Transcriptomic Response of Fusarium verticillioides to Variably Inhibitory Environmental Isolates of Streptomyces

Abstract

Fusarium verticillioides is a mycotoxigenic fungus that is a threat to food and feed safety due to its common infection of maize, a global staple crop. A proposed strategy to combat this threat is the use of biological control bacteria that can inhibit the fungus and reduce mycotoxin contamination. In this study, the effect of multiple environmental isolates of Streptomyces on F. verticillioides was examined via transcriptome analysis. The Streptomyces strains ranged from inducing no visible response to dramatic growth inhibition. Transcriptionally, F. verticillioides responded proportionally to strain inhibition with either little to no transcript changes to thousands of genes being differentially expressed. Expression changes in multiple F. verticillioides putative secondary metabolite gene clusters was observed. Interestingly, genes involved in the fusaric acid gene cluster were suppressed by inhibitory strains of Streptomyces. A F. verticillioides beta-lactamase encoding gene (FVEG_13172) was found to be highly induced by specific inhibitory Streptomyces strains and its deletion increased visible response to those strains. This study demonstrates that F. verticillioides does not have an all or nothing response to bacteria it encounters but rather a measured response that is strain specific and proportional to the strength of inhibition.

Keywords: Fusarium verticillioides; Streptomyces; fungal beta-lactamase; microbial interactions; mycotoxins; transcriptome.

Figure 1

Dual-culture inhibition assays of the four Streptomyces strains used in this study. Strains spanning the full range of inhibition phenotypes were selected from an initial 9 tested. The lower blue sharpy lines indicate the locations of Streptomyces strain inoculation, grown for two days before inoculation of F. verticillioides. The central dot (25mm from the line) indicates where F. verticillioides spores were inoculated. Images captured 7 days after fungal spore inoculation.

Figure 2

Fusarium verticillioides transcriptomic response to four Streptomyces strains. Venn diagrams representing the overlap of F. verticillioides DEGs over all timepoints for each Streptomyces strain dual-culture. The s2832 diagram does not include the 8 h timepoint since no statistically significant DEGs were detected.

Figure 3

Fusarium verticillioides response to Streptomyces at 2 h. Venn diagram showing the overlap of DEGs from all 4 F. verticillioides – Streptomyces dual-cultures at 2 h.

Figure 4

SMGCs induced at 2 h after exposure to Streptomyces. Heat maps of F. verticillioides RPKM values of fusaric acid and fusarin clusters and SMGC 7, 9 18, and 22 at 2 h. RPKM values were logarithmically transformation before being visualized by R package “pheatmaps”. The scale bars represent these logarithmic values. The data are presented for the 2 h exposures with Streptomyces strains s2825, s2831, and s2832. The s2827 interaction is not reflected in this figure as no genes (except FVEG_03697) were differentially expressed with respect to secondary metabolism gene clusters.

Figure 5

SMGCs in response to dual-culture with Streptomyces strain s2831. Heat maps of RPKM values of SMGC 7, 9, 16, and fusaric acid clusters showing expression by F. verticillioides in response to s2831. RPKM values underwent a logarithmic transformation to be visualized by R package “pheatmaps”. The scale bars represent these transformed values.

Figure 6

Fusarium verticillioides genes expressed in response to Streptomyces strain s2831 that cause strong inhibition. The number of DEGs was highest at 2 h for the Fv-s2832 interaction and 8 and 24 h with strain s2831. Comparisons to find similarly expressed genes were made and visualized by Venn Diagrams. Additional comparisons were made to parse gene encoding for transcription factors, secondary metabolite gene clusters, and secretome encoding genes.

Figure 7

Effect of ΔFVEG_13172 on inhibition caused by dual culture with Streptomyces strain s2831 or s2832. (A) Interactions of F verticillioides with s2831. (B) Interactions of F verticillioides with s2831Streptomyces strain s2381 and s2832 were inoculated and grown for two days before inoculation with F verticillioides 25 mm from initial Streptomyces inoculation. The M3125 WT strain, two ΔFVEG_13172 strains (11/13 & 11/15) and one ectopic transformant (11/17) were used in this experiment. Images captured 7 days after fungal spore inoculation.