Identification of Effector Genes in Foph

To validate the presence of homologous effectors (i.e., SIX, Ave1, and FOXM_16303), identified in our previous Foph-MAP5 transcriptomic analysis, we carried out two search strategies of the homolog effectors in a database that included the 22 genome sequences of the ff. spp. of F. oxysporum used for comparative genomics of Foph. The first strategy consisted in a tBlastn search. The hits with an e-value <0.0001 and identity higher than 50%, in the 50% of the length of the sequence query, were selected for further analysis. In the second strategy, a Blastx search was performed to identify all possible putative peptides of the homologous effectors in the F. oxysporum genome database. The best hits with an e-value <0.0001 were selected for further analysis.

To identify de novo candidate effector genes in Foph, the secretome and effectorome were predicted from the proteome of Foph_MAP5, using the software SignalP v5.0 (Almagro Armenteros et al., 2019) and EffectorP v2.0 (Sperschneider et al., 2018), respectively. In order to discard homologous sequences in other ff. spp., The two BLAST search strategies mentioned above were performed using the protein sequences positive for signal peptide and effector structure (i.e., <300 aa in length and cysteine rich) as a query. An additional search of Miniature Impala Transposable Elements (mimp) was performed in the UTR of the transcripts predicted of Foph, with the regular expression “NNCAGT[GA][GA]G[GAT][TGC]GCAA[TAG]AA,” using a customized Perl script as described by Schmidt et al. (2013) and van Dam and Rep (2017), to determine whether or not the novel candidate could correspond to SIX type genes.