAxML was used to create a maximum likelihood phylogenetic tree of PKS protein sequences, using the PKS sequences described previously [18] and others including the monodictyphenone-producing MdpG from A. nidulans, the cladofulvin-producing ClaG from C. fulvum, and the PKS8-1 homolog sequences identified from P. musae and P. eumusae. The clade containing PKS8-1, its P. musae and P. eumusae homologs, the monodictyphenone-producing PKS from A. nidulans, and the cladofulvin-producing PKS from C. fulvum is shown with a red box. Bootstrap values are indicated on the tree, and the scale bar indicates substitutions per site. An = Aspergillus nidulans; Ap = Aspergillus parasiticus; As = Alternaria solani; At = Aspergillus terreus; Bm = Bipolaris maydis; Bo = Bipolaris oryzae; Cf = Cladosporium fulvum; Cg = Colletotrichum graminicola; Cn = Cercospora nicotianae; Czm = Cercospora zeae-maydis; Ed = Exophiala dermatitidis; Fm = Fusarium moniliforme; Fv = Fusarium verticillioides; Gl = Glarea lozoyensis; Pc = Penicillium citrinum; Pg = Penicillium griseofulvum; Pe = Pseudocercospora eumusae; Pf = Pseudocercospora fijiensis; Pm = Pseudocercospora musae.
Fig 3.
Comparison of P. fijiensis PKS8-1 cluster, the cladofulvin-producing cluster from C. fulvum, and monodictyphenone-producing cluster from A. nidulans.
For the PKS8-1 cluster, the cladofulvin-producing cluster from C. fulvum, and the monodictyphenone-producing cluster from A. nidulans, each gene is shown in the genome with an arrow indicating its direction. Gene names, where applicable, are shown above the arrows, and descriptions of the putative function of each gene are shown below the arrows. Putative orthologs are shown with the same color arrow, and other genes are shown with white arrows. Details on cluster gene comparisons are shown in S2 Table.
Fig 4.
RNA-Seq analysis of PKS8-1 cluster genes.
PKS8-1 and adjacent genes in the P. fijiensis genome are represented by arrows indicating their direction, with descriptions of putative protein functions indicated below the arrows. Blue arrows = genes predicted by Noar and Daub [18] to be part of the PKS8-1 gene cluster; Gray arrows = genes predicted by Noar and Daub [18] to flank the PKS8-1 gene cluster. Scale bar indicates 5 kb. Black bars are proportional to log2FC values of expression in leaf tissue vs. culture medium. Single asterisks represent significant expression differences at p<0.05, and double asterisks represent significant expression differences at p<0.01. NE = no expression detected.
Fig 5.
Time course of expression of PKS8-1 and cluster genes in banana inoculated with wild-type P. fijiensis isolate 10CR1-24.
Cluster genes assayed are those shown in Fig 4. Fold-change gene expression is shown relative to expression in conidia, set to 1. Tissue was harvested and assayed weekly starting at 2 weeks post-inoculation through 9 weeks. Results for each gene are shown as a bar graph in log scale, with error bars indicating standard error from three biological replicates. ND = not detected; W = week.
Fig 6.
RT-qPCR assays of PKS8-1 cluster genes in overexpression transformants of the aflR-like gene and vector controls relative to wild-type expression in culture.
The wild type, two transformants of vector control (VC-1, VC-2), and two transformants of the aflR-like transcription factor overexpression (OX-4, OX-8) were grown in PDB medium. RT-qPCR assays were performed on these samples for the PKS8-1 cluster genes. Fold-change gene expression is shown on a log scale relative to wild type set to 1. Error bars indicate standard error from six biological replicates. Dotted bar: wild type; hatched bars: vector controls; black bars: overexpression strains.
Fig 7.
Symptoms on banana following inoculation with the 10CR1-24 wild type, wild type transformed with the vector construct (VC-2) and the overexpression transformant of the aflR-like gene (OX-4).
Figure shows symptoms on representative leaves from 2 weeks post-inoculation to 9 weeks post-inoculation. There were no differences in disease severity or timing of symptom development between the three treatments.
Fig 8.
Time course of PKS8-1 cluster gene expression in the overexpression strain of the aflR-like gene during disease development in banana.
A. Cluster gene expression in the overexpressor and in a vector-transformed control relative to expression in conidia (set to 1) of the respective strains. Black bar = overexpressor strain #4 (OX); hatched bar = vector control #2 (VC). B. Same data as in 8A, but cluster gene expression of the overexpressor is shown relative to expression in the vector-control strain (set to 1). Cluster genes assayed are those shown in Fig 4. Tissue was harvested and assayed weekly starting at 2 weeks post inoculation through 9 weeks. Results are shown as a bar graph in log scale. Error bars indicate standard error from three biological replicates. ND = not detected; W = week.