Contrasting transcriptional responses to Fusarium virguliforme colonization in symptomatic and asymptomatic hosts

Abstract

The broad host range of Fusarium virguliforme represents a unique comparative system to identify and define differentially induced responses between an asymptomatic monocot host, maize (Zea mays), and a symptomatic eudicot host, soybean (Glycine max). Using a temporal, comparative transcriptome-based approach, we observed that early gene expression profiles of root tissue from infected maize suggest that pathogen tolerance coincides with the rapid induction of senescence dampening transcriptional regulators, including ANACs (Arabidopsis thaliana NAM/ATAF/CUC protein) and Ethylene-Responsive Factors. In contrast, the expression of senescence-associated processes in soybean was coincident with the appearance of disease symptom development, suggesting pathogen-induced senescence as a key pathway driving pathogen susceptibility in soybean. Based on the analyses described herein, we posit that root senescence is a primary contributing factor underlying colonization and disease progression in symptomatic versus asymptomatic host-fungal interactions. This process also supports the lifestyle and virulence of F. virguliforme during biotrophy to necrotrophy transitions. Further support for this hypothesis lies in comprehensive co-expression and comparative transcriptome analyses, and in total, supports the emerging concept of necrotrophy-activated senescence. We propose that F. virguliforme conditions an environment within symptomatic hosts, which favors susceptibility through transcriptomic reprogramming, and as described herein, the induction of pathways associated with senescence during the necrotrophic stage of fungal development.

Figure 1

Fusarium virguliforme growth on maize and soybean. (A) Plant growth and development over 14 days (numbered) post inoculation with F. virguliforme. Soybean is on the left and maize is on the right in each pair. Bar = 4 cm. (B) F. virguliforme DNA in planta quantification from mock and inoculated soybean and maize roots. F. virguliforme levels were determined using a TaqMan-based quantitative PCR assay. Values shown are the average of three biological replicates, each of which contained two plants, ± sem (n = 6).

Figure 2

Temporal pattern of defense gene expression in maize and soybean. (A and B) The number of significantly DEGs between mock and inoculated maize with log₂(FC) > 1, respectively, of early differentially regulated genes in maize or soybean from six timepoints over the colonization timecourse. (C and D) Heatmap of log₂-fold gene expression changes among transcripts cataloged in ontologies significantly enriched in upregulated genes across pooled timepoints for maize (n = 266) and soybean (n = 5,643). (E and F) Heatmap of significantly enriched gene ontologies from F. virguliforme-induced downregulated genes across pooled timepoints for maize (n = 336) and soybean (n = 5,325).

Figure 3

An analysis of putative orthologous host processes reveals differential patterns of induced defense responses. (A and B) Heatmap of expression of significantly (log2(FC) > 1) upregulated (n = 3,003) and downregulated (n = 1,660) genes from mock- and fungal-inoculated samples. Gray indicates orthologous genes from soybean and maize are significant, blue indicates orthologous genes from only soybean are significant, and green illustrates orthologous genes from maize that were uniquely significantly DE. (C and D) Mean expression patterns of log₂(FC) > 1 gene expression profiles from among significantly up- and downregulated orthologous genes, respectively, at a single timepoint in both hosts over the colonization timecourse. Error bars indicate one standard deviation of the mean.

Figure 4

Analysis of nonorthologous host processes reveals host-specific patterns of induced defense responses that are consistent with disease and pathogen tolerance. (A and B) Heatmap of expression of log₂(FC) > 1 genes from a list of significantly upregulated (n = 180) and downregulated (n = 199) genes at a single timepoint from maize, and significantly upregulated genes (n = 3,347) and downregulated genes (n = 3,068) from soybean at a single timepoint. Orange indicates significantly upregulated DE nonorthologous genes and blue indicates significantly downregulated DE nonorthologous genes. (C and D) Heatmap of significantly enriched gene ontologies from upregulated genes across pooled timepoints for maize and soybean. (E and F) Heatmap of significantly enriched gene ontologies from downregulated genes across pooled timepoints for maize and soybean, respectively, following F. virguliforme inoculation. (G) Mean expression patterns of log₂(FC) > 1 of significantly upregulated and downregulated genes, respectively, at a single timepoint in both hosts, over the colonization timecourse. Error bars indicate one standard deviation of the mean.

Figure 5

Processes unique to maize are associated with host immune responses following F. virguliforme inoculation. (A) Mean expression patterns of log₂(FC) > 1 of significantly upregulated nonorthologous genes from at least a single timepoint in both hosts over the colonization timecourse. Genes for analysis were selected based on those identified within the same gene ontology categories, but themselves are nonorthologous. Error bars indicate one standard deviation of the mean. (B) Gene expression profile of maize root defense markers that do not share a soybean orthologous group across the colonization timecourse.

Figure 6

Conservation of defense gene expression patterns preceding inoculation with F. virguliforme. Line graph of expression patterns of orthogroups that are uniquely upregulated in maize when compared to soybean log₂(FC) > 1, and corresponding regulation when maize and soybean are colonized by F. virguliforme. Individual green lines represent individual orthogroups. Solid green or brown lines represent the mean of orthogroups from mock- or F. virguliforme-inoculated.

Figure 7

Divergence of defense expression patterns of orthologous transcription factors. (A) Heatmap of log₂(FC) > 1 of significantly upregulated genes at a single timepoint in at least one host between mock and inoculated (n = 215). Gray indicates orthologous genes from soybean and maize were significant, blue indicates orthologous genes from only soybean were significant, and green indicates that maize orthologous genes were uniquely significantly DE. Black dashed boxes highlight changes in transcription factor expression between hosts. (B) Heatmap of positive-regulatory TFs in maize (n = 9) and soybean (n = 4), and negative regulatory TFs in maize (n = 5) and soybean (n = 3). (C) Representation of reactive oxygen species induced NO APICAL MERISTEM (NAM), ATAF1/2, CUP-SHAPED COTYLEDON-2 (CUC2) pathways in soybean that exhibited differential expression patterns of log₂(FTAbleC) > 1 between mock and pathogen-inoculated samples. The corresponding orthologs or homologs in maize associated with senescence and root vascular development. Each gene heatmap illustrates temporal changes from 0 to 14 DAI in soybean (blue) and maize (green). Green font indicates orthologous genes.

Figure 8

Gene expression patterns of regulatory pathways that control senescence-associated processes. (A and B) Gene expression pattern of orthologous genes in maize (103) and soybean (376). (C and D) Gene expression pattern of nonorthologous genes in maize (159) and soybean (668).