The rust transferred proteins-a new family of effector proteins exhibiting protease inhibitor function

Abstract

Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N-terminus and a conserved and structured C-terminus. The characterization of Uf-RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C-terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf-RTP1p and Us-RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi-specific family of modular effector proteins comprising an unstructured N-terminal domain and a structured C-terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.

Figure 1

Schematic diagram of the RTP exon/intron structure and domain structure of the rust transferred proteins (RTPps). The variable exon 1 may be interrupted by additional introns (A, B) and codes for the structurally disordered N‐terminus. Exons 2–6 code for the C‐terminal globular domain. Black arrows indicate the positions of the four conserved cysteine residues (C1–C4). Grey arrows represent the seven β strands (β1–β7) of the globular domain.

Figure 2

Phylogenetic tree of the N‐ versus C‐terminus of the rust transferred proteins (RTPps). Colours are selected according to TribeMCL analyses. Clades of RTPp N‐termini and C‐termini show co‐evolution, except for three genes that show divergence between N‐ and C‐termini (indicated by blue connection bars). Tree based on a neighbour‐joining (NJ) analysis. All bootstrap counts refer to 1000 replications.

Figure 3

Rust transferred proteins (RTPps) show signs of purifying selection. Numerous C‐terminal residues of RTPs show signatures of purifying selection. In particular, conserved cysteine residues (C1–C4) are under purifying selection. X, value on Selecton selection scale, where ‘1’ indicates positive selection and ‘7’ indicates purifying selection.

Figure 4

Western blot analysis of Uf‐RTP1p cysteine mutants. Mutated proteins were heterologously expressed in Pichia pastoris. P, pellet; S, culture supernatant; wt, wild‐type Uf‐RTP1p. Uf‐RTP1p cysteine residues C104, C117, C147 and C179, corresponding to the conserved cysteine residues C1, C2, C3 and C4, respectively, were replaced by serine residues. Differences in the molecular weight of the Uf‐RTP1p variants originate from altered N‐glycosylation of the mutated proteins.

Figure 5

Alignment of selected rust transferred protein (RTPp) globular domain sequences and sequences of eukaryotic and bacterial cysteine protease inhibitors. Pb‐ICP, Py‐ICP, Tc‐ICP (chagasin), Cb‐ICP and Ps‐ICP are chagasin‐like cysteine protease inhibitors, whereas Pi‐EPIC2A is cystatin‐like. Cb, Coxiella burnetti; Pb, Plasmodium berghei; Pgt, Puccinia graminis f.sp. tritici; Pi, Phytophthora infestans; Ps, Pseudomonas syringae; Py, Plasmodium yoelii; Tc, Trypanosoma cruzi; Ua, Uromyces appendiculatus; Uf, Uromyces fabae. Pink arrows and sequences mark the positions of the seven β strands of Uf‐RTPp. The positions of β sheets of Tc‐ICP and Pb‐ICP according to Rennenberg et al. (2010) are marked by blue arrows and sequences. Green arrows, positions of β sheets specific for Pb‐ICP; yellow, sequence motifs that are known to be involved in cysteine protease binding (Rennenberg et al., 2010); red, conserved glycine residues.

Figure 6

Inhibition of proteolytic activity by Uf‐RTP1p and Us‐RTP1p. Gelatin‐based zymograms show proteolytic activity in Pichia pastoris supernatants from strains expressing Uf‐RTP1p, Us‐RTP1p, U. fabae invertase (INV) or no heterologous protein (pPIC3.5). Supernatant (20 μL) was loaded onto lanes 1–6. The addition of phenylmethanesulfonyl fluoride (PMSF) (10 mm), Uf‐RTP1p or Us‐RTP1p (equal amount as sample volume) to supernatants from INV and pPIC3.5 strains abolished proteolytic activity (lanes 5 and 6, and 11–14, respectively). The intensity of the proteolytic activity depends on the amount of the loaded supernatant (lanes 7–10).