Fusarium graminearum causes head blight in wheat and barley. Disease symptoms can spread to neighboring spikelets through the rachis in wheat heads facilitated by the type B mycotoxin deoxynivalenol (DON). In contrast, barley spikes exhibit native resistance to disease spread and this resistance is associated with the activity of a UDP-glycosyltransferase (UGT) gene, HvUGT13248. Utilizing a TILLING mutant of UGT13248 (H369Y) and a Fusarium reporter strain with TRI5prom::GFP and gdpAprom::dsRed, we performed confocal microscopy to monitor trichothecence biosynthesis (using GFP expression) and fungal growth in planta (using dsRed expression). Fungal infection progressed faster and DON production was consistently higher at 2, 4, and 6 days post inoculation (dpi) in the H369Y mutant compared to its wildtype sibling-line. At 4 dpi, GFP expression was more extensive in the rachis node in the mutant spike. At 6 dpi, the GFP signal was much reduced in the rachis node in the mutant compared to the wildtype. In wildtype, both the GFP and dsRed signal were confined in the rachis node while in the mutant the fungus progressed through rachis node and GFP and dsRed signals can be observed in the vascular vessels of the internode. Taken together, F. graminearum infection in the mutant progressed at a faster pace and spread through rachis node into vascular vessels. Trichothecene production was strongly induced at 4 dpi in rachis node and at 6 dpi in internode as the fungus spread over the spike. To understand the transcriptomic changes that underlie these infection differences, we performed an RNA-Seq experiment on the infected floret and rachis tissues at 2, 4 and 6 dpi in the mutant and wildtype. Standard bioinformatics workflow was applied to the raw data to remove sequencing adaptors, align to the MorexV3 genome and count reads. Principal component analysis (PCA) at the sample level revealed that tissue type explained variation on PC1 (38.7%) and treatment on PC2 (29.3%). Differentially expressed genes (DEGs) were identified using DESeq2. In the floret, more DEGs were identified at 2 and 4 dpi in H369Y than in wildtype but it was reversed for 6 dpi. In the rachis, more DEGs were identified in H369Y through all timepoints. The list of DEGs contained transcripts that have been shown to respond to FHB infection, including UGT13248, ABC transporters, GSTs, receptor-like kinases, as well as novel transcripts (e.g., Fusarium resistance orphan protein). Over-representation and pathway analyses of the DEGs are being conducted to reveal enrichment of gene sets and pathways and to construct a gene regulatory network.
Acknowledgement and Disclaimer:
This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No 59-0206-2-140. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.