Role of the Arginyl-Glycyl-Aspartic Motif in the Action of Ptr ToxA Produced byPyrenophora tritici-repentis 

Abstract

A fundamental problem of plant science is to understand the biochemical basis of plant/pathogen interactions. The foliar disease tan spot of wheat (Triticum aestivum), caused by Pyrenophora tritici-repentis, involves Ptr ToxA, a proteinaceous host-selective toxin that causes host cell death. The fungal gene ToxA encodes a 17.2-kD pre-pro-protein that is processed to produce the mature 13.2-kD toxin. Amino acids 140 to 142 of the pre-pro-protein form an arginyl-glycyl-aspartic (RGD) sequence, a motif involved in the binding of some animal proteins and pathogens to transmembrane receptor proteins called integrins. Integrin-like proteins have been identified in plants recently, but their role in plant biology is unclear. Our model for Ptr ToxA action predicts that toxin interacts with a putative host receptor through the RGD motif. Mutant clones of a ToxA cDNA, created by polymerase chain reaction such that the RGD in the pro-toxin was changed to arginyl-alanyl-aspartic or to arginyl-glycyl-glutamic, were expressed in Escherichia coli. Extracts containing mutated forms of toxin failed to cause host cell death, but extracts from E. coli expressing both a wild-type pro-protein cDNA and a control mutation away from RGD were active in cell death development. In competition experiments, 2 mm RGD tripeptide reduced the level of electrolyte leakage from wheat leaves by 63% when co-infiltrated with purified Ptr ToxA (15 g mL Ϫ1 ) obtained from the fungus, but the control peptide arginyl-glycyl-glutamyl-serine provided no protection. These experiments indicate that the RGD motif of Ptr ToxA is involved with toxin action, possibly by interacting with a putative integrin-like receptor in the host.One of the fundamental problems of plant biology is to understand the molecular and biochemical basis of plant disease caused by microbes. Some of the best models for understanding fungal pathogenicity and host susceptibility are those systems that involve pathogen-produced metabolites called host-selective toxins (HSTs). In many of these systems, host sensitivity to a toxin has been genetically associated with disease susceptibility. Further, toxin production by the pathogen has been associated with pathogenicity (the ability to cause disease) or with enhanced virulence (disease severity; Scheffer and Livingston, 1984; Walton and Panaccione, 1993). The simplest model for HST action predicts that toxin perception by the host is mediated through a host receptor (Scheffer and Livingston, 1984). Toxin/receptor interactions have been demonstrated for some HSTs, such as victorin, produced by Cochliobolus victorae, causal agent of victoria blight of oats (Avena sativa; Wolpert and Macko, 1989), and with Hmt toxin, the HST produced by Cochliobolus heterostrophus race T, the Southern corn (Zea mays) leaf blight pathogen (Braun et al., 1990). Toxin perception activates biochemical, physiological, and molecular events in the host that are associated with plant disease.Pyrenophora tritici-repentis (Died.) Dreschs. is the causal agent in the...

Structure of Ptr ToxA: An RGD-Containing Host-Selective Toxin fromPyrenophora tritici-repentis

“…To date, published mutation analyses of ToxA include 20 single mutations (Tuori et al, 2000;Meinhardt et al, 2002;Manning et al, 2004) that targeted the residues in the RGDcontaining segment (Asn136 through Phe147) and residues that represented potential phophorylation sites (Thr residues 115, 123, 126, 132, 139, and 167) or myristoylation sites (Gly residues 62 and 93) ( Table 2). In addition to these mutants, we present here data for a further three mutated residues (see Table 2 and Methods).…”

“…In the absence of structural or sequence conservation information to guide mutagenesis experiments, sequence motif searches were performed to find potential functional sites. The searches revealed putative sites of phosphorylation (Thr residues 115,123,126,132,139,and 167) (Ciuffetti et al, 1997;Zhang et al, 1997), of myristoylation (Gly residues 62 and 93) (Ciuffetti et al, 1997), and of cell attachment (an Arg-GlyAsp [RGD]-containing motif; residues Asn136 through Phe147) (Ciuffetti et al, 1997;Zhang et al, 1997;Meinhardt et al, 2002). Mutagenesis studies have identified a number of residues, including the RGD motif, that may be important for function (Tuori et al, 2000;Meinhardt et al, 2002;Manning et al, 2004), but without structural information it was not possible to conclusively deduce which residues were important for structural reasons and which might be directly involved in recognition events.…”

High affinity recognition of a Phytophthora protein by Arabidopsis via an RGD motif

“…Such recognition is a general strategy that allows microorganisms to modulate infectivity and to regulate toxicity towards host cells [5]. Recent observations report that RGD peptides interfere with the activation of plant defence responses [13][14][15]. Thus, RGD recognition through plant proteins may occur and RGD-containing proteins from phytopathogenic microorganisms may act as pathogenicity factors.…”

“…So far, very few RGD-containing proteins from plant pathogens have been described. We know of one virus protein, the precursor of G1 and G2 glycoproteins of the tomato spotted wilt virus [30]; two fungal proteins, ECP5 from Cladosporium fulvum [31] and Ptr ToxA from Pyrenophora tritici-repentis [14]; and one oomycete protein, IPI-O from Phytophthora infestans [19]. ECP5, Ptr ToxA and IPI-O are secreted proteins and also G1 and G2 are located outside the virus namely in the virus envelope membrane.…”

“…Integrins are also chosen by pathogens as targets for binding, and the RGD motif is present in surface proteins of several pathogens that attach to mammalian cells [5]. In plants, synthetic peptides containing the RGD motif can disrupt the adhesion between plasma membrane and cell wall [6][7] and can interfere with physiological processes thereby affecting, e.g., development [8][9][10][11], mechano-perception [12] and the interaction with microorganisms [13][14][15]. In particular, expression of cell wall associated defence responses triggered by fungal pathogens is dependent on adhesion between the plasma membrane and the plant cell wall [15].…”