Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families

Abstract

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales- or P. pachyrhizi-specific families (tribes). Members of some families were strongly up-regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.

Keywords: ETI suppression; LCM; P. pachyrhizi; effectors; secretome; soybean rust; transcriptome.

Figure 1

Differential gene ontology (GO) term distribution between the Phakopsora pachyrhizi transcriptome (36 350 contigs) and its predicted secretome (851 contigs). This graph was automatically generated after the enrichment analysis of the Blast2GO tool (Fisher's exact test, P ≤ 0.05).

Figure 2

Properties of TribeMCL families. (A) Venn diagram showing the set of all tribes with at least two members of Phakopsora pachyrhizi, our predicted secretome and P. pachyrhizi contigs that were similar to the rust tribes found by Link et al. (2014) or Saunders et al. (2012). Rust‐specific families were found in intersections 37, 40, 61 and 186 (Tribe_2, 3, 21, 72 and 301). Phakopsora pachyrhizi‐specific families were found in intersections 37 and 186 (Tribe_18, 142 and 300). The basidiomycete Tribe_12 grouped into intersections 37 and 61. (B) Number of contigs associated with subcellular compartments for each of the 13 selected tribes. (C) MEME logos of conserved motifs identified in Tribe_1, 2 and 3. Pp, Phakopsora pachyrhizi.

Figure 3

Phylogenetic trees of Pucciniales secreted tribes. Trees for Tribe_1, 2 and 3 were generated using neighbour‐joining analysis (mega6, 1000× bootstrap) of the protein alignment generated from sequences in each tribe using ClustalW.

Figure 4

Heat map and hierarchical clustering of the expression profiles of 22 selected Phakopsora pachyrhizi contigs over a 10‐day infection time course. Fourteen contigs were from general tribes (Tribe_232, 204, 216, 383, 19, 20 and 6), one was not grouped into any tribe (de_novo_3696), six were not secreted (NS) and eight were from Tribe_1 and 3. Clustering was performed using Cluster 3 software. The fold values of the normalized expression data were used in clustering. The colour scale bar is shown at the bottom.

Figure 5

Immune suppression and subcellular localization of candidate effectors selected from Tribe_1 and 3. (A) In vivo test of immune suppression [effector‐triggered immunity (ETI) suppression] using the Pseudomonas syringae (Pst) DC3000 delivery assay on Nicotiana benthamiana leaves. Pst DC3000 transformed with empty vector [DC3000 + empty vector (ev)] was infiltrated on the left side of tobacco leaves, where a hypersensitive response (HR) could be detected. On the right side of the leaves, Pst DC3000 expressing Phakopsora pachyrhizi effector candidates fused to the type III secretion signal was infiltrated using the same suspension concentrations. The sequence not predicted to be secreted, de_novo_3823, was used as a negative control for ETI suppression. (B) Subcellular localization of P. pachyrhizi effector candidates using multiple fluorescent markers in tobacco leaves. Green fluorescence indicates the location of candidate effector proteins fused to green fluorescent protein (GFP), red fluorescence indicates the location of mCherry protein in the cytoplasm and the nucleus (RFP), and blue fluorescence indicates 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclei. The fluorescent images were detected using a confocal microscope. Bars, 20 µm. Arrows indicate nuclei.