ancestor of animals, plants, and fungi that produce these proteins nowadays (247). It is significant to note, however, that numerous typical small cysteine-rich pathogen effectors adopt AMP-like confirmations and that tertiary structures of many AMP families strongly resemble each and every other (27, 28). Hence, ERα custom synthesis structure prediction can very easily lead to false-positive classifications as AMP or allocation towards the wrong AMP family. CS defensins, or so-called cis-defensins, owe their structure to extremely conserved cis-orientated disulfide bonds that establish an interaction amongst a double- or triple-stranded antiparallel -sheet with an -helix (25, 27). To validate the prediction of VdAMP3 as a member of this ancient antimicrobial protein family members, we aligned its amino acid sequence using the antibacterial CS defensins plectasin and eurocin, from the saprophytic Ascomycete species Pseudoplectania nigrella and Eurotium amstelodami (formerly Aspergillus amstelodami), respectively (291). Despite the fact that the biological relevance of these defensins for the respective fungi remains unclear, their antibacterial activity and protein structure have already been properly characterized, which led to their recognition as genuine CS defensins (291). Even though the all round identity in between the 3 proteins was rather low (25 to 40 ), protein sequence alignment revealed that VdAMP3 contains the six very conserved cysteine residues which are regarded as crucial for the structure of CS defensins (Fig. 1B) (27). To further substantiate the emerging picture that VdAMP3 belongs to this unique protein family members and that the detected similarities with plectasin and eurocin are certainly not the outcome of convergent protein evolution, weAB CFig. 1. The V. dahliae effector VdAMP3 evolved from an ancient fungal protein. (A) VdAMP3 (Left) is predicted to adopt a CS defensin-like fold. The structure with the CS defensin plectasin (Right) of the fungus P. nigrella is integrated as CLK Formulation reference. The disulfide bonds stabilizing the antiparallel -sheets along with the -helix are highlighted in yellow. Positively and negatively charged amino acid residues are highlighted in blue and red, respectively. (B) Protein sequence alignment with CS defensins plectasin and eurocin (E. amstelodami) supports the structure prediction of VdAMP3. (C) VdAMP3 homologs are widespread inside the fungal kingdom. Protein sequence alignment of VdAMP3 using a subset of its homologs identified in higher (Ascomycota and Basidiomycota) and lower fungi (Mucoromycotina and Zoopagomycota). The alignment as shown in B and C displays one of the most conserved area from the CS defensin protein loved ones and was performed using HMMER and visualized with Espript3. The hugely conserved cysteine and glycine residues that contribute for the CS defensin structure are highlighted by yellow and red backgrounds, respectively. The numbers on best on the alignment indicate the corresponding residue numbers of VdAMP3. The homologs displayed in C had been identified employing blastP within the predicted proteomes from the respective fungi included in the JGI 1000 Fungal Genomes Project (32).two of 11 j PNAS doi.org/10.1073/pnas.Snelders et al. An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulationqueried the predicted proteomes of the fungi from the Joint Genome Institute (JGI) 1000 Fungal Genomes Project (32) for homologs of VdAMP3 with greater sequence identity and integrated a subset of these within the protein alignment (Fig. 1C). Interestingly, apart from homolog
