Transcriptome analyses suggest a disturbance of iron homeostasis in soybean leaves during white mould disease establishment

Authors: Calla, Bernarda; BLAHUT-BEATTY, LAUREEN - Agriculture Canada; KOZIOL, LISA - Agriculture Canada; SIMMONDS, DAINA - Agriculture Canada; Clough, Steven

Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2013
Publication Date: 6/11/2014
Publication URL: http://handle.nal.usda.gov/10113/60184
Citation: Calla Zalles, B., Blahut-Beatty, L., Koziol, L., Simmonds, D.H., Clough, S.J. 2014. Transcriptome analyses suggest a disturbance of iron homeostasis in soybean leaves during white mould disease establishment. Molecular Plant Pathology. 15(6):576-588. DOI:10.1111/mpp.12113.

Interpretive Summary: All soybean varieties planted throughout the world are susceptible to the pathogen Sclerotinia sclerotiorum. A key factor to this pathogen's success is its ability to produce a chemical called oxalic acid (OA) that pre-conditions the plant tissue to infection. Without OA, S. sclerotiorum is a very weak pathogen. We examined soybean gene expression in response to purified OA infiltrated into leaves. Our analyses revealed that OA affects the status of iron as indicated by differential expression of iron responsive genes. The results suggest that S. sclerotiorium benefits from the release of OA into the leaf tissue as it binds iron strongly. This binding of iron would presumably allow uptake of iron into the fungus, and would remove iron from plant tissue.

Technical Abstract: Sclerotinia sclerotiorum is a serious pathogen of numerous crops around the world. The major virulence factor of this pathogen is oxalic acid (OA). Mutants that cannot produce OA do not cause disease, and plants that express enzymes that degrade OA, such as oxalate oxidase (OxO) are very resistant to S. sclerotiorum. To look at the effect that OA has on plants, we infiltrated soybean leaves with 5 mM OA and examined gene expression changes 2 hours post infiltration. By comparing gene expression levels between leaves of a transgenic soybean carrying an OxO gene (OxO) and its parent AC Colibri (AC) infiltrated with OA (pH 2.4) or water (pH 2.4 or 5.5), we were able to compare the effects of OA dependent or independent of its pH. Gene expression by microarray analysis identified 2,390 genes that were changing in expression as determined by using overall F-tests p-value cut off of 0.001. The additional requirement that at least one pairwise t-test fdr corrected p-value be less than 0.001 reduced the list of most highly significant differentially expressed genes to 1054. Independent of pH, OA altered expression levels of 78 genes, with ferritin showing the strongest induction by OA. The combination of OA plus its low pH caused 1,045 genes (99% of all the significant genes) to be differentially expressed, with many of the up-regulated genes being related to basal defense, such as genes of the phenylpropanoid pathway and various cytochrome P450s. RNA-seq was also conducted on four samples: OxO or AC Colbri genotypes infiltrated with either OA pH 2.4 or water pH 2.4. The RNA-seq analysis also identified ferritin paralogs as strongly induced by OA. As expression of ferritin, a gene that encodes for an iron-storage protein, is induced by free iron, these results suggest that S. sclerotiorum is benefitting from the ability of OA to free iron from plant proteins, as this would induce host cell death, and to also uptake and assimilation the iron for its own metabolic needs.