The haustorial transcriptomes of Uromyces appendiculatus and Phakopsora pachyrhizi and their candidate effector families

Abstract

Haustoria of biotrophic rust fungi are responsible for the uptake of nutrients from their hosts and for the production of secreted proteins, known as effectors, which modulate the host immune system. The identification of the transcriptome of haustoria and an understanding of the functions of expressed genes therefore hold essential keys for the elucidation of fungus-plant interactions and the development of novel fungal control strategies. Here, we purified haustoria from infected leaves and used 454 sequencing to examine the haustorial transcriptomes of Phakopsora pachyrhizi and Uromyces appendiculatus, the causal agents of soybean rust and common bean rust, respectively. These pathogens cause extensive yield losses in their respective legume crop hosts. A series of analyses were used to annotate expressed sequences, including transposable elements and viruses, to predict secreted proteins from the assembled sequences and to identify families of candidate effectors. This work provides a foundation for the comparative analysis of haustorial gene expression with further insights into physiology and effector evolution.

Figure 1

Purification of haustoria and haustorial RNA. (a) Micrographs of haustoria preparations. Haustorial suspensions after elution from the concanavalin A (Con‐A) column were examined microscopically. Haustoria (H) and chloroplasts (C) are indicated, as well as two representative haustorial necks (N). Bar, 10 μm. (b) Counts of haustoria and chloroplasts in preparations. The graphs show means of 23 haustoria preparations each. (c) Agarose gel electrophoresis of total RNA prepared from haustoria. Visible bands represent rRNA from haustoria and chloroplasts, and degraded RNAs can also be seen. nt, nucleotide.

Figure 2

Work flow for the identification of putative secreted proteins expressed in haustoria of Phakopsora pachyrhizi and Uromyces appendiculatus. The BESTORF program was used to predict the open reading frames present in high‐confidence fungal contigs. Amino acid sequences of proteins predicted by BESTORF were used in the subsequent analyses with SignalP to predict secreted proteins. The prediction of proteins most likely to be secreted outside of fungal cells was further refined using the TMHMM, TargetP and PredGPI programs.

Figure 3

Annotation of the Phakopsora pachyrhizi and Uromyces appendiculatus transcriptomes: (a) P. pachyrhizi haustoria; (b) P. pachyrhizi urediospores and germ tubes; (c) U. appendiculatus haustoria. For the two P. pachyrhizi stages, the over‐represented gene ontology (GO) categories are emphasized by offset slices.

Figure 4

Summary of properties of the 16 largest clusters of proteins with predicted secretion motifs. Single linkage clustering was used to group the contigs, and the cluster ID, corresponding to Table S5, is provided to the left of the graph. The graph shows the numbers of genes (unique contigs) per cluster, which is subdivided into those predicted to be soluble secreted proteins (black) and all others (white). Roman numbers indicate the specificities of the clusters: I, specific to rust fungi; II, specific to Pucciniaceae; III, specific to Uromyces. The columns on the right give additional information. (H), Hacquard et al. (2012); (S), Saunders et al. (2012).

Figure 5

Gene family cluster 112. (a) Conserved profile of the central 94 amino acids (WebLogo). (b) Phylogeny: neighbour‐joining analysis of protein alignment; consensus tree of 100 bootstraps; only bootstrap values greater than 50 are shown (PAUP*). *up, among the 100 most strongly up‐regulated genes in infected leaves versus urediospores (Duplessis et al., 2011). Mellp, Melampsora larcici‐populina; PGTG, Puccinia graminis f. sp. tritici; Pp, Phakopsora pachyrhizi; Ua, Uromyces appendiculatus.

Figure 6

Gene family cluster 398. (a) Conserved profile of 137 central amino acids (WebLogo). (b) Phylogeny: neighbour‐joining analysis of protein alignment (PAUP*); consensus tree of 100 bootstraps; only bootstrap values greater than 50 are shown. *a, abundant transcripts; *up, among 100 most strongly up‐regulated genes in infected leaves versus urediospores; *a*up, abundant and up‐regulated in haustorium against pre‐haustorial cell types (Duplessis et al., 2011; Link and Voegele, 2008). Mellp, Melampsora larcici‐populina; PGTG, Puccinia graminis f. sp. tritici; Pp, Phakopsora pachyrhizi; Ua, Uromyces appendiculatus; Uf, Uromyces fabae. Pst sec. prot., secreted protein from Puccinia striiformis f. sp. tritici.