The effect on the transcriptome of Anemone coronaria following infection with rust (Tranzschelia discolor)

Abstract

In order to understand plant/pathogen interaction, the transcriptome of uninfected (1S) and infected (2I) plant was sequenced at 3'end by the GS FLX 454 platform. De novo assembly of high-quality reads generated 27,231 contigs leaving 37,191 singletons in the 1S and 38,393 in the 2I libraries. ESTcalc tool suggested that 71% of the transcriptome had been captured, with 99% of the genes present being represented by at least one read. Unigene annotation showed that 50.5% of the predicted translation products shared significant homology with protein sequences in GenBank. In all 253 differential transcript abundance (DTAs) were in higher abundance and 52 in lower abundance in the 2I library. 128 higher abundance DTA genes were of fungal origin and 49 were clearly plant sequences. A tBLASTn-based search of the sequences using as query the full length predicted polypeptide product of 50 R genes identified 16 R gene products. Only one R gene (PGIP) was up-regulated. The response of the plant to fungal invasion included the up-regulation of several pathogenesis related protein (PR) genes involved in JA signaling and other genes associated with defense response and down regulation of cell wall associated genes, non-race-specific disease resistance1 (NDR1) and other genes like myb, presqualene diphosphate phosphatase (PSDPase), a UDP-glycosyltransferase 74E2-like (UGT). The DTA genes identified here should provide a basis for understanding the A. coronaria/T. discolor interaction and leads for biotechnology-based disease resistance breeding.

Fig 1. Healthy and Tranzschelia discolor infected plant.

In comparison to healthy plant (A), during biotropich relationship, infected plant displays plethoric vegetation with robust leaf stems and curly leaf lamina. Flowering is strongly repressed.

Fig 2. GO multilevel-Pie.

Pie chart representation of Gene Ontology classification of (A) biological process (B) cellular component (C) molecular function, using a sequences cutoff of 5.0.

Fig 3. Phylogenetic tree and aligment of thaumatin-like protein.

(A) Anemone coronaria thaumatin-like protein (contig 13789) cluster with Puccinia graminis and Melampsora larici-populina proteins. TLPs of no-rust fungal taxa cluster in a separate group. Bootstrap values are indicated in relevant nodes. Arabidopsis thaliana TLP was used as out-group. (B) Amino acid sequence alignment of five rust TLPs. The conserved 16 cystein residues are highlighted in the boxes.

Fig 4. KEGG pathway of α-linoleic acid metabolism.

Anemone coronaria transcrips involved in jasmonic acid metablolic pathway are highlighted by grey tone. 12-oxophytodienoate reductase 2 (EC: 5.3.99.6) and acyl-CoA oxidase (ACX) are upregulated during Tranzschelia discolor infection.

Fig 5. Phylogenetic tree of PGIP.

Anemone coronaria PGIP (contig 21204) clusters with those of Monocotyledon species. Dicotyledon PGIPs cluster in two separate groups. Bootstrap values are indicated in relevant nodes. Entamoeba invadens PGIP protein was used as out-group.

Fig 6. Phylogenetic tree of chitinases.

Analysis resolves family 18 chitinases into two main branches: the first includes plant chitinases and the second bring together fungal chitinases. Escherichia coli chitinase was used as out-group.

Fig 7. Aligment of NDR1 proteins.

Anemone coronaria contigs 6057 and 9288 are aligned with selected member of NDR1 proteins. Motif 1, motif 2 and 3 of NDR1/HIN-like (NHL) protein superfamily are highlighted in the boxes.

Fig 8. Gene expression in Anemone coronaria infected with Tranzschelia discolor.

Expression analysis was conducted among sample A (plants analyzed by pyrosequencing) and samples B and C, each composed of five distinct uninfected and infected plants. Ten plant DTAs genes (five up- and five down-regulated) putatively involved in the response to Tranzschelia discolor infection and three fungal genes, were tested. The data were normalized using Anemone coronaria 18s rRNA gene as the reference. Expression analysis was performed in triplicate on three biological replicates. Transcript abundance data were expressed in the form mean ± standard error (SE).