Computational protocol: An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor

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Protocol publication

[…] ATG8 interacting motif (AIM), also known as LC3 interacting region (LIR), mediates interaction of cargo receptors or adaptors with ATG8 proteins anchored in autophagosome membranes (). It follows the W/F/Y-xx-L/I/V amino acid consensus. Initially, we identified two AIM candidates in PexRD54, AIM1 and AIM2 based on the manual search of the consensus sequence mentioned earlier. Then, we confirmed these AIM candidates using the recently published iLIR software (). This software assigned AIM1 with an iLIR score of 12 (1.1e-1) and AIM2 with an iLIR score of 23 (3.2e-3). [...] To determine the ATG8 variant(s) specifically targeted by PexRD54 among various host ATG8 family members, we performed a BLASTP search () against solanaceous plant proteomes including Solanum tuberosum (potato), Solanum lycopersicum (tomato) and Nicotiana benthamiana using Arabidopsis thaliana ATG8C as the query protein sequence. We found nine ATG8 members in S. tuberosum, seven in S. lycopersicum, and eight in N. benthamiana. To verify the gene calls and open reading frame predictions of the putative orthologs in these three species, we performed a sequence alignment of the family members in each species using the Clustal X program (v2) () and compared it to the published A. thaliana ATG8 sequences. We found evidence of misannotation for two S. lycopersicum sequences (Solyc10g006270 and Solyc08g078820) and three others in N. benthamiana (NbS00015425g0005, NbS00003316g0005 and NbS00003005g0010). The two S. lycopersium sequences and the NbS00003005g0010 sequence from N. benthamiana carried extra nucleotides before the likely start codon, which were corrected accordingly. A TBLASTN search against the N. benthamiana scaffolds followed by a BLASTP search of the different exons allowed curation of the two other sequences. Clustal X program was used for multiple sequence alignment of ATG8 variants. Boxshade server (http://embnet.vital-it.ch/software/BOX_form.html) was used to visualize the sequence alignment. [...] The phylogenetic tree of ATG8 homologs in plants was constructed with the neighbor-joining method using ATG8-like curated proteins from S. tuberosum, S. lycopersium, N. benthamina, and A. thaliana. The phylogenetic tree was constructed using MEGA5 () with bootstrap values based on 1000 iterations. ATG8CL variants in N. benthamiana and potato have identical amino acid sequences. […]

Pipeline specifications

Software tools iLIR, BLASTP, Clustal W, TBLASTN, BoxShade, MEGA
Organisms Solanum tuberosum, Phytophthora infestans
Diseases Communicable Diseases