Solution structure of the phytotoxic protein PcF: The first characterized member of the Phytophthora PcF toxin family

Abstract

The PcF protein from Phytophthora cactorum is the first member of the ''PcF toxin family'' from the plant pathogens Phytophthora spp. It is able to induce withering in tomato and strawberry leaves. The lack of sequence similarity with other proteins hampers the identification of the molecular mechanisms responsible for its toxicity. Here, we show that the six cysteines form a disulphide pattern that is exclusive for PcF and essential for the protein withering activity. The NMR solution structure identifies a novel fold among protein effectors: a helix-loop-helix motif. The presence of a negatively charged surface suggests that it might act as a site of electrostatic interaction. Interestingly, a good fold match with Ole e 6, a plant protein with allergenic activity, highlighted the spatial superimposition of a stretch of identical residues. This finding suggests a possible biological activity based on molecular mimicry.

Comparative structural and functional characterization of putative protein effectors belonging to the PcF toxin family from Phytophthora spp

“…As expected, they share with PcF its all-alpha architecture with a helix-loop-helix core domain rigidly held in place by a peculiar disulfide bridging pattern, flanked by unstructured and flexible stretches at both N-and C-termini. 22 The Ramachandran plots of refined model structures, as evaluated by PROCHECK, showed overall good quality. For SCR91 indeed 71.4% residues resulted in the most allowed regions, 25% in the additionally allowed regions, and 1.8% in the generously allowed and disallowed regions.…”

“…1, top), interestingly including all six SSbridged cysteines of PcF recognized as essential for structuring its helix-loop-helix core domain. 22 Notably, only the SCR74 species possess two extra cysteines and thus one presumed additional SS bridge, not shared by other family members 20 ( Fig. 1, bottom).…”

“…22 The molecular characteristics predicted for each selected recombinant member of the PcF Toxin Family are summarized in Table I. In accordance with the present strategy, the protein effectors were subsequently obtained from overexpressing bacteria as detailed in ''Materials and Methods'' section.…”

“…Further IS-MS analyses also revealed no mass shifts after alkylating treatment carried out without prior protein reduction (not shown), thus confirming that six and eight SS-bridged cysteines, respectively, were originally present in SCR91 and SCR74 species, and involved in the formation of the three or four expected SAS bridges. Hence, due to cysteine conservation within the family and the obligatory integrity of disulfides for PcF function, 21,22 the disulfide linkages of PcF were taken as template constraints for structural prediction of PcF's homologs ( Fig. 1, bottom).…”

“…17 Its 3D structure recently solved by NMR unraveled a peculiar disulfide-stabilized helix-loophelix fold with a negative surface spot, highly suggestive of electrostatic interaction with yet unidentified target(s). 22 The protein behaves as a necrosis-inducing factor, that is it triggers HR symptoms of localized cell-death when applied on leaves of both strawberry and tomato. 17 In tomato, it also triggers intracellular induction of the key defense-related enzyme PAL 21 and other PR genes, as well as extracellular leakage of electrolytes (Orsomando & Ruggieri, unpublished).…”